Can Mycotoxins Effect Gastro- intestinal Tract (GIT) Function?

Ochratoxins: Damaged kidneys

T-2 Toxin: Oral and dermal lesions Aflatoxin B1: Fatty liver

Can Mycotoxins Effect Gastro-intestinal Tract (GIT) Function?

Todd J. Applegate

Do Mycotoxins affect the Gastro-intestinal Tract (GIT)?

• Plausible change in nutrient content of feedstuffs contaminated with mold

• Feed intake responses to growth vs physiology

• Differentiation between “post-GIT” metabolic inefficiencies vs physiological responses

• Differences in absorption (some rapid vs virtually non-absorbed)

Reduction in nutritional quality of corn

0

1

2

3

4

5

6

7

8

9

10

Energy Protein Fat

Control Moldy

% -or

kcal/g

Tindall (1983)

Feedstuff Nutrient/Energy

Change in nutritional quality of wheat

Dänicke et al. (2007)

Feedstuff Nutrient/Energy

CW = control wheat diet*

FIW = Fusarium infected diet

(~ 1.4 mg/kg DON)

+/- endo-1,4-β-xylanase

*Control wheat 1% less CP

Differences in non-starch

polysaccharide content???

Ochratoxins: Damaged kidneys

T-2 Toxin: Oral and dermal lesions

Aflatoxin B1: Fatty liver

GIT & Functional “Upkeep”

• GIT consumes approximately 20% of dietary energy

• Protein turn-over rate of 50 to 75% per day (Cant et al., 1996)

• ~25% of daily protein synthesis can be secreted into the gut (Simeon et al., 1983)

NUTRITION

DEFENSE

STUDIES

Grenier and Applegate, 2013

Mycotoxin

Absorption rate

Pig Poultry

AF >80% >80%

OTA 65% 40%

DON 55% 5-20%

FB 3-6 % 1%

du

od

en

um

jeju

nu

mje

jun

um

ileu

m

Most of the absorbed dose

(80-90%) occur in the upper

part of the GIT

DON

ZEA

AF

OTA

Enterohepatic circulation

may increase the exposure

all along the GIT

DON

T-2 toxin

ZEA

FB

OTA


Mycotoxin

Absorption rate

Pig Poultry

AF >80% >80%

OTA 65% 40%

DON 55% 5-20%

FB 3-6 % 1%

du

od

en

um

jeju

nu

mje

jun

um

ileu

m



part of the GIT

DON

ZEA

AF

OTA



all along the GIT

DON

T-2 toxin

ZEA

FB

OTA



PSA meeting (1975)


PSA meeting (1975)

Realistic AFLA = < 0.3 mg/kg

Occasional AFLA = > 0.3 to 2 mg/kg

Unrealistic AFLA = > 2 mg/kg

Are results applicable from

“Unrealistic doses” to “realistic /

occasional doses”

Liver

Osborne et al. (1982)

Max. “Occasional” doses

Aflatoxin = Malabsorption???

Liver


Vs.Pancreas




D. Ledoux

Max. “Occasional” doses

Mycotoxins

Clinical Mycotoxicosis:Lesions

Sub -Clinical Mycotoxicosis:Impaired immune function,

performance loss, FCR reduction

Unknowns…..

Mycotoxin interactions???

Mycotoxins

Performance

Biomarkers (physiology,

toxicology, immunology,

nutritional consequences…)

Unknowns…..

Mycotoxin interactions???

Performance is

2400

2600

2800

3000

3200

0 0.6 1.2

kca

l /

kg

AFLA (mg/kg)

AMEn ADE

Hen Energy Utilization

0.6 and 1.2 mg/kg ↓ ADE (10%) and AMEn (4%)Applegate et al., 2009

NUTRITION

0

500

1000

1500

2000

2500

3000

Control 1.5 ppm AFB

2051

2619

Endogenous N loss (mg/kg DM intake)

P-value 0.09

28%

Chen et al., 2015

Dietary Protein & Aflatoxin

Small intestinal Morphology

-200

0

200

400

600

800

1000

0 0.6 1.2 2.5

AFLA B1 (mg/kg)

He

igh

t o

r d

ep

th, m

icro

ns

Villus length; no effect

Crypt depth; linear increase (P < 0.016)

Goblet cells; no effect (number or number / villus length)

PHYSIOLOGY

0.05 0.06 0.04

0.19

0.13

0.06

0.00

0.05

0.10

0.15

0.20

18 22 26

Dietary CP concentration (%)

Lactulose/Rhamnose Ratio

0 ppm AF

1.5ppm AF

AFB P=0.04

L: R ratio = Impaired barrier integrity

Chen et al., 2015Dietary Protein & Aflatoxin: Gut Permeability (dual sugar test)

MYCOTOXIN DOSE SPECIES OUTCOMES

Aflatoxin Realistic Duck & HenModulation of activity of digestive enzymes

(protease, amylase, trypsin and chymotrypsin)

Aflatoxin Realistic Duck Reduced apparent digestibility of crude protein

Aflatoxin ModerateChicken &

Hen

Reduced apparent digestibility, digestible &

metabolizable energy

INTESTINE: FIRST TARGETNUTRITION

DIGESTIVE PROCESSES


Mycotoxin

Absorption rate

Pig Poultry

AF >80% >80%

OTA 65% 40%

DON 55% 5-20%

FB 3-6 % 1%

du

od

en

um

jeju

nu

mje

jun

um

ileu

m



part of the GIT

DON

ZEA

AF

OTA



all along the GIT

DON

T-2 toxin

ZEA

FB

OTA


IMPACT OF THE

NON-ABSORBED PART ??

MYCOTOXIN DOSE SPECIES OUTCOMES

Aflatoxin Realistic Duck & HenModulation of activity of digestive enzymes

(protease, amylase, trypsin and chymotrypsin)

Aflatoxin Realistic Duck Reduced apparent digestibility of crude protein

Aflatoxin ModerateChicken &

Hen

Reduced apparent digestibility, digestible &

metabolizable energy

Fusariotoxins Moderate ChickenIncreased protein digestibility & net protein

utilization

Fumonisin Moderate Rat & Pig Reduced nutrient digestibility

INTESTINE: FIRST TARGETNUTRITION

DIGESTIVE PROCESSES


Grenier et al. 2015

Spinganine (Sa):

Sphingosine (So)

ratio – Fumonisin

Effect in Broilers

↑ inflammatory

markers

Voss e

t al.,

2007

Micro-villi

Paracellular route

Apical surface

Basolateral surface

ZO-1ZO-1 Occludin

Claudin

Claudin

E-cadherin

Transcellular route

Na+/K+ Pump

Na+

SGLT1

GLUT2

Glucose

Glucose

Na+

Na+

Glucose

K+

K+

Lower uptake of nutrients, such as glucoseMalabsorption of water

IF IMPAIRMENT:

Investigations on

permeability:

mostly with DON

reduced TEER,

expression of claudin

family and ZO-1

Higher translocation of luminal antigens- commensal flora- pathogens- food antigens- toxins and mycotoxins

IF IMPAIRMENT:

INTESTINE: FIRST TARGETPERMEABILITY


Effects of DON and FUM in broilers challenged with Coccidiosis vaccine

Treatments (4 diets x cocci challenge/no challenge):

Control feed,

DON (2 mg/kg),

FUM (20 mg/kg),

DON+FUM (2 and 20 mg/kg)

Challenge: Coccivac-B 25X (mix of 4 strains of Eimeria) at day 14

Grenier et al., 2015

DON & cellular barrier integrity in IPEC-J2 cells

0

20

40

60

80

100

120

140

1h 24h 48hTE

ER

(%

of

con

tro

l)

1000 ng/mL DON 2000 ng/mL DON 4000 ng/mL DON

aa

b

ab

c

0

500

1000

1500

2000

0 4000

FIT

C-d

extr

an f

lux

ng

/cm

2 ·h…

DON concentration ng/mL

b

a

• DON impaired cellular barrier integrity in IPEC-J2 by decreasing TEER value and increasing FITC-dextran passage.

Zhang & Applegate, unpublished

0

2

4

6

8

10

0 5 10

Re

lati

ve m

RN

A e

xpre

ssio

n o

f C

LDN

1

Time (h)

control

DON

*

****

1 2 3 4 5 6 12 24

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6

Re

lati

ve d

en

sity

of

CLD

N1

Time (h)

Control

DON

1 2 3 4

** DON ↓ CLDN1 Protein (3h)

prior to ↑of mRNA (4h) in

IPEC-J2 cells.

(translation of RNA can not

keep pace with protein

degradation)

Zhang & Applegate, unpublished

mRNA

Protein

ZO-1ZO-1 Occludin

Claudin

Claudin

E-cadherin

DON & Claudin1 (CLDN1)

Major findings: LESION SCORE & OOCYST COUNTS

0

1

2

3

Ctrl DON FUM D+F

0 50000 100000 150000 200000 250000

Ctrl

DON

FUM

D+F

0 1000000 2000000 3000000

Ctrl

DON

FUM

D+F

Lesion score in ceca Number of oocysts in mucosa

Number of oocysts in excreta

Combinded effects: FUM+DON+Eimeria

DON (2 mg/kg),

FUM (20 mg/kg),

DON+FUM (2 and 20 mg/kg)Grenier et al., 2015

Major Findings: Spinganine (Sa): Sphingosine (So) ratio

0.0

0.5

1.0

1.5

2.0

Ctrl DON FUM D+F Ctrl DON FUM D+F Ctrl DON FUM D+F

Sa/S

o r

atio

Liver Serum Ileum

***

**

*** **

*

Unchallenged birds

Challenged birds

Coccidial challenge compromised the intestinal barrier (confirmed by histology with destruction of the epithelium) and facilitated the passage of FUM


Combinded effects: FUM+DON+Eimeria

Ctrl DON Ctrl DON

NON-CHAL. CHAL.

CD4+CD25+

(cecal tonsil, n=5)

CD4+CD25+

Treg lymphocyte, role in regulation of inflammation

Major findings: Treg lymphocytes

Assumption: challenged birds fed DON required more Treg

to control inflammation?

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Lym

phocyte

s (

%)

a a

b

c


Combinded effects: DON+Eimeria

DON increases Incidence & Severity of Necrotic Enteritis

0

10

20

30

40

50

60

Negativecontrol

DON alone CP CP+DON

% birds with necrotic lesions

Antonissen et al., 2014; PLOS One 9:e108775

DON=3.8 to 4.4 mg/kg

CP=Clostridium perfringenes

DON increases Incidence & Severity of Necrotic Enteritis

0

10

20

30

40

50

60

Negativecontrol

DON alone CP CP+DON

% birds with necrotic lesions

Antonissen et al., 2014; PLOS One 9:e108775

DON=3.8 to 4.4 mg/kg

CP=Clostridium perfringenes

Deoxynivalenol predisposes for C. perfringensinduced necrotic enteritis

Antonissen G, Van Immerseel F, Pasmans F, Ducatelle R, et al. (2014) PLoS ONE 9(9): e108775.

PARASITE

Fusariotoxins (chicken):

- impaired recovery of duodenal villi from coccidial

lesions,

- upregulation of IFN-γ expression in CT,

- delayed recruitment of CD4+ and CD8+ cells in

jejunum

OTA (turkey, chicken):

- bloody diarrhea,

- higher lesions and oocyst in intestine,

- duodenal hemorrhages

BACTERIA

FB1 (pig):

- increased intestinal colonization by E. coli,

- affect APC maturation, T cell stimulatory capacity,

specific Ig in PP

DON (porcine cells & ileal loop):

- enhanced S. typhimurium invasion and

translocation,

OTA (chicken):

- higher number of S. typhimurium in duodenum &

cecum, acute enteritis

INTESTINE: FIRST TARGETDEFENSE

HIGHER SENSITIVITY WHEN COMBINED WITH MYCOTOXINS

COMBINATION WITH DIGESTIVE PATHOGENS


Do Mycotoxins affect the GIT?• Yes….but it depends on:

– Which mycotoxin

– Concentration (realistic, occasional, unrealistic)

– Route of effect

• Not easy to elucidate as research is inherently confounded:

– Changes to nutrient/energy content of feedstuffs

– Changes to feed intake, metabolism

• Particular unknowns:

– Interactive effects of multiple mycotoxins

– Interactive effects of DON/FUM with intestinal pathogens

Poultry Production & Management Systems

Bird Health &

Well-Being

Bird Physiology, Metabolism, and

Genomics

Safeguarding & Sustaining “Our World”

Department of Poultry Science

Dr. Todd J. Applegate

[email protected]

Poultry SciencePreparing future leaders

Fostering INNOVATION for the poultry industry for over 100

years…

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Can Mycotoxins Effect Gastro- intestinal Tract (GIT) Function?