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Afghanistan
John F. Leslie
Andreia Bianchini
Debra Frey
Five Agriculturally Most Important (Regulated) Mycotoxins
o Aflatoxins
o Fumonisins
o Deoxynivalenol and other trichothecenes, e.g., T-2
o Zearalenone
o Ochratoxin
What Was Done?
• Afghanistan – MAIL and Deb Frey– Trained staff – Established and equipped a functional lab– Collected samples from across Afghanistan– Assayed raisins, nuts & wheat with Romer test kits
• Italy – ISPA– UPLC and LC-MS assays for trichothecenes in wheat– Mycological analyses of flour
• Austria – BOKU– Quadripole MS assay for 650 different metabolites
• USA – K-State and Univ. Nebraska-Lincoln– Assay nuts and wheat with Romer test kits– Test kits from Vicam and Neogen– Mycological analyses of flour– Synthesize results– Test reliability of test kits
OchratoxinsAspergillus ochraceus
coffee-ota.orgStructure of Ochratoxin A Cl
O OH O
O
HOOC
N
HH
H
CH3
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emsl.comKidney failureCacaoNutsGrapesCoffeeWheat
Trichothecenes
• Two classes – A & B, strains make only one type
• Both inhibit protein synthesis
• Most common in grains
• Type A – very toxic – T-2, HT-2 & DAS– US Select agent list
– Purportedly used for biological warfare
• Type B – not as toxic – DON, NIV– More widespread, especially on wheat
• Can be taken up through skin or intestinal mucosa
• Cause vomiting, diarrhea, & immune suppression
T-2 – Killed 1000s in Russia after WWIIDON – Becoming very widespread in US
and Europe, especially where wheat and maize are grown
DON is changing the economic landscape of the US Great Plains
Fusarium is the main producer on grains, but other fungi and some plants also synthesize
Ergot Alkaloids
• Small Grains – Wheat, rye, barley & oats
• An unexpected finding by Austrian group
• Not highly regulated (animal feed only)
• In small doses – hallucinations (LSD)
• In other cases – neuropathy and gangrene
• Gnostics and ancient Greeks may have used them to help people have visions
• Controlled by sorting ergot bodies from the grain before processing
Some Ergot Epidemics-600 – Assyria
857 – Germany945 – France
1093 – France1692 – USA 1926 – Russia1929 – Ireland1953 – France1958 – India1973 – Ethiopia
Test Kit Performance
• Romer kits for aflatoxin, ochratoxin and deoxynivalenol (DON or vomitoxin) were reliable for all tested substrates
• Vicam kits for ochratoxin and DON were reliable for wheat
• Neogen tests for aflatoxin and ochratoxin were reliable for all tested substrates
• Vicam kit for aflatoxin did not work with wheat• Romer test for T-2 toxin was erratic• Neogen and Romer tests for T-2/HT-2 toxins gave many
(Romer) and exclusively (Neogen) false positives in wheat
Nuts – Results
• Aflatoxin
– Almonds – 15/81 at export limiting level
– Pistachios – 19/40 at export limiting level
– Walnuts – 8/25 at export limiting level
• Ochratoxin
– Almonds – no contamination detected
– Pistachios – 2/40 at export limiting levels
– Walnuts – no contamination detected
0
10
20
30
40
50
60
70
80
90
100
110
120
Sattarbai Shokorbai Abdul Wahidi Qambari Sangaki andMurawaji
Others
Afl
ato
xin
(µ
g/kg
)
Sample Type
Total SamplesSattarbai 13Shokorbai 10Abdul Wahidi 9Qambari 13Sangaki and Murawaji 15Others 12
EU Regulations
USA Regulations
0
10
20
30
40
50
60
70
80
90
100
110
120
Balkh Herat Kabul Kandahar Samangan
Afl
ato
xin
(µ
g/kg
)
Province
Total SamplesBalkh 22Herat 9 Kabul 31Kandahar 4Samangan 6
EU Regulations
USA Regulations
Almonds – Aflatoxins
0
10
20
30
40
50
60
70
80
90
100
110
120
Korak Pushdara Khandan-e-safid Others
Afl
ato
xin
(µ
g/kg
)
Sample Type
Total SamplesKorak 20 Pushdara 2 Khandan-e-safid 3Others 3
EU Regulations
USA Regulations
0
10
20
30
40
50
60
70
80
90
100
110
120
Balkh Herat Kabul Samangan
Afl
ato
xin
(µ
g/kg
)
Province
EU Regulations
USA Regulations
Total SamplesBalkh 7 Herat 4 Kabul 10Samangan 6
Pistachios – Aflatoxins
0
10
20
30
40
50
60
70
80
90
100
110
120
Zard Mazaari Takhari Korak Kaghazi Others
Afl
ato
xin
(µ
g/kg
)
Sample Type
EU Regulations
USA Regulations
Total SamplesZard 8Mazaari 1Takhari 1Korak 2Kaghazi 10Others 14
0
10
20
30
40
50
60
70
80
90
100
110
120
Balkh Herat Kabul Samangan
Afl
ato
xin
(µ
g/kg
)
Province
EU Regulations
USA Regulations
Total SamplesBalkh 7 Herat 4 Kabul 22Samangan 3
Walnuts – Aflatoxins
Austrian Screen – Nuts Fusarium Alternaria Aspergillus Penicillium
Butenolide Alternariol Cyclopiazonic Acid Andrastin A
EpiequisetinAlternariol methyl ether Aflatoxin Andrastin B
Equisetin Altersetin Asperfuran Agroclavine
Fusaric acid Infectopyron Kojic acid Chanoclavin
HT-2 toxin Macrosporin Malformin A Epoxyagroclavin
T-2 toxin Tentoxin Malformin A2 Festuclavine
Zearalenone Tenuazonic acid Malformin C Mycophenolic acid
α-Zearalenol NigragillinMycophenolic acid IV
β-Zearalenol 3-Nitropropionic acid Penitrem A
Ochratoxin
Paspalin
Raisins – Results
• Aflatoxins could limit exports in 43/89 samples
• Ochratoxin could limit exports in 25/80 samples
• Raisin type and drying method can be important
• Afghanistan and Austria results are discordant
• Choice of country to export to may depend on level of contamination
0
10
20
30
40
50
60
70
80
90
100
110
120
M.Q. RoundGreen
M.Q. LongGreen
H.Q.Shundurkhani
M.Q. Red Sun DriedShomali
Sun DriedGhazni
Sun DriedTayefee
Small Red
Afl
ato
xin
(µ
g/kg
)
Sample Type
Total SamplesMQ Round Green 26MQ Long Green 14HQ Shundurkhani 25MQ Red 17Sun Dried Shomali 3 Sun Dried Ghazni 11 Sun Dried Tayefee 2Samll Red 4
EU Regulations
USA Regulations
0
10
20
30
40
50
60
70
80
90
100
110
120
Herat Kabul Kandahar
Afl
ato
xin
(µ
g/kg
)
Province
EU Regulations
USA Regulations
Total SamplesHerat 20Kabul 67Kandahar 15
Raisins – Aflatoxins
0
10
20
30
40
50
60
70
80
90
100
110
120
M.Q. RoundGreen
M.Q. LongGreen
H.Q.Shundurkhani
M.Q. Red Sun DriedShomali
Sun DriedGhazni
Sun DriedTayefee
Small Red
Och
rato
xin
(µ
g/kg
)
Sample Type
Total SamplesMQ Round Green 27MQ Long Green 14HQ Shundurkhani 26MQ Red 17Sun Dried Shomali 3 Sun Dried Ghazni 11 Sun Dried Tayefee 2Samll Red 4
EU Regulations
0
10
20
30
40
50
60
70
80
90
100
110
120
Herat Kabul Kandahar
Och
rato
xin
(µ
g/kg
)
Province
EU Regulations
Total SamplesHerat 20Kabul 68Kandahar 16
Raisins – Ochratoxin
Austrian Screen – RaisinsFusarium Alternaria Aspergillus Penicillium
Fumonisins Alternariol Aflatoxin Andrastin A
Alternariol methyl ether Aurasperon B Andrastin B
Altersetin Aurasperon C Andrastin C
Altertoxin-I Aurasperon G Chanoclavin
Macrosporin Fonsecin Festuclavine
Tentoxin Malformin A Mycophenolic acid
Tenuazonic acid Malformin A2 Mycophenolic acid IV
Malformin C Penitrem A
Nigragillin Quinolactacin A
Ochratoxin
Pyranonigrin
Wheat – Results • International standards may be too high for Afghanistan safety because of
the large amount of wheat consumed daily (500 g/person/day
• Aflatoxins – detectable in 23/151 samples– Not a field contaminant of wheat– Contamination likely occurs in storage
• DON – 3/185 above international guidelines– Not a major problem, but exists– Weather and storage dependent
• Ochratoxin – detectable in 36/181– Common problem in northern Europe– Needs attention– May carry over to meat
• T-2 and HT-2 – Not reliably detected• Ergot – detectable 51/151
– High incidence– Easily remedied by cleaning grain
0
10
20
30
40
50
60
70
80
90
100
110
120
Balkh Herat Kandahar Parwan Samangan
Afl
ato
xin
(µ
g/kg
)
Province
EU Regulations
USA Regulations
Total SamplesBalkh 29Herat 22 Kandahar 7Parwan 25Samangan 42
0
10
20
30
40
50
60
70
80
90
100
110
120
Asiabs Mill Grist Mill Warewhouses Naan Bakeries CommercialFlour Mills
Pakistan Flour TurkmenistanFlour
Others
Afl
ato
xin
(µ
g/kg
)
Sample Type
EU Regulations
USA Regulations
Total SamplesAsiab Mills 45Grist Mills 37Warehouses 4Naan Bakeries 22Commercial Flour Mills 3Pakistan Flour 10Turkmenistan Flour 3Others 2
Wheat - Aflatoxin
0
10
20
30
40
50
60
70
80
90
100
110
120
Asiabs Mill Grist Mill Warewhouses Naan Bakeries CommercialFlour Mills
Pakistan Flour TurkmenistanFlour
Others
Och
rato
xin
(µ
g/kg
)
Sample Type
EU Regulations
Total SamplesAsiab Mills 32Grist Millls 37Warehouses 4Naan Bakeries 22Commercial Flour Mills 3Pakistan Flour 10Turkmenistan Flour 3Others 2
0
10
20
30
40
50
60
70
80
90
100
110
120
Balkh Herat Kandahar Parwan Samangan
Och
rato
xin
(µ
g/kg
)
Province
EU Regulations
Total SamplesBalkh 18Herat 21 Kandahar 7Parwan 25Samangan 42
Wheat – Ochratoxin
Austrian Screen – Wheat
Fusarium Alternaria Aspergillus Penicillium ClavicepsBeauvericin Alternariol Aflatoxin Agroclavine Ergocristine
Enniatin AAlternariolmethyl ether Averantin Chanoclavine Ergocristinine
Enniatin A1 Altersetin Averufin Citrinin Ergometrine
Enniatin B Altersolanol Cycloaspeptide A Chrysogine Ergometrinine
Enniatin B1 Altertoxin-I Kojic acid Elymoclavine Ergosin
Epiequisetin MacrosporinMethoxysterigmatocystin
Mycophenolic acid Ergosinin
Equisetin Tentoxin3-Nitropropionic acid
QuestiomycinA Ergotamine
HT-2 toxinTenuazonic acid Norsolorinic acid
Quinolactacin A Ergotaminine
T-2 toxin OchratoxinSecalonic acid D
Zearalenone Sterigmatocystin
Conclusions
• The test kit used matters• Sampling procedures are critical• Pre- and Post-harvest processes both matter• Nuts and aflatoxins – Pistachios > Walnuts > Almonds• Raisins – Aflatoxins > Ochratoxin
– Type of raisin and drying method important
• Wheat– Need to evaluate safety levels for Afghan diets– Aflatoxin is a storage issue– Fusarium toxins (T-2, HT-2, DON & Zearalenone) are minimal– Citrinin + ochratoxin could enhance kidney problems– Ergot – high frequency, but relatively easy to fix
Questions?
“Where waters are
murky, crocodiles lurk!”
- Old African saying