mycotoxins and animal health: focus on intestinal health

Prof. dr. Siska CROUBELS

[email protected]

Department of Pharmacology, Toxicology and Biochemistry

Faculty of Veterinary Medicine – Ghent University

IPV VVZ – 04.09.2014

MYCOTOXINS and ANIMAL HEALTH:focus on intestinal health

www.mytox.be

mailto:[email protected]

Mycotoxins:

• 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

OUTLINE

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

LABORATORY OF PHARMACOLOGY & TOXICOLOGY

OCCURRENCE OF MYCOTOXINS IN EUROPEAN CEREALS

Streit et al., 2013

Analysis with LC-MS/MS 83 samples of feed and feed raw materials

OCCURRENCE OF MYCOTOXINS IN EUROPEAN CEREALS

Global surveyfeed and feed ingredients (2004-2011)

55% DON54% 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 ruminants

5.0 0.25 0.50 50 0.02(dairy 0.005)

---

problem of co-contamination?

Pig reduced performancereduced feed utilization and efficiencyimpaired fertilityskin lesions

Poultry reduced performance“fatty liver” syndromedecreased egg productionpoor carcass quality

Cattle reduced milk yieldimpaired fertilitymastitisketonuria

Horse neurotoxic syndrome

Subclinical doses: immunosuppressive effects

CLINICAL EFFECTS IN ANIMALS

Dependent on

- type and dose

- duration of exposure

- species, gender, age, health status

- synergistic effects (single vs. multiple toxins)

www.efsa.eu.intwww.knowmycotoxins.com

www.thepoultrysite.comwww.mycotoxins.info

CLINICAL EFFECTS IN ANIMALS

http://www.thepoultrysite.com/



http://www.mycotoxins.info/

EFFECTS OF MYCOTOXINS

www.mycotoxins.info

META-ANALYSIS FOR INTERACTIONS OF MYCOTOXINS WITH PERFORMANCE IN BROILERS

Andretta et al., 2011

98 papers between 1980-2009, in total 1,401 diets and 37,371 birds

Mycotoxins Ochratoxins Aflatoxins Deoxynivalenol (DON)

Feed intake reduction

12% 17% 11%

Weight gain reduction

14% 20% 11%

Mortalityincrease

2.8x 8.5x

Mycotoxins influence broiler performance, productive indices and organ weight, magnitude dependent on:

type and concentrationduration of exposureanimal age

nutritional factors

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

AFLATOXINS

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

AFLATOXINS

Symptoms and lesionsA. 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 impairmentimmunosuppression (Salmonella, Coccidia, …)

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)

Residues in food

2013-2014

TRICHOTHECENES: DON and T2-TOXIN ToxicokineticsOral bioavailability DON: 20-25% (broiler chicken) and 55-80% (pigs)Biotransformation: de-epoxidationExcretion: urinary and biliary

T2-toxin DON

ToxicityInhibition protein synthesisIncrease cytokine release (IL-6)Increase IgA, decrease IgG, IgMIARC class 3

Symptoms

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?)

TRICHOTHECENES: DON

Symptoms

“Alimentary toxic aleukia (ALTA)”

radiomimetic effectleukopenia, thrombocytopenia, cell necrosis(dermatitis, conjunctivitis, stomatitis, gastro-enteritis) hemorrhagic diathesisimmunosuppression

TRICHOTHECENES: T2-TOXIN

Bron: Prof. J. Fink-Gremmels

Fusarium graminearum, F. culmorumMaize, 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

ZEARALENONE

ToxicokineticsOral 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

FUMONISINS

PathogenesisOral 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

Symptoms

1. horseequine leucoencephalomalacia (ELEM)necrosis in cerebrum(ataxia, paresis, apathia, coma)+ liver pathology

2. pigpulmonary porcine edema (PPE)

lung edema, hydrothorax + liver- and pancreas pathology

3. broiler chickensmotor disturbances, immunosuppressionLOAEL: 2 mg/kg BW/day for broilers

FUMONISINS

ELEM horse

OCHRATOXINS: OCHRATOXIN A

ToxicokineticsOral bioavailability: +/- 65 %96 to 99.8 % plasma protein bindingOTA 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 synthesisrenal pathologies, e.g. Balkan endemic nephropathy (BEN)

urinary tract tumours, nephritis, renal karyomegaly

Small intestine:• Absorption of water and nutrients• Dynamic barrier

GASTROINTESTINAL TRACT: FIRST TARGET FOR MYCOTOXINS

inte

stin

al

epit

hel

ium

lamina propria

gut lumen

intestinal permeability

↑ by DON, FB1

cell proliferation↓ by DON, T-2, FB1,

OTA and AFB1mucus production

↓ by DON + T-2 + ZEN↑ ZEN alone

sIgA

IEC goblet cell paneth cell

plasmocyte secreting Ig

IgA production↑ by DON and

nivalenol, ↓ by T-2 antimicrobial peptidesunknown

alter cytokines productionDON, T-2, nivalenol, FB1,

ZEN, patulin, gliotoxin

Antonissen et al., 2014Bouhet and Oswald, 2005

MODULATION OF INTESTINAL FUNCTIONS BY MYCOTOXINS: META-ANALYSIS

Grenier and Applegate, 2013

about 100 papers, in total 7 intestinal processes were investigated

Mycotoxins, in particular DON, at realistic doses can compromise digestion, absorption, permeability and defense

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

Mycotoxin T-2 DON ZEA FB1

Cytotoxic concentration

3 ng/ml 2.5 µg/ml 9 µg/ml 20 µg/mlmost toxic

IN VITRO VIABILITY OF ENTEROCYTES

Goossens et al., 2012

Concentration-dependent effect on viability of intestinal porcine epithelial cells derived from the jejunum

Importance: cell death may result in damage to intestinal barrier

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

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/kg3 weeks feeding trial

Osselaere et al., 2013;Awad et al., 2006

Duodenum

*

DON DECREASES VILLUS HEIGHT IN TURKEYS

DON ± 5 mg/kg (2006/576/EC)12 weeks feeding trial

Devreese et al., 2014

*

T2-TOXIN DECREASES VILLUS HEIGHT IN PIGS

T2 ≤ 250 µg/kg (2013/165/EC)3 weeks feeding trial


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:

Osselaere et al., 2013

DON DECREASES INTESTINAL EXPRESSION OF TIGHT JUNCTION PROTEINS IN PIGS

Pinton et al., 2009Oswald, 2013

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?

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. Leen Timbermont

Fig. Gunther Antonissen

Antonissen et al., 2014

Experimental groups:

1.C. perfringens + control diet2.C. perfringens + fumonisins3.C. perfringens + fumonisins + DON4.C. perfringens + DON

Concentration DON: +/- 4 mg/kg feed and fumonisins +/- 20 mg/kg feed Fig. Leen Timbermont

Fig. Gunther Antonissen

predisposing for necrotic enteritis?RESULTS



Influence of mycotoxin contamination on intestinal protein availability for clostridial proliferation


Fusarium mycotoxins intestinal and systemic phase of the pathogenesis of Salmonella Typhimurium

gut lumen

lamina propria

1

2

3

4

inte

stina

l ep

ithel

ial

cell

mac

roph

age

DON and T-2

3 ↑ macrophage invasion

4no effect intracellular bacterial proliferation

Vandenbroucke et al., 2009; Verbrugghe et al., 2012

1↑ intestinal Salmonella invasion

2↑ passage Salmonella across epithelium

CONSEQUENCES: SUSCEPTIBILITY TO INFECTIONS

42

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 308224 chicks

4 groups(56 birds/group)

CONSEQUENCES: ABSORPTION OF NUTRIENTS

b0,+AT Na+-independent neutral and dibasic amino acid transporterrBAT protein related to b0,+ATB0AT Na+-dependent neutral amino acid transporterEAAT3 excitatory amino acid transporter-3ASCT1 alanine, serine, cysteine and threonine transporter-1CAT1 cationic amino acid transporter-1CAT2 cationic amino acid transporter-2LAT1 L type amino acid transportery+LAT1 y+ L amino acid transporter-1y+LAT2 y+ L amino acid transporter-2PepT1 peptide transporter-1APN aminopeptidase NSI sucrase isomaltaseGLUT2 glucose transporter-2GLUT5 glucose transporter-5SGLT1 sodium glucose transporter-1NPT2b type II sodium-dependent phosphate cotransporterZNT1 zinc transporter-1

qRT-PCR expression of genes encoding: •digestive enzymes •amino acid transporters•peptide transporters•sugar transporters•mineral transporters

transporters located atbrushborder basolateral membranes of enterocytes


*

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)


N = 6

blankfeed

100 µg/kg T-2 contaminated feed

(<250 µg/kg (2013/165/EC))

N = 6

3 weeks

1 week acclimatisation

CONSEQUENCES: ABSORPTION OF ANTIBIOTICS

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

CONSEQUENCES: ABSORPTION OF ANTIBIOTICS

Plasma concentration-time profile of chlortetracycline


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?

↓

TRANSFER OF MYCOTOXINS TO ANIMAL FOOD PRODUCTS

TRANSFER FACTORS IN FOOD-PRODUCING ANIMALS

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?

Fate of masked mycotoxins: in vivo hydrolysis of these conjugated forms?

Effect of other mycotoxins and co-contaminants on the gastro-intestinal tract

DON-3-glucoside

•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

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

TAKE-HOME MESSAGES

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http://www.vetft.ugent.be/

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mycotoxins and animal health: focus on intestinal health

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