CBT in Malaria and Cassava Research addresses 6/8 MDGs Thomas G. Egwang, PhD ED, African Academy of...
-
Upload
luis-marshall -
Category
Documents
-
view
216 -
download
0
Transcript of CBT in Malaria and Cassava Research addresses 6/8 MDGs Thomas G. Egwang, PhD ED, African Academy of...
CBT in Malaria and Cassava Research addresses 6/8 MDGs
Thomas G. Egwang, PhD
ED, African Academy of Sciences, Nairobi, Kenya
DG/SD, Med Biotech Laboratories, Kampala, Uganda
Science and Technology for Sustainable
Development in Africa
AAAS Boston 17th Feb 2008
Cassava Food to> 500 million
Roots high in caloriesLeaves :Vitamins A and BStalks : propagation
Hardy and drought-resistantPoor soils, marginalized landNo fertilizers/pesticides
Animal feed Industrial applications
Sugar, acetone, alcohol
Food security to the poor
Cassava Family:EuphorbiaceaeGenus: ManihotGenus: Manihot esculentaPloidy: Allotetraploid (n=9)Genome size: 770MbpCassava Genetic Resources: -5700 acc. (CIAT Gene Bank) -2,000 (IITA Gene bank) -2,500 (EMBRAPA)98 wild Manihot relativesMonoecious Dicot
Source: FAOSTAT
Cassava: A ‘Shining Example’ of African Agriculture
Production in Tons/Ha of the three principal producing regions
2004
Important issues
• Diseases– Cassava Mosaic Disease (CMD)
• Starch content– Amylose versus amylopectin content
• Bitter taste: bitter strains, sweet strains• Glycoside of HCN• V. bitter forms > 100 pmm cyanide
S&T and cassava issues
• Molecular markers for breeders
• Transgenic cassava– modified starch content: low amylopectin
The Cassava Mosaic Disease (CMD)
A viral disease endemic in SS-Africa
Vectored by the white fly (Bemisia tabaci)
Est > $1 B/y Control: development of CMD
resistant varieties: 6-10 years Molecular markers
CMD-resistant CMD-susceptibleMultiple resistance to diseases and pests
Improved multiple disease and pest resistant clones showing high levels of resistance to CMD
Multiple Sources of Resistance to CMD
Targeted training in Cassava Genomics
Elizabeth Balyejusa KizitoElizabeth Balyejusa KizitoYona BagumaYona Baguma
Scientists at NARO (Uganda)-developingSLU
CIAT (Colombia) -advanced
Cassava Biotechnology Research: CBT needs
Tissue Culture
Genetic Transformation
Embryo rescue
Micro-propagation
Tissue Culture
Conservation and exchange
Molecular Markers
BAC Libraries
BAC Clone screening and fingerprinting
Genetic Markers Positional Cloning
Genomic/cDNALibraries
Genetic Mapping
QTL Mapping
Molecular Breeding
Applications of Molecular Markers in Cassava
BreedingIncrease heritability in the identification of
superior genotypes Follow specific chromosome segments from
unadapted wild relatives in breeding schemesReduce population sizes for more precise
field evaluationsDelineate heterotic pools or to choose
parents that maximize additive varianceAccelerate inbred line development
Molecular Marker Resources for Cassava at
CIAT 5 small insert (1-2kb) genomic libraries3000 low-copy RFLP marker probes3 genomic SSR sequence enriched libraries16 partial and full-length cDNA libraries 850SSR markers from genomic and cDNA libraries Two diversity array technology (DArT) chip with 1500
and 10,000 polymorphic clones respectivelyUnigene chip of 6,000 ESTsWeb-based data base www.cassavagenome.org
being prepared for easy access to these global public goods (GPG)
HDist MarkercM Name
GY8513.1
OJ1
20.4
SSRY521.6SSRY1211.2SSRY48
14.5
rGY19912.6
rGY17010.3
rGY1046.4
GY127
19.7
rGY211
19.7
rSSRY91
24.3
SSRY178
16.9
SSRY6311.2
SSRY164
rSSRY31
Dist MarkercM Name
rSSRY527.2
rSSRY12
12.4
rQ11
19.0
rGY99
11.1
rGY8-1
12.0
GY85-19.1
nGY116.0
rSSRY178
37.4
rSSRY164
H
ADist MarkercM Name
ACP-19.2
rGY208
15.7
SSRY98
11.4
rGY759.3
GY128.2
GY286.8
GY2135.1
GY209
rSSRY4
Dist MarkercM Name
rSSRY4
16.4
rGY22-1
19.5
rGY75-17.3
rSSRY988.8
rBEST-28.8
GY687.0
GY288.9
GY32
A
Dist MarkercM Name
rGY1553.9rGY192
9.5GY154
14.6
rSSRY111
15.3
SSRY591.8SSRY10
11.1
rGY215
19.3
L7
28.1
AC-1
M
CDist MarkercM Name
rGY1442.5rGY232.5GY816.2rGY89
14.0
rGY177
12.0
O11a
16.1
GY174
9.8
rSSRY1836.4
rSSRY454.4
rSSRY95
17.8
rSSRY1095.8
SSRY32
Dist MarkercM Name
rK9a
20.1
rNI1.C27.6
GY23-1
13.4
rGY182.8rGY26
8.2rGY89-1
8.2SSRY171
9.5
GY429.5
GY447.0
GY546.2
GY81-1
CDist MarkercM Name
rA16
13.2
rGY191
17.5
rV17a6.5
GY1976.7
rGY1475.6
rSSRY50
26.0
GY207
BDist MarkercM Name
rAi14a
17.2
GY72
10.9
rSSRY619.4
rGY655.0
rPASK1
17.1
SSRY1131.8SSRY21
BDist MarkercM Name
rF19b
15.0
rGY118
12.7
GY217
11.0
rS24.4
rSSRY161
11.6
rGY176
20.3
rGY190
E
SSRY84
Dist MarkercM Name
rSSRY180
17.2
GY83
28.7
GY1712.3
SSRY7211.0
nGY162
15.6
rAE15
23.7
GY58
21.1
CDY123a11.5
GY11810.0
rI4a
17.5
G13
E
SSRY84
DDist MarkercM Name
rGOT-28.8
rGY1675.0
rGY1801.2GY2221.2GY1811.2GY423.7GY2193.7GY503.7GY1253.7GY1793.8rON17.0SSRY30.0SSRY1203.6rSSRY40
SSRY9
D
rSSRY169
10.6
GY4
14.8
SSRY1087.6
rGY246.5
rGY597.3
GY78
Dist MarkercM Name
rGY67H
15.8
rI18b
20.1
rJ1a
19.0
rGY57
20.5
rGY25 SSRY9SSRY120SSRY23
SSRY3
Dist MarkercM Name
rSSRY55
10.7
rJ72.5rG11
20.7
GY1108.9
rSSRY1007.9
SSRY88
19.2
Ai11b
LDist MarkercM Name
rSSRY55
19.4
N15b
12.1
GY537.4
rSSRY888.0
rGY110
LDist MarkercM Name
rSSRY172
22.0
rE14b
11.9
rJ9c7.5
rCDY744.4
CDY767.5
AD1c
14.4
K2a
27.0
GY349.4
SSRY1018.6
SSRY2 SSRY5
J
SSRY47
Dist MarkercM Name
O20
11.3
AE10a
11.8
CDY76
17.1
rSSRY172
10.9
rM8b9.5
GY7
10.6
GY34-1
25.9
K10
J
Dist MarkercM Name
rGY93
16.9
GY781.2GY976.8SSRY1352.5rSSRY30
22.4
rSSRY387.8
rGY41
16.7
CYP79(152)
GGDist MarkercM Name
rSSRY135
23.4
rGA-13110.4
rGY948.8
AM1812.6
rK16d13.3
rSSRY38
20.8
rCPY79(152)7.8
rGY137
23.5
AB9a
24.8
SKDH-114.6
rAGPaseB16.0
rSSRY1068.2
rSSRY10315.2
SSRY83
rSSRY30
FDist MarkercM Name
GY1966.4
GY2035.1
GY2183.8GY122
7.5CDY1073.7GY372.5GY2042.5GY1945.0GY186
5.8SSRY179
17.0
SSRY68
rSSRY31
Dist MarkercM Name
Z11b
12.5
GY2111.5
SSRY6811.4
GY376.7
rCDY107-110.1
CDY123b4.6 GY80
21.0
rV20a
28.3
H14c
20.3
GY16
23.0
rGY55
F
I
rAC1
11.1
GY130
11.9
R1
13.6
rW3
19.4
rGY201-1a
13.1
rD5a
11.5
rB3a
H14b
15.0
GY128
17.4
GY143
cMDist Marker
Name
I
CDY29
22.6
CDY68
12.1
CDY128
rGY19
10.0
rSSRY8
14.5
rS12c
23.8
rU2a
MarkerNamecM
Dist
Dist MarkercM Name
rSSRY122
37.0
nrGY673.9rGY363.0rSSRY86
9.4SSRY105
13.3
rGY87
15.6
W19b8.8
GY1055.9
SSRY1778.4
GY55.7
SSRY99
14.6
rK3
UDist MarkercM Name
K17
18.0
SSRY67.3
SSRY1229.0
SSRY42
18.7
rSSRY86
34.6
GY1382.5rGY1643.7GY2234.5SSRY170
U
SSRY105
Dist MarkercM Name
CDY131-16.5
rGY113H6.3
rGY927.4
SSRY1852.3SSRY20
21.5
U2c
XDist MarkercM Name
rM5a7.0
CDY131
13.5
nCBB
10.0
rSSRY208.8
rAF14a
13.3
SSRY110
X
N
rCDY99
11.9
rGBSSII
8.6SSRY13
11.1
rCDY445.6
rSSRY518.4
GY206.1
rGY106.8
GY1086.2
GY525.3
GY13
14.6
Z18b
GY88
17.3
rGY474.0nGY143
cMDist Marker
Name
N
rGY109
9.4
rGY1485.3
rGY1452.4
GY201-1b
11.9
SSRY13
18.8
AD4b
SSRY35
6.7
rGY1085.1
GY182
GY 124
MarkerNamecM
Dist
Dist MarkercM Name
rAF17
19.4
SSRY1077.2
GY1717.6
rGY2105.5
ri4b
11.0
rA18
GY131
13.9
rSSRY83
MarkerNamecM
DistDist MarkercM Name
GY66
13.2
rSSRY34
9.9
SSRY1022.6SSRY1825.4GY39
28.6
AM6
Dist MarkercM Name
rGY1157.9
rGY9
15.6
rGY1
16.1
rSSRY28
11.3
Ai19
PO2
16.8
SSRY7
MarkerNamecM
Dist
F19a4.4
AC11a3.8
GY711.2
GY18312.8
i16b
RMarkerNamecM
Dist
V
J13c
8.7
GY51
25.0
F15b
18.6
rSSRY19
12.1
rG45.4
rK11c7.5
rAC57.6
rR13b
MarkerNamecM
DistV
rP1a
10.9
rGY156
21.2
GY134
9.9
rGY220GY63
6.0rSSRY19
16.0
rAC9a
MarkerNamecM
DistR
GY100
31.4
GY71
25.9
rGY48
12.3
AE2
24.8
U1
MarkerNamecM
Dist
rGY163
16.4
rGA-127
15.2
CDY106
27.8
rSSRY49
12.4
rGY119
KDistcM
MarkerName Dist Marker
cM Name
rGY117
17.3
rSSRY1751.5rSSRY856.9GY82
8.3CDY106
14.0
W16b
14.1
AC9b
10.0
GY119
19.0
rI2
K
SSRY174rSSRY82
Cassava Map 2004: 202 SSRs, 307 RFLPs, 100 RAPDs, 3 isozymes
RFLP Marker Segregation in an F1 Cross of Non-inbred
Parents
Three different CMD Resistance genes have been mapped and Can now be pyramided using molecular markers
Dist MarkercM Name
rGY1157.9
rGY915.6
rGY116.1
rSSRY2811.3
Ai19
CMD2
R
Gene Tagging of Multiple Sources of CMD Resistance Genes
DY66
18.5rI18b
20.5rJ1a
20.0rGY57
21.0rGY25
21.2SSRY9
23.9
SSRY316.2
SSRY23CMD1CMD1
Fregene et al. 2000
Akano et al. 2001
M
SSRY182
SSRY102
24.4
SSRY230
16.6
Ns9059.1
GY3911.2
SSRY299
NS170
SSRY182
SSRY102
24.4
SSRY230
16.6
Ns9059.1
GY3911.2
SSRY299
SSRY102
24.4
SSRY230
16.6
Ns9059.1
GY3911.2
SSRY299
CMD3
Hurtado et al. 2007
High resolution map around CMD2
A C
250
R A G G G T C A A A
260
A C M A A A G C T T
270
C G A T C C C C G C
280
C A A A C C C T A T
290
T G T T G G T A C T
300
A G T T C A C G G G
310
T C A T T C T G C T
320
A T G C A G G A T
Candidate BAC Sequence
Contigs of BAC clones around theCMD2 locus
Positional Cloning of CMD2
Susceptible to CMDHigh Starch
TMS30555
Female Parent
1288 F1s
Resistant to CMD(CMD2 gene)
X
Male ParentTME3
HindIII partialdigestion
Purification andcloning
Ligation and Sizing of clones
Cloning vector
388kb
194kb
48.5kb
Size selection
Sizing of clones
3 BAC libraries with 5X, 10X, and 11X coverage of the genome respectively
Clone picking byRobotics
Mega base pair-sized DNA in Agarose Plugs
Cassava BAC Libraries
97kb
6.9kb
48.5kb
BAC Clone Fingerprinting
Digestion with Hind III
Construction of a Contig Around CMD2
CM
D2
NS
15
8
RM
E--
1
RM
E--
2
SS
RY
-28
BA
C 3
3
Contig construction and genetic mapping of the ends of a BAC cone (BAC-33b)
4cM7cM
Bringing the Benefits of Modern Science to Farmer’s
FieldsMolecular markers can help reduce breeding populations
Proof of concept: Farmer participation demonstrate quickly superiority of new germplasm
Social scientists/mol biologists
Two scientists
• Elizabeth Balyejusa: molecular markers– PhD – Grant awards– MBL and NARO
• Yona Baguma: modified starch content– PhD– Heading Biotechnology Division, NARO
CIAT-Outstanding Principal Staff Achievement Award (OPSA) granted to Segenet Kelemu
CIAT-Outstanding Young Scientist-of-the-Year Award (OYSYA)granted to Marcela Quintero.
CIAT-Outstanding Research Publication Award (ORPA) , granted to Elizabeth Balyejusa Kizito, Linley Chiwona-Karltun, Thomas Egwang, Martin Fregene, and Anna Westerbergh.
Achievement Award in Bean (Phaseolus) Improvementgranted by the Bean Improvement Cooperative (BIC) to Matthew Blair.
Hereditas. 2007 Sep;144(4):129-36
Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots
Balyejusa Kizito E, Rönnberg-Wästljung AC, Egwang T, Gullberg U, Fregene M, Westerbergh A.
Genetic diversity and variety composition of cassava on small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews.
Balyejusa Kizito E, Chiwona-Karltun L, Egwang T, Fregene M, Westerbergh A.
Genetica. 2007 Jul;130(3):301-18. Epub 2006 Nov 3.
Plant Science 164: 833-839, 2003
Expression patterns of the gene encoding starch branching enzyme II in the storage roots of cassava (Manihot esculenta Crantz)
Baguma Y, Sun C, Ahlandsberg S, Mutisya J, Palmqvist S, Rubaihayo PR, Magambo MJ, Egwang TG, Larsson H, Jansson C. .
Malaria
Malaria kills 110 Ugandan children <5 years old annually
Pregnant women (1st pregnancies) bear the brunt:
•Poor pregnancy outcomes
•Maternal anemia
•Maternal deaths
Malaria controlDrug treatment: RESISTANCE
Insecticide treated bed nets: RESISTANCE
Vector control IRS: RESISTANCE; LOGISTICS
Vaccines: future ray of hope
Targeted training
•Malaria drug resistance monitoring
•Malaria vaccine studies
Malaria vaccines Activity phase
VaccineType
Development Clinical trials Total
PreErythrocytic 13 4 27
Asexual 37 9 46
Transmission-Blocking
2 1 3
Combination 6 2 8
Total 58 16 74
Merozoite
RBC membrane
SERA
Localization of SERA (serine repeat antigen) in P. falciparum infected RBC by immuno-gold staining
Longitudinal study School children Age 7-16 years
Study Populations
Case-control study of severe & mild malaria Age 6-59 months
Apac, Uganda transmission : perennial
After processing
Before processing
NS H
Recombinant proteins
Serine repeats (35)23 191-225
I III II
17 382
△193-225
P47 P50 P18
SE47’
SE36
989
P6
図 1
P50A
0 1 2 3 4
0
2
4
6
8
10
12
14
0 1 2 3 4
0
2
4
6
8
10
12
14
0
1
2
3
4
0
0.5
1.0
2.0
1.5
Correlation between anti-SERA IgG and malaria symptom or blood parasitemia
No Fever < 37.5 ℃
Fever> 37.5 ℃
anti-SE47' IgG3 (OD at 405 nm)
anti-SE50A IgG1 (OD at 405 nm)
Anti-SERA IgG and Fever Anti-SERA IgG and blood parasitemia
Pa
ras
ite
mia
(1
00
0/
μl
blo
od
)
No Fever < 37.5 ℃
Fever> 37.5 ℃
An
ti-S
E4
7' I
gG
3 E
LIS
A O
D
An
ti-E
50
A I
gG
1 E
LIS
A O
D
Anti-SE47’ Anti-SE50A Anti-SE47’ Anti-SE50A
Mean ODs for IgG subclass responses to SE47’ and SE50A as a function of age in Apac District, Northern Uganda
IgG 1 IgG 2 IgG 3 IgG 4
11-
14
0
2.0
1.5
1.0
0.5
0
1.0
0.75
0.5
0.25
Anti-SE47' IgG Anti-SE50A IgG0
- 1
2 -
5
15 -
20
6 -
8
21 -
30
9 -
10
31 -
40
> 4
0
11-
14
0 -
1
2 -
5
15 -
20
6 -
8
21 -
30
9 -
10
31 -
40
> 4
0
Age group Age group
EL
ISA
OD
EL
ISA
OD
Parasite growth inhibition by human serum
75
50
25
0
Honduras ー 1
FCR3
Par
asit
e G
row
th i
nh
ibit
ion
(%
)
IgG3 ELISA titer against SE47’
105102 103 104
K1
Patient Serum
Parasite Growth Inhibition Assay after 24 h.
Parasite culture
Recombinant 47’ neutralizes the inhibitory effect of patient serum on in vitro parasite
growth
SE50A
SE47’
SE36
Concentration of recombinant protein for neutralization (μg/ml)
50
40
30
20
10
0Pa
ras
ite
Gro
wth
Inh
ibit
ion
(%
)
0 10 20 30 40
8
SMSM
MM
Median SE36 titre compared with level of pigmented leucocytes
IQR 7,000 6,500 1,500
GMP production facilities for SE36 located in the Kanonji institute of The Research Foundation for Microbial Diseases of Osaka University
(BIKEN)
Production of malaria vaccine SE36 under GMP environments for clinical trials
P. falciparum: chloroquine resistance
Countries with at least one study indicating chloroquine total failure rate > 20%
No recent data available
Countries with at least one study indicating chloroquine total failure rate > 10%
P. falciparum: mefloquine treatment failure
Countries with at least one study indicating mefloquine total failure rate > 20%
No failure reported
Mefloquine total failure rate < 10%
No recent data available
Countries with at least one study indicating mefloquine total failure rate > 10%
P. falciparum: sulfadoxine-pyrimethamine
resistance
Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 20%
No failure reported
Sulfadoxine-pyrimethamine total failure rate < 10%
No recent data available
Countries with at least one study indicating sulfadoxine-pyrimethamine total failure rate > 10%
P. vivax: chloroquine prophylactic
or treatment failure
P. vivax prophylactic or treatment failure
Sample ID
Molecular monitoring of drug resistance
-Finger prick blood spotted onto filter paper
-Sample was air-dried
-Storage at room temperature
-Shipment in luggage or by regular mail
-DNA extracted by simple methods for molecular analysis
Mutation Sites in the PfCRT Transmembrane Protein
NH2
COOH
K76
N75M74
C72
H97
A220
N326
Q271 R371
I356
TCRP1
TCRP2
TCRP3
TCRP4-wTCRP4-m
Nested Mutation Specific PCR of pfcrt
Primary amplification PCR1: TCRP1 + TCRP2
Diagnostic PCR PCR2: TCRP2 + TCRP4-w or TCRP4-m
K76T
M S R S R S R S R S R
ctrl H2ODd2 3D7 106/1
pfcrt diagnostic PCR
Molecular Diagnosis of Chloroquine Resistance in the Field
PCR detection of pfcrt K76T polymorphisms
P1
P2
P3
P4-wP4-m
D1
D2
Apo1
DIAGNOSTIC PCR PCR1: P1+P2 PCR2: P3+P4-w or P4-m
RESTRICTION PCR1: P1+P2PCR2: D1+D2Digestion with Apo1
Restriction digestion analysis of pfcrt K76T
M 1 2 3 4 5 Dd2 3D7 C M
300bp
Distinguishing Recrudescent from reinfection with Microsatellite ta99
6 9 19 33 35 53 Dd2 Hb3 CM
Pteridine
Dihydrofolate
Tetrahydrofolate
pABA dihydropteorate synthase(DHPS)
Thimidine, Methionine synthesis
dihydrofolate reductase(DHFR)
Pyrimethamine (PM)
Sulfadoxine (SD)
GTP
Mode of action of antifolates
Relevance of Malaria Studies to MDG
•Change in CQ/SP drug policy
•Phase I-II trials of SERA5
•IPTp assessment of Coartem-on-going
•Collaboration with Control Pgm
Targeted CBT to meet MDGs• Cassava: Molecular markers
– Elizabeth Bayejusa Kizito
• Cassava: Modified starch content– Yona Baguma
• Malaria: Vaccine studies– Brenda Okech
• Malaria Drug resistance– Anne Nalunkuma– Connie Agwang
Female scientists trained• Elizabeth Balyejusa Kizito
– PhD Ag biotech– Best CIAT paper 2006– Woman science fellowship-USA 2007
• Brenda Okech– PhD Malaria vaccine studies– Career development award TDR/GSK– Management of malaria vaccine clinical trials– UK and Japan
• Anne Kazibwe: MSc malaria drug resistance– PhD drug resistance
• Connie Agwang: DipBSc drug resistance– Best AMANET student paper 2006
• Prossy Namuwulya– Short term training Plasmodium transfection: LUMC
Gender equality/equity issues addressed?
Not entirely
Other training in malaria• Group workshops
– Bioinformatics 2002 HHMI– Drug resistance genotyping MIM/DTR, IAEA– Molecular biology primer WHO /TDR– > 70 Africans from> 12 countries
• IAEA training for Latin America– Panama, Colombia, Peru
• Malaria/HIV interactions– Mentored clinical fellow under ILA EGPAF– International Scholars Award EGPAF
• Media malaria advocacy and outreach– Uganda Media for Health (UM4H)– Malaria Consortium
CBT in Malaria and Cassava Research addresses 6/8 MDGs
Science and Technology for Sustainable
Development in Africa
Acknowledgement• HHMI• Elizabeth Glaser
Pediatric AIDS Fdn• WHO/TDR• SIDA/SAREC• IAEA• MIM/TDR• NIH• EU• Govt of Japan• Malaria Consortium
• NARO• CIAT• MBL staff • Martin Fregene• Ann Westerbergh• Urban Gullberg• Christian Johannson• LindleyChiwona-Karltun• Eleanor Riley• Lisa Ranford-Cartwright• Cally Roper• Toshihiro Horii• Andy Waters• Christine Clayton• Alan Thomas
Thank you