MYCOTOXINS

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MYCOTOXINS

• DAVID R. LEDOUX
• Fusarium \ Poultry Research Laboratory
• University of Missouri-Columbia USA

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What are Mycotoxins

• Toxic fungal secondary
• metabolites
• Acute levels disease and death
• Chronic levels reduced efficiency
  of production

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ST. ANTHONYS FIRE

• Ergot poisoning Rye Bread
• Assyrian tablet - 600 BC
• Ergot bodies Claviceps purpurea
• Monastery Flame-Red Walls
• Fiery pain of peripheral
• vasospasm dry gangrene
• Migraines
• Ergotamine Methysergide

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Ergot can Infect all Cereal Grains and Grasses
wheat barley rye oats triticale
Quack grass
Reed canary grass
Orchard grass
Kentucky blue grass
Crested wheat grass
Timothy
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CONSEQUENCES OF ST. ANTHONYS FIRE
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Salem Witch Trials

• 1692-Salem Massachusetts
• 250 people were arrested for supposed witchcraft
  (several were tried and hung)
• Rye was grown that year and had been overtaken
  with claviceps purpurea
• Suffered hallucinations feeling of being
  pricked burning sensation and temporary blindness

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Contamination and costs

• 25 worlds crops affected
• USA Mean 932 M (CAST 2003)
• Range 418M 1.66B
• Mitigation Livestock losses (472 M)
• Human health Costs

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Mold vs. Mycotoxins

• Presence of mold does not always mean toxins are
  present
• Presence of toxins does not always mean molds are
  still present

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Mycotoxins of Importance

• Zearalenone
• Ergot alkaloids
• Moniliformin

• Aflatoxins
• Fumonisins
• Ochratoxin A
• Trichothecenes

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MYCOTOXIN PRODUCTION
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Preparing Flasks
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Inoculating Flasks
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Growing Cultures
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FUMONISIN B1 CULTURE(5 week culture)
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Fumonisin Culture Material
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AFLATOXINS

• Turkey X disease (1960)
• Brazilian groundnuts
• A. flavus A. parasiticus
• Corn peanuts (groundnuts) CSM
• Field transportation storage

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AFLATOXIN
4 naturally occurring toxins Fluoresence
B1 B2 G1 G2 8-9 double bond forms
epoxide Nucleus of hepatocytes
targeted liver toxin Epoxide binds to DNA
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100
50
10
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AF binds to DNA
CAST 1989
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Effects of low levels of AF in broilers (Doerr et
al. 1983)
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Effects of low levels of AF in broilers (Doerr et
al. 1983)
AF Live Wt
Dressed Wt Chilled EWt (ppm)
-------------------------- (g)
------------------------
0 2024a 1914a 1490a 0.30 1932a 1850a 1414b 0.
90 1998a 1882a 1466ab 2.70 1671b 1583b 1213c abcP
lt 0.05
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Liver pathology - Gross
Control
Aflatoxin
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Effects of Aflatoxin B1 (AFB1) on Liver of
Broilers (Severe periportal cytoplasmic
vacuolation of hepatocytes)
Control
AFB1
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Effects of AF on lipase and amylase activity of
chickens
Activity (Units/g dry wt)
AF Dose (ppm)
Osborne et al. 1982
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Effects of AF on trypsin and RNase activity of
chickens
Activity (Units/g dry wt)
AF Dose (ppm)
Osborne et al. 1982
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Effects of aflatoxin on pigmentation
(Aflatoxin -)
(Aflatoxin )
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HSCAS and AFB1 Dairy cows (Harvey et al. 1991)
AFB1 HSCAS Milk AFM1 Decrease (ppb) ()
(ppb) () 100 0 0.91a
----- 100 1.0 0.51b 43.6
200 0 1.85A ----- 200
0.5 1.41B 23.7 abABP lt 0.01
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AFM1 reduction in milk after withdrawal of AFB1
in feed (McKinney et al. 1973)
1-2 of dietary dose
Time (hr)
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a
a
b
b
-------IM--------
-------Oral-------
Atef et al. 2002
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Effects of AF on antibody titers against
infectious bursal disease (IBD) and ND
(Newcastle disease) in broilers at 6 weeks
a
a
Antibody Titers
b
c
b
c
d
c
Swamy and Devegowda 1998
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Ochratoxin A

• Discovered in 1965
• Isocoumarin derivatives linked to phenylalanine
• 7 metabolites but only OA widespread contaminant
• Hydrolysed to ochratoxin alpha and phenylalanine
• Phenylalanine moiety causes enzyme inhibition

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Ochratoxins

• Aspergillus ochraceus (alutaceous)
• Penicillium verrucosum
• Penicillium purpurescens
• Moisture 18.5 - 40 Temperature 4 - 37 C
• Corn barley wheat oats and rye
• Moldy coffee beans peanuts Brazil nuts beans
  hay surface of hams red pepper and black pepper

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Effects of OA on chick growth
OA
Control
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Effects of OA on body weight gain of broilers
a
a
ab
bc
de
Body weight gain (g)
f
c
g
Huff and Ruff 1982
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Effects of OA on kidney weight gain of broilers
a
ab
Kidney weight (g/100g BWT)
c
de
de
e
Huff and Ruff 1982
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Effects of OA on plasma carotenoids of broilers
a
a
ab
b
Plasma Carotenoids (eq/ml)
de
de
Huff and Ruff 1982
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Effects of OA on Pigmentation
OA
OA -
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Fumonisin

• Fusarium moniliforme
• Substrate - corn
• Moisture - gt 23
• Temp - 28 C

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Fumonisins

• Discovered in 1988
• FB1- FB4 A1 A2
• Fusarium verticilioides
  F. proliferatum
• Corn and corn products
• Moisture gt23 Temp 28C
• Water soluble
• ELEM PPE esophageal cancer

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Fumonisin B1 and Leukoencephalomalcia
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PPE - LUNGS
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EFFECTS OF FB1 ON 7 WEEK BROILER PERFORMANCE
FB1 INTAKE GAIN FC mg/kg kg
kg kgkg 0 5.83 3.05 1.91
25 5.84 3.03 1.93 50 5.81
3.00 1.93 SE .04 .03 .01 21
day 450 mg/kg
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EFFECTS OF FB1 ON 14 WEEK TURKEY PERFORMANCE
FB1 INTAKE GAIN FC mg/kg kg
kg kgkg 0 21.36A
8.27a 2.58ab 25 21.80A 8.23ab 2.65a
50 20.39B 7.98b 2.55b SE .30
.09 .03 ABDIFFER SIGNIFICANTLY Plt.05 abDIFFER
SIGNIFICANTLY Plt.06 21 day 325 mg/kg
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Effect of FB1 on ducklings
Control 400 mg/kg
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STRUCTURE OF FB1 SPHINGANINE AND SPHINGOSINE
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Mechanism of Toxic Action of FB1 - disruption of
sphingolipid biosynthesis
FB1
acyltransferase
acyltransferase
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Effects of FB1 on liver SASO ratio (Broomhead et
al. 2002)
FB1 BROILER TURKEY mg/kg
SASO SASO 0
0.15B 0.18C 25 0.52A 0.62B
50 0.59A 1.21A SE .03
.11 ABCDIFFER SIGNIFICANTLY Plt.05
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SPHINGOLIPIDS

• FOUND IN MEMBRANES BRAIN NERVES
• FUNCTIONS INCLUDE
• INTERNALIZATION OF LIPIDS PROTEINS
• CELL-CELL COMMUNICATION
• REGULATION OF CELL SURFACE RECEPTORS
• REGULATION OF ION PUMPS

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Fumonisin B1 and Folic acid
FB1 shown to indirectly inhibit folate
uptake Folate is a vitamin involved in single
carbon metabolism and in metabolism of amino
acids and nucleotides Folate receptor found to
be associated with membrane domains rich in
sphingolipids and cholesterol FB1
decreased sphingolipid uptake in both whole cells
and domains rich in sphingolipids 20

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Fumonisin B1 and Folic acid
FB1 treated cells 5-methyl-FH4 uptake
inhibited 90 5-methyl-FH4 predominant form
of folate transported to peripheral tissues
from liver Folate deficiciency during first
trimester neural tube defects in
fetuses FB1 implicated in neural tube defects
in Texas Hispanic
population consuming corn (1989 1991) High
incidence of leukencephalamalacia (1989)
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IMPLICATED IN OTHER HUMAN DISEASES

• Neural tube birth defects
• Esophageal cancer (Asia SA)
• Hepatocellular carcinoma (HCC) in Asia and
  Africa

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TRICHOTHECENES
T-2 toxin
Fusarium tricinctum HT-2 toxin Neosolaniol
(NEO) 8-Acetylneosolaniol (8-Ac-NEO)
4-Deacetylneosolaniol (4-DN) 415-Diacetoxyscirp
enol (DAS) 15-Monoacetoxyscirpenol (MAS)
Nivalenol (NIV) 4-acetylnivalenol
(Fusarenone-X) Deoxynivalenol (DON Vomitoxin)
Fusarium graminearum 3-Acetyldeoxynivalenol
(3-Ac-DON)
Leeson et al. 1995
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TRICHOTHECENES

• Most potent small molecule inhibitors of protein
  synthesis known
• Cellular level Primary inhibition of protein
  synthesis
• Secondary disruption of DNA and RNA synthesis
• Reactivity associated with 1213 epoxide
• Oral lesions poor growth abnormal feathering
  decreased egg production poor egg shell quality
  regression of bursa of Fabricius

Vomitoxin (DON)
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Effects of DON on broiler performance
a
a
a
Body Weight (g)
b
c
Moran et al. 1982
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Effects of AF and DON in broiler chicks
AF DON GAIN FC
--(mg/kg)--- (g) (gg)
0 0 626a 1.66b 2.5 0
521c 1.70b 0 16 571b 2.04a
2.5 16 488d 2.17a Huff et al.
1986
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Effects of T-2 on Growth
CAST 1989
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Oral Lesions Caused by T-2 toxin
Primary effect inflammatory response in the
mouth which progresses to necrosis and invasion
by normal microbial flora
CAST 1989
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ZEARALENONE
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Zearalenone

• First described in US 1920s
• F-2 toxin
• Fusarium roseum (graminearum)
• F. tricinctum F. gibbosum
• F. oxysporum F. moniliforme
• Substrate - corn wheat barley oats sorghum
  sesame seed corn silage

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Zearalenone

• Moisture - gt 23 45 optimum
• Temp 12 - 25 C 27 C optimum
• Zearanol (-zearalenol) Ralgro
• Phenolic compound
• Chemically modified to produce Ralgro (saturation
  of 1 double bond and conversion of aldehyde to
  alcohol)
• Activity associated with 6 carbonyl and 4
  phenolic hydroxide

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Effects of pure ZEN or pure F. roseum cultures in
Gilts (Sundlof and Strickland 1986)

• Dose Clinical Lesions
• 1 mg/day Vulva swelling
• 5 mg/day Enlarged mammary glands
• Enlarged teats prolapsed vagina
• Ovarian atrophy enlarged uterus
• Metaplasia of cervix and vagina
• 6 mg/day Smaller litter size
• 12.6 mg/day Persistent corpora lutea
• Prevention of early fetal development

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Estrogenic Effects in Pigs
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ESTROGENIC EFFECTS IN PIGS
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RESIDUES

• Hyperestrogenism in humans associated with
  consumption of meat and poultry products
• Prepubertal children most affected
• Premature sexual development Italy
• Since 1972 many cases in Puerto Rico
• Residues of ZEN and Zeranol

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RESIDUES

• Pharmokinectic data does not support theory
• Muscle contains few binding sites for ZEN
• Eggs shown to accumulate ZEN (yolk)
• Possibility of withdrawal period (65 d) being
  ignored
• Excessive doses used (gt 36 mg/animal)

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PROBLEMS ASSOCIATED WITH INTAKE OF
ENDOPHYTE- INFECTED TALL FESCUE
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Tall fescue is now grown in countries such as
France USSR Argentina Australia Chile and
New Zealand. Most research has focused on
endophytes of tall fescue and perennial ryegrass
in northern Europe New Zealand and southeast
US.
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• Four distinct problems are associated
• with animals grazing endophyte-infected
• tall fescue
• Fescue Foot
• Fescue Toxicosis
• Agalactia
• 4. Fat Necrosis

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Fescue Foot occurs at cold temperatures First
described by Cunningham (New Zealand
1949) Observed dairy cows grazing tall fescue
1. Rear limb stiffness 2. Swelling / reddness of
hoof and coronary band upwards 3. Necrosis and
loss of extremities 4. Possible animal death
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Fescue Toxicosis Summer Slump Summer
Syndrome Poor growth Low milk production
Increased sensitivity to heat during summer
heat Reduced milk production calf gain stocker
gain and conception rate Hair coats dull
long and resist shedding Reduced serum
prolactin
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Fescue Toxicosis Summer Slump Summer
Syndrome Major problem for producers grazing
cattle southeastern quarter US Hoveland
(1991) estimated that cattle losses from
grazing EIF exceeds 600 million/year with 354
million from reproductive loss and 255 million
from decreased weight gain. This is
conservative. Related to ambient temperature
gt 90F
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Measures to combat mycotoxicoses
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Measures to combat Mycotoxicoses

• Mold Inhibitors Nutritional supplements
• Microbial Thermal inactivation
• Irradiation
• Ammoniation Ozonation
• Solvent extraction Mechanical seperation
• Molecular biology techniques
• Adsorbents

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First reports of efficacy of adsorbents

• Phillips et al. 1987
• J. Amer. Vet. Med Assoc.
• HSCAS effective in reducing toxicity
• of AF in poultry diets

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Si
Al
(Taylor 2000)
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Effects of HSCAS and AFB1 in Broilers (Ledoux et
al. 1999)
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HSCAS and AFB1 Dairy Goats (Smith et al. 1994)
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HSCAS and AFB1 Dairy cows (Harvey et al. 1991)
AFB1 HSCAS Milk AFM1 Decrease (ppb) ()
(ppb) () 100 0 0.91a
----- 100 1.0 0.51b 43.6
200 0 1.85A ----- 200
0.5 1.41B 23.7 abABP lt 0.01
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(Taylor 2000)
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Proposed mechanism for chemisorption of AFB1 to
HSCAS (Phillips et al. 1990)
HSCAS
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Other Adsorbents

• Activated charcoal
• Anion exchange resins
• Cholestyramine
• Dinvinylbenzene-styrene polymer
• Esterified glucomannans

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Advantages of adsorbents

• Cost
• Safety
• Easy to use
• GRAS up to 2 diet

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Concerns with respect to adsorbents

• Not all equally effective
• Some impair nutrient utilization
• Some not tested for in vivo efficacy
• Clay adsorbents not effective against Fusarium
  and OA

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THANK YOU