Prof. dr. Siska CROUBELS Department of Pharmacology, Toxicology and Biochemistry Faculty of Veterinary Medicine – Ghent University

1. MYCOTOXINS and ANIMAL HEALTH:
focus on intestinal health
Prof. dr. Siska CROUBELS
Siska.Croubels@ugent.be
Department of Pharmacology, Toxicology and Biochemistry
Faculty of Veterinary Medicine – Ghent University
IPV VVZ – 04.09.2014
www.mytox.be

2. Mycotoxins:
OUTLINE
• Introduction
• Occurrence in animal feed
• Effects in animals
• General
• Specific (aflatoxin B1, DON, T2-toxin, zearalenone,
fumonisins, ochratoxin A)
• Effects on intestinal barrier function
• Consequences of impaired barrier function
• Interaction with intestinal pathogens
• Absorption of nutrients
• Absorption of veterinary drugs (antibiotics)
• Transfer factors of residues to animal food products

3. LABORATORY OF PHARMACOLOGY & TOXICOLOGY
Mycotoxin research:
• Toxicity (cell viability)
• Toxicokinetics (ADME) of (masked) mycotoxins
• Tissue residues
• Interaction with pathogens
• Salmonella Typhimurium
• Clostridium perfringens
• Escherichia coli
• Interaction with pharmacokinetics of veterinary drugs
• Efficacy testing of mycotoxin binders

4. 2012

5. OCCURRENCE OF MYCOTOXINS IN EUROPEAN CEREALS
Analysis with LC-MS/MS 83 samples of feed and feed raw materials
Streit et al., 2013

6. OCCURRENCE OF MYCOTOXINS IN EUROPEAN CEREALS
Analysis with LC-MS/MS 83 samples of feed and feed raw materials
Streit et al., 2013

7. OCCURRENCE OF MYCOTOXINS IN EUROPEAN CEREALS
Global survey
feed and feed ingredients (2004-2011)
55% DON
54% fumonisins
36% ZEN
most of samples comply with EU regulations or recommendations
on the maximal tolerable concentration
(2002/32/EC, 2006/576/EC and 2013/165/EC)
mg/kg feed DON T-2 and HT-2 ZEN FB1+FB2 AFB1 OTA
Poultry 5.0 0.25 --- 20 0.02 0.10
Pigs 0.9 0.25 0.10 5 0.02 0.05
Calves 2.0 0.25 0.50 20 0.02 ---
Adult
5.0 0.25 0.50 50 0.02
---
ruminants
(dairy 0.005)
problem of co-contamination?

8. CLINICAL EFFECTS IN ANIMALS
Pig reduced performance
reduced feed utilization and efficiency
impaired fertility
skin lesions
Poultry reduced performance
“fatty liver” syndrome
decreased egg production
poor carcass quality
Cattle reduced milk yield
impaired fertility
mastitis
ketonuria
Horse neurotoxic syndrome
Subclinical doses: immunosuppressive effects

9. Dependent on
CLINICAL EFFECTS IN ANIMALS
- type and dose
- duration of exposure
- species, gender, age, health status
- synergistic effects (single vs. multiple toxins)
www.efsa.eu.int
www.knowmycotoxins.com
www.thepoultrysite.com
www.mycotoxins.info

10. EFFECTS OF MYCOTOXINS
www.mycotoxins.info

11. EFFECTS OF MYCOTOXINS
www.mycotoxins.info

12. EFFECTS OF MYCOTOXINS
www.mycotoxins.info

13. META-ANALYSIS FOR INTERACTIONS OF MYCOTOXINS
WITH PERFORMANCE IN BROILERS
98 papers between 1980-2009, in total 1,401 diets and 37,371 birds
Mycotoxins Ochratoxins Aflatoxins Deoxynivalenol
Andretta et al., 2011
(DON)
Feed intake
reduction
12% 17% 11%
Weight gain
reduction
14% 20% 11%
Mortality
increase
2.8x 8.5x
Mycotoxins influence broiler performance, productive indices
and organ weight, magnitude dependent on:
type and concentration
duration of exposure
animal age
nutritional factors

14. AFLATOXINS
Aflatoxin B1 most toxic (blue fluorescence, UV-light)
carcinogen (a.o. rat, duck, trout fish)
IARC class 1 - Ames test and
inactivation of tumour suppressor p53 gene
(mutation at codon 249)
Aflatoxin B2 dihydro derivative, less toxic (+/- 20%)
potentialises B1

15. AFLATOXINS
Toxicokinetics
Oral bioavailability: about 100%
Biotransformation: AFB-8,9 epoxidation (liver enzymes CYP450)
Elimination: urinary, biliary and milk (1-6%)

16. AFLATOXINS
Symptoms and lesions
A. Acute aflatoxicosis
massive intake
anorexia, depression, malabsorption, bloody stools,
acute liver degeneration
 icterus and secundary hemorrhages (vit. K1 treatment)
B. Chronic exposure
production losses
icterus, ‘pale bird syndrome’
hepatocellular carcinoma
liver function impairment
immunosuppression (Salmonella, Coccidia, …)
Residues in food
milk: aflatoxin M1 toxicity ~ aflatoxin B1
EU: 0.05 μg/kg AFM1 in raw milk, heat-treated milk and
milk manufactured for milk-based products
(ALARA, Commission Regulation (EU) No 165/2010)

17. 2013-2014

18. TRICHOTHECENES: DON and T2-TOXIN
Toxicokinetics
Oral bioavailability DON: 20-25% (broiler chicken) and 55-80% (pigs)
Biotransformation: de-epoxidation
Excretion: urinary and biliary
T2-toxin DON
Toxicity
Inhibition protein synthesis
Increase cytokine release (IL-6)
Increase IgA, decrease IgG, IgM
IARC class 3

19. Symptoms
DON
TRICHOTHECENES: DON
in pigs “Abdominal distress syndrome”
vomiting and/or anorexia, diarrhea (from 2000 ppb)
feed refusal (from1200 ppb)
reduced feed intake (from1000 ppb)
immunosuppression (from100 ppb?)

20. Symptoms
TRICHOTHECENES: T2-TOXIN
“Alimentary toxic aleukia (ALTA)”
radiomimetic effect
leukopenia, thrombocytopenia, cell necrosis
(dermatitis, conjunctivitis, stomatitis,
gastro-enteritis) hemorrhagic diathesis
immunosuppression
Bron: Prof. J. Fink-Gremmels

21. ZEARALENONE
Fusarium graminearum, F. culmorum
Maize, wheat, corn cob mix
Symptoms
hyperoestrogenism, swollen edematous vulva, uterus prolapse,
rectal prolapse, vulvovaginitis, atrophy of the ovaries, ...
 mainly reproduction problems
Sensitive species: pig, sheep, dog, cattle

22. Toxicokinetics
Oral bioavailability: 80-85%
Biotransformation: reduction to ,-zearalenol, further to -
zearalanol (zeranol) and -zearalanol (taleranol)
Excretion: urinary and biliary
Binds on oestrogen receptors ER and ER
-zearalenol factor 138 uterotrophicity
-zearalenol factor 0.5
IARC class 3
ZEARALENONE

23. FUMONISINS
Pathogenesis
Oral bioavailability: 3-6%
1. Inhibition sfinganine N-acyltransferase (ceramid synthase)
2. Increase sfinganine and sfinganine/sfingosine ratio
(Sa/So in serum, urine and tissues)
IARC class 2B, oesophageal and hepatic cancer

24. Symptoms
1. horse
FUMONISINS
equine leucoencephalomalacia (ELEM)
necrosis in cerebrum
(ataxia, paresis, apathia, coma)
+ liver pathology
2. pig
pulmonary porcine edema (PPE)
lung edema, hydrothorax
+ liver- and pancreas pathology
3. broiler chickens
motor disturbances, immunosuppression
LOAEL: 2 mg/kg BW/day for broilers
ELEM horse

25. OCHRATOXINS: OCHRATOXIN A
Toxicokinetics
Oral bioavailability: +/- 65 %
96 to 99.8 % plasma protein binding
OTA is substrate for oatp (renal accumulation)
Excretion: urinary and biliary,
milk (monogastric animals and humans, cattle < 1%)
IARC class 2B
pig (MPN = mycotoxic porcine nephropathy)
inhibition of protein synthesis
renal pathologies, e.g. Balkan endemic nephropathy (BEN)
urinary tract tumours, nephritis, renal karyomegaly

26. GASTROINTESTINAL TRACT: FIRST TARGET FOR
MYCOTOXINS
Small intestine:
• Absorption of water and nutrients
• Dynamic barrier

27. gut lumen
intestinal
permeability
↑ by DON, FB1
intestinal
epithelium
cell proliferation
↓ by DON, T-2, FB1,
OTA and AFB1
lamina propria
IgA production
↑ by DON and
nivalenol, ↓ by T-2 antimicrobial
mucus production
↓ by DON + T-2 + ZEN
↑ ZEN alone
sIgA
IEC goblet cell paneth cell
plasmocyte
secreting Ig
peptides
unknown
alter cytokines production
DON, T-2, nivalenol, FB1,
ZEN, patulin, gliotoxin
Antonissen et al., 2014
Bouhet and Oswald, 2005

28. MODULATION OF INTESTINAL FUNCTIONS BY
MYCOTOXINS: META-ANALYSIS
about 100 papers, in total 7 intestinal processes were investigated
Mycotoxins, in particular DON, at realistic doses can compromise
digestion, absorption, permeability and defense
Grenier and Applegate, 2013

29. EFFECTS ON INTESTINAL BARRIER FUNCTION
 Analyze the effects on the intestinal barrier function
- gut wall morphology
- measurement of the transepithelial electrical resistance (TEER)
 Identify the underlying mechanisms
- tight junction proteins
- oxidative stress markers
- inflammatory proteins
- nutrient and xenobiotic transporter proteins
- biotransformation enzymes
 Consequences of an altered barrier function
- passage of macromolecules and bacteria
→ results obtained from swine and poultry

30. IN VITRO VIABILITY OF ENTEROCYTES
Importance: cell death may result in damage to intestinal barrier
Mycotoxin T-2 DON ZEA FB1
Cytotoxic
concentration
3 ng/ml 2.5 μg/ml 9 μg/ml 20 μg/ml
most toxic
Goossens et al., 2012
100
80
60
40
20
0
Viability
0 1 2.5 5 7.5 10
Percentage viable cells (%)
Concentration DON (μg/ml)
Concentration-dependent effect on viability of intestinal porcine
epithelial cells derived from the jejunum

31. DON DECREASES VILLUS HEIGHT IN BROILERS
Control DON
Duodenum
(μm)
1734 ± 26 a 1449 ± 31 b
Jejunum
(μm)
1343 ± 37 a 1184 ± 48 b
Ileum
(μm)
596 ± 30 a 616 ± 38 a
Duodenum
*
DON ≤ 5 mg/kg (2006/576/EC)
3 weeks feeding trial
Antonissen et al., 2014;
Böhm et al., 2006; Yunus et al., 2012
DON > 5 mg/kg
3 weeks feeding trial
Osselaere et al., 2013;
Awad et al., 2006

32. DON DECREASES VILLUS HEIGHT IN TURKEYS
2300
2200
2100
2000
1900
1800
1700
1600
1500
Duodenum: villus height
Control Contaminated
Villus height (μm)
DON ± 5 mg/kg (2006/576/EC)
12 weeks feeding trial
Devreese et al., 2014
*
*
3100
3000
2900
2800
2700
2600
2500
Control Contaminated
Villus surface area (mm2)
Duodenum: apparent villus
surface area

33. T2-TOXIN DECREASES VILLUS HEIGHT IN PIGS
700
600
500
400
300
200
100
0
Blank feed Blank feed +
Alphamune®
T2 ≤ 250 μg/kg (2013/165/EC)
3 weeks feeding trial
Goossens et al., 2013
T-2 T-2 +
Alphamune®
Villus length (μm)
Villus Duodenum
Crypth

34. DON DECREASES INTESTINAL GENE EXPRESSION OF
TIGHT JUNCTION PROTEINS IN BROILERS
Protocol
•one-day-old broilers fed for 3 weeks with DON at 7.5 mg/kg, incl. adsorbing agent (binder)
•sections from duodenum, jejunum and ileum
•qRT-PCR analysis for CLDN1, CLDN5, ZO1, ZO2
Tight junctions:
•Duodenum: no effects
•Jejunum:
30
25
20
15
10
5
0
Control Adsorbent DON DON +
Adsorbent
CLDN1
CLDN5
ZO1
ZO2
Osselaere et al., 2013

35. DON DECREASES INTESTINAL EXPRESSION OF TIGHT
JUNCTION PROTEINS IN PIGS
Pinton et al., 2009
Oswald, 2013

36. MYCOTOXINS IMPAIR INTESTINAL BARRIER
Altered intestinal functions:
CONSEQUENCES?
Enhanced susceptibility to enteric infections?
Altered digestion and absorption of nutrients?
Altered absorption of veterinary drugs and feed
additives?

37. CONSEQUENCES: SUSCEPTIBILITY TO ENTERIC
INFECTIONS
Deoxynivalenol and also fumonisins predispose for the development of
necrotic enteritis in broilers
Experimental in vivo model for subclinical necrotic enteritis
Fig. Gunther Antonissen
Fig. Leen Timbermont
Antonissen et al., 2014
Experimental groups:
1. C. perfringens + control diet
2. C. perfringens + fumonisins
3. C. perfringens + fumonisins + DON
4. C. perfringens + DON
Concentration DON: +/- 4 mg/kg feed and
fumonisins +/- 20 mg/kg feed

38. CONSEQUENCES: SUSCEPTIBILITY TO ENTERIC
INFECTIONS
predisposing for necrotic enteritis?
RESULTS

39. CONSEQUENCES: SUSCEPTIBILITY TO ENTERIC
INFECTIONS
Influence of mycotoxin contamination on intestinal protein availability
for clostridial proliferation
Antonissen et al., 2014

40. CONSEQUENCES: SUSCEPTIBILITY TO ENTERIC
INFECTIONS
Antonissen et al., 2014

41. CONSEQUENCES: SUSCEPTIBILITY TO INFECTIONS
Fusarium mycotoxins  intestinal and systemic phase of the
pathogenesis of Salmonella Typhimurium
gut lumen
lamina propria
1
2
3
4
intestinal
epithelial
cell
macrophage
DON and T-2
1
↑ intestinal
Salmonella invasion
2
↑ passage
Salmonella across
epithelium
3 ↑ macrophage invasion
4
no effect intracellular
bacterial proliferation
Vandenbroucke et al., 2009; Verbrugghe et al., 2012

42. 42
CONSEQUENCES: ABSORPTION OF NUTRIENTS
control diet DON diet
≈ 5 mg/kg
fumonisins
diet
≈ 20 mg/kg
FB1+FB2+FB3
DON + fumonisins
diet
≈ 5 mg/kg and 20
mg/kg
Ross 308
224 chicks
4 groups
(56 birds/group)

43. CONSEQUENCES: ABSORPTION OF NUTRIENTS
b0,+AT Na+-independent neutral and dibasic amino acid transporter
rBAT protein related to b0,+AT
B0AT Na+-dependent neutral amino acid transporter
EAAT3 excitatory amino acid transporter-3
ASCT1 alanine, serine, cysteine and threonine transporter-1
CAT1 cationic amino acid transporter-1
CAT2 cationic amino acid transporter-2
LAT1 L type amino acid transporter
y+LAT1 y+ L amino acid transporter-1
y+LAT2 y+ L amino acid transporter-2
PepT1 peptide transporter-1
APN aminopeptidase N
SI sucrase isomaltase
GLUT2 glucose transporter-2
GLUT5 glucose transporter-5
SGLT1 sodium glucose transporter-1
NPT2b type II sodium-dependent phosphate cotransporter
ZNT1 zinc transporter-1
qRT-PCR expression of
genes encoding:
• digestive enzymes
• amino acid
transporters
• peptide transporters
• sugar transporters
• mineral transporters
transporters located at
 brushborder
 basolateral
membranes
of enterocytes

44. CONSEQUENCES: ABSORPTION OF NUTRIENTS
*
ZINC transporter -1
- located at basolateral
membrane
- exports zinc: cell 
circulation
ZINC
= essential micronutrient
 enzyme activities
 DNA and protein synthesis
 inhibitor of apoptosis and
oxidative stress
↓ expression
 intracellular zinc concentration is preserved during e.g.
oxidative stress evoked by DON (Osselaere et al., 2013)

45. CONSEQUENCES: ABSORPTION OF ANTIBIOTICS
N = 6
blank
feed
N = 6
100 μg/kg T-2
contaminated feed
(<250 μg/kg (2013/165/EC))
3 weeks
1 week
acclimatisation
Oral bolus chlortetracycline (20 mg/kg BW)
Blood samples: 0 - 0.5 – 1 - 1.5 – 2 – 3 – 4 – 6 – 8 – 12 - 24 h post
administration and analysis with HPLC-UV

46. CONSEQUENCES: ABSORPTION OF ANTIBIOTICS
Plasma concentration-time profile of chlortetracycline
Goossens et al., 2013
2.00
1.50
1.00
0.50
0.00
0 4 8 12 16 20 24
Concentration CTC (μg/ml)
Time (hours p.a.)
Blank feed
T-2
Significant increased plasma concentrations after intake of
T-2 contaminated feed for 3 weeks
↓
Effect on tissue concentrations of antibiotics?
Therapeutic efficacy and public health?

47. TRANSFER OF MYCOTOXINS TO ANIMAL FOOD
PRODUCTS

48. TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

49. TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

50. TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

51. TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

52. TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

53. FURTHER NEEDS
Effect on oral
absorption of drugs,
coccidiostats and
other mycotoxins:
increased passage?
Altered
intestinal
functions
Effect of mycotoxins
on intestinal
microbiota and
pathogens?
Effect of other
mycotoxins and co-contaminants
on the
gastro-intestinal tract
0 50 100 150 200
Fate of masked
30.000
25.000
20.000
15.000
10.000
5.000
0.000
mycotoxins: in vivo
hydrolysis of these
conjugated forms?
DON-3-glucoside
3ADON PO
DON
3ADON

54. TAKE-HOME MESSAGES
• Feed contamination: 1 mycotoxin << multimycotoxin
(1+1 = sometimes 3)
• Effect of high contamination levels of the feed (rare) on animal health
is mostly known, however the effects of low to moderate
contamination levels (frequently occurring) is less well-known
• Even low contamination levels (< max. EU guidance levels) can have a
negative effect on poultry, pigs, …
• Economic impact of mycotoxins ≠ animals with clinical mycotoxicosis

55. TAKE-HOME MESSAGES
• Negative impact on the intestinal morphology and function:
- enhanced susceptibility for infectious diseases
- altered digestion and absorption of nutrients
- altered absorption of veterinary drugs
• Modulate the immune response
• (Partial) hydrolysis of masked DON and release of native DON in
poultry and pigs
• Limited transfer of residues to animal food products, except e.g. for
aflatoxin B1 (M1) to milk and ochratoxin A to kidney tissue

56. More information:
www.vetftb.ugent.be
www.mytox.be