Post on 12-Aug-2015
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aflasafe
Development for Aflatoxin
Mitigation in Tanzania
Ranajit Bandyopadhyay: IITA
Peter Cotty: USDA-ARS
On behalf of the Aflasafe Team
National Steering Committee for Mycotoxins in Tanzania,
Dar-Es-Salaam, 1 Aug 2014
• Highly toxic metabolite produced by the ubiquitous Aspergillus flavus fungus
• The fungus resides in soil and crop debris, infects crops and produces the toxin in the field and in stores
• Death, liver cancer, immune-suppression, stunted growth
• Impacts animal productivity
• Negatively impacts trade
• Fungus carried from field to store
• Contamination possible without visible signs of the fungus
Aflatoxin Facts
acute
acute hepatic necrosis, cirrhosis,
carcinoma
Death; 108 in 1974 in Gujarat, 250
to 15,000 ppb aflatoxins in corn
chronic
carcinogenic
associated with stunting
anti-nutritional
immune-suppressive
gut integrity?
BBC 2004, Gong et al 2004, NIEHS 2010
underreported
unknown
Human Health Effects
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Aflatoxin Contamination Occurs in
Two Phases
Phase I: Before Crop Maturity
Developing crops become infected.
Associated with crop damage (insect, bird, stress).
Favored by high temperature (night) and dry conditions.
Phase II: After Crop Maturity
Aflatoxin increases in mature crop.
Seed is vulnerable until consumed.
Rain on the mature crop increases contamination.
Associated with high humidity in the field & store, insect
damage, and improper crop storage or transportation.
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Pre-Harvest Problem
Aflatoxin (ppb)ppb)
Peanut (n = 188) Maize (n = 241)
Distribution (% samples)
> 4 54 70
> 10 41 52
> 20 29 24
Descriptive statistics (ppb)
Minimum < LOD < LOD
Maximum 3487 838
Mean 111 33
LOD = Limit of Detection; 1 ppb
Aflatoxin in Groundnut and Maize at Harvest
Increases in store
Kenya (CDC and Kenyan Ministry of Health 2004)
District Samples
% samples with aflatoxin levels (ppb)
<20 21-99 100-1,000 >1,000
Makueni 91 35 13 40 12
Kitui 73 38 21 32 10
Machakos 102 49 25 23 3
Thika 76 66 17 13 4
Total 342 47% 19% 27% 7%
Tanzania (IITA & partners, unpublished, 2013)
Aflatoxins in Markets
Crops Samples % samples with aflatoxin levels (ppb)
<10 11-100 101-1,000 >1,000
Groundnut 180 89 11 0 0
Maize 287 71 15 10 4
Cassava 405 86 10 4 0.2
Aflatoxin and Poultry (Broilers)
Aflatoxin
levels in
feeds in
Nigeria
Aflatoxin level (ppb) Samples (%)
<20 (safe) 38
>20 to 100 (up to 5x) 14
>100 to 500 (up to 25x) 41
>500 to 1,000 (up to 100x) 7
AF-free diet 500 ppb AF diet
AF-free
diet
500 ppb AF diet
~40% reduction in live weight (8 weeks)
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EPA approved 2 products
AF36
Afla-guard
More than 1 million hectares treated
annually in the US!
Production Room
Atoxigenic Strain Manufacturing Facility
Arizona Cotton Research & Protection Council
(Funded and Governed by the Farmers of Arizona),
Phoenix, Arizona
It Works in Africa Too
Biocontrol Works!
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Biocontrol Principles
In nature, some strains produce a lot
(toxigenic), and others no aflatoxin
(atoxigenic) (Donner, Soil Biol Biochem
2009)
Atoxigenic strains are already present on
the crop (Atehnkeng et al., IJFM, 2008)
Increase the frequency of atoxigenic
strains to competitively displace
toxigenic strains (Cotty & Bayman,
Phytopath 1993)
Thus, aflatoxin contamination reduced
Atoxigenic strains can be applied without
increasing infection and without
increasing the overall quantity of A. flavus
on the crop or in the environment (Cotty,
Phytopath 1994; Atehnkeng 2014)
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100Afl
ato
xin
B1 (
ng
/g X
10,0
00)
Isolates (%) in Applied Atoxigenic Strain
Strains move from
field to stores
Multiple year & crop
carry-over effect
(Jaime & Cotty,
Phytopath 2006)
We use only native
strains
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Aflatoxin & Aspergillus
A. flavus
L-strain
Aflatoxin: +/-
A. tamarii
Aflatoxin: -
A. flavus
S-strain
SBG
Aflatoxin: +++
A. parasiticus
Aflatoxin: ++++
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Atoxigenic Strain Identification
Collection/characterization Toxin assay
Field
efficacy
test
Lab
competition
assay
VCG/DNA characterization
cnx nia-D
Unknown 2
+
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Strain Selection Criteria
In the laboratory (~5,000 strains):
• Does not produce aflatoxin
• VCG/SSR group with
Wide geographic distribution
No toxigenic member
• Defective in >2 aflatoxin & CPA
genes
• Outcompetes toxigenic strains
After field application:
• Superior capacity to colonize,
multiply and survive in soil
• Superior frequency of isolation
from grains
• Superior capacity to reduce
aflatoxin 8-12 native strains
selected for field tests
4 native strains
formulated into
the final
product
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• IITA
• USDA
• AATF
• BMGF
• Doreo Partners
• National institutions
Strong Partnership
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Broadcast @ 10 kg/ha 2-3 weeks before flowering
Sporulation on moist soil
Spores
Insects
Aflasafe in 5 kg boxes
3-20 days
Wind
Soil colonization
30-33 grains m-2
Fungal network in killed grain
How Does aflasafe Work?
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Efficacy Trials: Data Collection
• All trials conducted in farmers’ fields on crops grown by farmers
• Aflasafe applied by farmers
• Soil sampled before treatment and grains at harvest:
– Aspergillus population density
– Aspergillus strain profile
– Incidence of aflasafe strains
• Aflatoxin concentration in grains at harvest and after poor storage
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Design & Analysis of Trials
• Field trial size: 0.25 to 15 ha
• Number of fields: 14 to 200 per year
• Paired plot: Each treated field with its own companion control field in close vicinity
• Each farmers’ field considered as a replicate
• Student’s t-test to compare treatment effects
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Nigeria: Efficacy on Maize
372
0
20
40
60
80
100
120
2009 2010 2011 2012
Aflasafe™ Control
0
100
200
300
400
500
600
2009 2010 2011 2012
82 94 83 86 82 93 89 90
51 14 199 38 51 14 166 38 Fields (#)
Less (%)
At Harvest After Storage
*All means of aflasafe and control pairs significantly different; Student’s t-test (P<0.05)
*
Aflato
xin
(ppb)
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0
10
20
30
40
50
60
2010 2011
Aflasafe™
Control
0
20
40
60
80
100
120
140
160
180
2010 2011
Aflasafe™
Control
82 94 82 93
16 82 16 81 Fields (#)
At Harvest After storage
*All means of aflasafe and control pairs significantly different; Student’s t-test (P<0.05)
Aflato
xin
(ppb)
Aflatoxin Reduction
(%)
*
Nigeria: Efficacy on Groundnut
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Basis of efficacy: species shift
Treatment (n = 14)
Aspergillus species/strain distribution (%) – MAIZE/NIGERIA
Soil before inoculation Grain at harvest L SBG parasiticus L SBG parasiticus
Aflasafe™ 90 aB 7 aA 3 aA 100 aB 0 bA 0 aA
Control 78 aB 15 aA 7 aA 83 bB 16 aA 0.3 aA
Means within the column with different lowercase letters are significantly different according to the t-
test at 5% level of probability. Means within the row with different uppercase letters are significantly
different according to the Fisher’s LSD test at 5% level of probability
Region
Treatment
Aspergillus Colony Forming Units/g – G-nut/Senegal 2010 (n = 20) 2011 (n = 17)
Soil Kernel Soil Kernel
Diourbel Control 2311 a 2912 a 474 a 3257 a
Aflasafe SN01 1793 a 3598 a 795 a 3965 a
Nioro Control 228 a 3367 a 369 a 3572 a
Aflasafe SN01 120 a 3189 a 470 a 4275 a
*All means of aflasafe and control pairs significantly different; Student’s t-test (P<0.05)
Aspergillus population does not increase due to aflasafe application
No change in Aspergillus Pop.
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Bars with same letter within the same
crop/year not significantly different (P<0.05)
Basis of Efficacy: Strain Shift
0
10
20
30
40
50
60
70
80
90
Soil Grain Soil Grain
2009 (n = 49) 2010 (n = 14)
Control Treated
0
10
20
30
40
50
60
70
80
90
Soil Grain Soil Grain
2009 (n = 2) 2010 (n = 16)
Proportion of 4 aflasafe™ strains in soil before treatment
and grains after harvest in control and treated fields
Afl
asafe
str
ain
s (
%)
a a a a a a a a a a a a
b b b
b
Carry-over of inoculum: 71, 52
and 28% after 1, 2, and 3 years
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Area Sample Treatment Mean
Aflatox (ppb)
Reduct. (%)
Mean Aflatox (ppb)
Reduct. (%)
Mean Aflatox (ppb)
Reduct. (%)
Diourbel
Harvest Treated 1.9
93 6.6
87 3.7
82 Control 29.7 50.1 20.3
Storage Treated 4.4
86 2.1
91 6.9
81 Control 31.3 22.1 35.5
Nioro
Harvest Treated 4.4
75 5.6
76 5.4
90 Control 17.6 23.1 55.7
Storage Treated 3.5
95 2.8
94 11.5
84 Control 52.1 46.7 72.5
*All means of aflasafe treated and control pairs significantly different; Student’s t-test (P<0.05)
Senegal: Efficacy of aflasafe SN01
2010 (n=40) 2011 (n=34) 2012 (n=71)
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Kenya: Efficacy of aflasafe KE01™
Area (fields) Control Treated Reduction
(%)
Hola (n = 20) 885 20 98
Bura (n = 16) 105 7 93
Makueni (n = 15) 85 1 99
Aflatoxin (ppb)
*All means of aflasafe treated and control pairs significantly different; Student’s t-test (P<0.05)
38
20
0
88
60
33
0
10
20
30
40
50
60
70
80
90
100Treated
Control
Fields (%) above 10 ppb in 3 areas
Fie
lds (
%)
Deadly (3,700 ppb & 2,270 ppb)
533 ppb
Hola
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Aflasafe Development in Tanzania
• 510 samples (260 maize grain and 250 groundnut kernel) samples from Tanzania
• 5017 isolates obtained from these samples. Happy Daudi (ARI-Naliendele) trained
• 818 atoxigenic isolates identified
• 690 isolates characterized by SSR and 136 by CAPs
• 20 atoxigenic isolates belonging to 12 haplotypes; 6 also occur in Kenya
• Testers developed for these 20 atoxigenic isolates
• Being complemented with 444 toxigenic and 284 atoxigenic nit- mutants
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Tanzania Haplotypes
Haplotype
Number of Samples
Tanzania
(239 samples)
Kenya
(357 samples)
Mozambique
(38 samples)
Zambia
(97 samples)
BOWAGA 40 4 0 0 BAQEGA 31 0 1 0 BOHEZI 16 0 0 0 BOPAJI 13 1 0 1
BOHAMA 11 1 0 0 BAQIFO 14 0 0 0 BOHEGI 7 8 0 1 BAQEZE 8 2 0 1 BOHAJU 6 0 0 1 BOHIPO 4 0 0 1 BAQALE 6 4 0 1 BAQESO 13 0 0 0
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Proposed Plan
• 8-12 isolates will be selected for first field trial in Tanzania
• To be conducted in 120 farmers’ fields in Mtwara, Dodoma and Manyara in 2014/15 season
• Field trials will be managed in collaboration with national partners (suggestions required) and private sector (e.g., Export Trading Group, BRITZ, and the Clinton Development Initiative) .
• Samples will be analyzed in IITA-Dar and IITA-Ibadan
• 4 most efficient isolates selected to constitute Aflasafe TZ01
• Large-scale evaluation of Aflasafe TZ01 in 2015 and 2016
• Registration, business plan, manufacturing from 2016 onwards
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Product Types National
Products
Products ready for registration
Products under testing
Strain development in progress
Aflasafe-NigeriaTM
Aflasafe-SenegalTM
Aflasafe-KenyaTM etc…
Aflasafe-WestTM
Aflasafe-EastTM
Aflasafe-SouthTM
Regional
Products Senegal
Mali
Burkina
Ghana
Nigeria
Kenya
Tanzania
Mozambique
Zambia
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Challenges
• Aflatoxin is a hidden problem
• Chemical analysis required
• Awareness is low
• Long incubation for expression of health impacts
• Regulations either non-existent or poorly enforced
• Market does not usually discriminate
• Demonstration of product value
• Lack of biopesticide manufacturers
The value of a technology on the shelf is as much as the cost of the space it occupies on the shelf.
Must translate knowledge into usable products and practices to benefit people
But……
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Integrated Management
The elements are:
• Technology
• Awareness – entire range of value chain participants
• Advocacy – regional, national, investors
• Training – farmers, transporters, traders, regulators, consumers
• Policies – standards, harmonization, trade
• Institutions – regulators, markets, testing, private sector
• Trade / Markets – food/feed processors, poultry/fish industry
• Public good – home consumption; urban and rural markets; government procurement, HGSF
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Farmer Training
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Markets
Doreo Partner analysis
Poultry industry
Export-oriented aggregators
Food processors
Large commercial farmers
Smallholder farmers
Market based
• Poultry feed
• Premium food
market
• Export
• AgResults (Incentive-
cum-market based)
• Public distribution
ma
rke
t d
em
an
d f
or
Afl
as
afe
• 60% maize consumed by farmers
• 40% sold in the market
Poultry Feeding Study
$3,200 net
profit from
10,000 birds
in 8 weeks
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Aflasafe maize feed Toxic maize feed
Pilot Implementation
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• Value chain-centric: Farmers’ and other value chain participants’ interest as the foundation of the action
• Public sector intervention with health perspectives for smallholders
• Action-oriented: Using practical methods to actively solve problems, not just talking about ideas, plans, or theories
• Innovation platform: problem solving by participants working regularly together to address common issues and challenges.
Innovation Platform
• Platform meetings with leadership and members of Poultry Association of Nigeria, feed manufacturers, maize aggregators, aflasafe farmers, vet professionals and regulators
• Poultry farmers to buy all aflasafe maize at a negotiated premium
• Agriculture ministry to fund NAFDAC to set up aflatoxin testing facilities in each state
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G-20 AgResults Aflasafe
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• Pull mechanism – Aflasafe is one of the first three pilots
• Provides incentives after demonstrating adoption
• Private sector driven, but focused on smallholder groups
• Implementers provide credit, inputs and technical services to increase yield
• Aflasafe purchased at cost to improve quality
• Maize tested for aflasafe strains; if present in large frequency, the implementers incentivized with $18.75/ton maize
• Implementers negotiate maize sale at premium
• Project provides aflatoxin awareness, training of implementers, and identifies potential market linkages
• Target: 260,000 ha in 4 years
AgResults Aflasafe Pilot -- 2013
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Some key statistics
• Number of implementers: 4
• Number of farmers: 1,015
• Treated area: 1,457 ha
• Average productivity: 4.3 tons/ha
• Maize aggregated for sale: 2,031 tons
• Samples with <4 ppb AF (n = 660): 99%
• Mean recovery of aflasafe strains from samples (n = 88): 72% to 89%
• Samples with >70% aflasafe strains
(n = 88): 65% to 100%
• Aflasafe maize kept for family (n = 60): 46%
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Aflasafe Return on Investment
Quantity sold
(tons) Premium
(%) Premium
(USD)
Aflasafe cost
(USD)
Finance cost
(USD)
Net profit (USD)
Seasonal RoI
AgResults Premium
(USD)
Total Profit (USD)
Total RoI
120.0 7.5% 3,000 836 146 2,017 241% 1,800 3,817 456%
150.0 7.5% 3,750 1,046 183 2,521 241% 2,250 4,771 456%
96.0 3.6% 1,200 669 117 414 62% 1,440 1,854 277%
128.0 13.2% 5,600 892 156 4,552 510% 1,920 6,472 725%
32.0 7.5% 800 223 39 538 241% 480 1,018 456%
30.1 1.8% 188 210 37 -58 -28% 452 393 187%
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Scaling-Out
• Nigeria: AgResults • Senegal: Area-wide treatment in
2013 • Kenya: Government buy-in;
excellent support • Zambia: Large-scale efficacy tests
and demonstration of product value with private sector
• Need for business plan, manufacturing capacity, marketing and distribution strategies
• Critical role of PACA and RECs
Coming in 2014-2015…
The Gambia, Benin,
Togo, Ivory Coast,
Uganda, Rwanda,
Burundi, Ethiopia, South
Sudan, Malawi
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Aflasafe Production in Lab 60 tons produced in 2011, 2012, and 2013 for deployment
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Aflasafe Manufacturing Facility
Large-scale: capacity 5 tons/hour
La
Capacity Development
Laboratory infrastructure
• Burkina Faso (Austria)
• Kenya (USDA/USAID)
• Zambia (USAID)
• Mozambique (USDA/USAID)
Human Technical capacity
• Senegal
• Ghana
• Cameroon
• Tanzania
• Nigeria
Training of African biocontrol researchers, USDA-ARS, Univ. of Arizona
(funded by USDA-FAS with a grant from USAID)
A Zambia technician
receiving training on
aflasafe production at
IITA, Ibadan (USAID)
• Aflatoxins in food and feed pervasive in Africa
• Biological control , as the foundation, with other practices can dramatically reduce aflatoxin contamination and improve food safety and security
• Efforts underway to pilot commercialization of aflatoxin biocontrol and develop regional strains
• The pilots need to be up-scaled and efforts to improve efficacy needs a fillip for wide-spread impact on health and trade in Africa
Summary
IITA
Tucson
USDA/ARS IITA, USDA, AATF & Doreo have Teamed up to Bring
Aflatoxin Prevention to Africa
Made Possible by Many National Partners in Ministries, Industry, and on the Farm
Nigeria
For more information about aflatoxin biocontrol for Africa, check out: www.aflasafe.com