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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,

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Presentation on theme: "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,"— Presentation transcript:

1 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 17 th Feb 2008

2 Cassava Food to> 500 million Roots high in calories Leaves :Vitamins A and B Stalks : propagation Hardy and drought-resistant Poor soils, marginalized land No fertilizers/pesticides Animal feed Industrial applications Sugar, acetone, alcohol Food security to the poor

3 Cassava Family: Euphorbiaceae Genus: Manihot Genus: Manihot esculenta Ploidy: Allotetraploid (n=9) Genome size: 770Mbp Cassava Genetic Resources: -5700 acc. (CIAT Gene Bank) -2,000 (IITA Gene bank) -2,500 (EMBRAPA) 98 wild Manihot relatives Monoecious Dicot

4 Source: FAOSTAT Cassava: A Shining Example of African Agriculture Production in Tons/Ha of the three principal producing regions

5 2004

6 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

7 S&T and cassava issues Molecular markers for breeders Transgenic cassava – modified starch content: low amylopectin

8 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

9 CMD-resistantCMD-susceptible Multiple 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

10 Targeted training in Cassava Genomics Elizabeth Balyejusa Kizito Yona Baguma Scientists at NARO (Uganda)-developing SLU CIAT (Colombia) - advanced

11 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 MarkersPositional Cloning Genomic/cDNA Libraries Genetic Mapping QTL Mapping Molecular Breeding

12 Applications of Molecular Markers in Cassava Breeding Increase heritability in the identification of superior genotypes Follow specific chromosome segments from unadapted wild relatives in breeding schemes Reduce population sizes for more precise field evaluations Delineate heterotic pools or to choose parents that maximize additive variance Accelerate inbred line development

13 Molecular Marker Resources for Cassava at CIAT 5 small insert (1-2kb) genomic libraries 3000 low-copy RFLP marker probes 3 genomic SSR sequence enriched libraries 16 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 respectively Unigene chip of 6,000 ESTs Web-based data base www.cassavagenome.org being prepared for easy access to these global public goods (GPG)

14 H DistMarker cMName GY85 13.1 OJ1 20.4 SSRY52 1.6 SSRY12 11.2 SSRY48 14.5 rGY199 12.6 rGY170 10.3 rGY104 6.4 GY127 19.7 rGY211 19.7 rSSRY91 24.3 SSRY178 16.9 SSRY63 11.2 SSRY164 rSSRY31 DistMarker cMName rSSRY52 7.2 rSSRY12 12.4 rQ11 19.0 rGY99 11.1 rGY8-1 12.0 GY85-1 9.1 nGY11 6.0 rSSRY178 37.4 rSSRY164 H A DistMarker cMName ACP-1 9.2 rGY208 15.7 SSRY98 11.4 rGY75 9.3 GY12 8.2 GY28 6.8 GY213 5.1 GY209 rSSRY4 Dist Marker cMName rSSRY4 16.4 rGY22-1 19.5 rGY75-1 7.3 rSSRY98 8.8 rBEST-2 8.8 GY68 7.0 GY28 8.9 GY32 A DistMarker cMName rGY155 3.9 rGY192 9.5 GY154 14.6 rSSRY111 15.3 SSRY59 1.8 SSRY10 11.1 rGY215 19.3 L7 28.1 AC-1 M C DistMarker cMName rGY144 2.5 rGY23 2.5 GY81 6.2 rGY89 14.0 rGY177 12.0 O11a 16.1 GY174 9.8 rSSRY183 6.4 rSSRY45 4.4 rSSRY95 17.8 rSSRY109 5.8 SSRY32 DistMarker cMName rK9a 20.1 rNI1.C2 7.6 GY23-1 13.4 rGY18 2.8 rGY26 8.2 rGY89-1 8.2 SSRY171 9.5 GY42 9.5 GY44 7.0 GY54 6.2 GY81-1 C DistMarker cMName rA16 13.2 rGY191 17.5 rV17a 6.5 GY197 6.7 rGY147 5.6 rSSRY50 26.0 GY207 B DistMarker cMName rAi14a 17.2 GY72 10.9 rSSRY61 9.4 rGY65 5.0 rPASK1 17.1 SSRY113 1.8 SSRY21 B DistMarker cMName rF19b 15.0 rGY118 12.7 GY217 11.0 rS2 4.4 rSSRY161 11.6 rGY176 20.3 rGY190 E SSRY84 Dist Marker cMName rSSRY180 17.2 GY83 28.7 GY17 12.3 SSRY72 11.0 nGY162 15.6 rAE15 23.7 GY58 21.1 CDY123a 11.5 GY118 10.0 rI4a 17.5 G13 E SSRY84 D DistMarker cMName rGOT-2 8.8 rGY167 5.0 rGY180 1.2 GY222 1.2 GY181 1.2 GY42 3.7 GY219 3.7 GY50 3.7 GY125 3.7 GY179 3.8 rON1 7.0 SSRY3 0.0 SSRY120 3.6 rSSRY40 SSRY9 D rSSRY169 10.6 GY4 14.8 SSRY108 7.6 rGY24 6.5 rGY59 7.3 GY78 DistMarker cMName rGY67H 15.8 rI18b 20.1 rJ1a 19.0 rGY57 20.5 rGY25 SSRY9 SSRY120 SSRY23 SSRY3 DistMarker cMName rSSRY55 10.7 rJ7 2.5 rG11 20.7 GY110 8.9 rSSRY100 7.9 SSRY88 19.2 Ai11b L DistMarker cMName rSSRY55 19.4 N15b 12.1 GY53 7.4 rSSRY88 8.0 rGY110 L DistMarker cMName rSSRY172 22.0 rE14b 11.9 rJ9c 7.5 rCDY74 4.4 CDY76 7.5 AD1c 14.4 K2a 27.0 GY34 9.4 SSRY101 8.6 SSRY2 SSRY5 J SSRY47 DistMarker cMName O20 11.3 AE10a 11.8 CDY76 17.1 rSSRY172 10.9 rM8b 9.5 GY7 10.6 GY34-1 25.9 K10 J DistMarker cMName rGY93 16.9 GY78 1.2 GY97 6.8 SSRY135 2.5 rSSRY30 22.4 rSSRY38 7.8 rGY41 16.7 CYP79(152) GG DistMarker cMName rSSRY135 23.4 rGA-131 10.4 rGY94 8.8 AM18 12.6 rK16d 13.3 rSSRY38 20.8 rCPY79(152) 7.8 rGY137 23.5 AB9a 24.8 SKDH-1 14.6 rAGPaseB 16.0 rSSRY106 8.2 rSSRY103 15.2 SSRY83 rSSRY30 F DistMarker cMName GY196 6.4 GY203 5.1 GY218 3.8 GY122 7.5 CDY107 3.7 GY37 2.5 GY204 2.5 GY194 5.0 GY186 5.8 SSRY179 17.0 SSRY68 rSSRY31 DistMarker cMName Z11b 12.5 GY21 11.5 SSRY68 11.4 GY37 6.7 rCDY107-1 10.1 CDY123b 4.6 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 cM DistMarker Name I CDY29 22.6 CDY68 12.1 CDY128 rGY19 10.0 rSSRY8 14.5 rS12c 23.8 rU2a Marker NamecM Dist Marker cMName rSSRY122 37.0 nrGY67 3.9 rGY36 3.0 rSSRY86 9.4 SSRY105 13.3 rGY87 15.6 W19b 8.8 GY105 5.9 SSRY177 8.4 GY5 5.7 SSRY99 14.6 rK3 U DistMarker cMName K17 18.0 SSRY6 7.3 SSRY122 9.0 SSRY42 18.7 rSSRY86 34.6 GY138 2.5 rGY164 3.7 GY223 4.5 SSRY170 U SSRY105 DistMarker cMName CDY131-1 6.5 rGY113H 6.3 rGY92 7.4 SSRY185 2.3 SSRY20 21.5 U2c X DistMarker cMName rM5a 7.0 CDY131 13.5 nCBB 10.0 rSSRY20 8.8 rAF14a 13.3 SSRY110 X N rCDY99 11.9 rGBSSII 8.6 SSRY13 11.1 rCDY44 5.6 rSSRY51 8.4 GY20 6.1 rGY10 6.8 GY108 6.2 GY52 5.3 GY13 14.6 Z18b GY88 17.3 rGY47 4.0 nGY143 cM DistMarker Name N rGY109 9.4 rGY148 5.3 rGY145 2.4 GY201-1b 11.9 SSRY13 18.8 AD4b SSRY35 6.7 rGY108 5.1 GY182 GY 124 Marker NamecM Dist Marker cMName rAF17 19.4 SSRY107 7.2 GY171 7.6 rGY210 5.5 ri4b 11.0 rA18 GY131 13.9 rSSRY83 Marker NamecM Dist Marker cMName GY66 13.2 rSSRY34 9.9 SSRY102 2.6 SSRY182 5.4 GY39 28.6 AM6 DistMarker cMName rGY115 7.9 rGY9 15.6 rGY1 16.1 rSSRY28 11.3 Ai19 PO2 16.8 SSRY7 Marker NamecM Dist F19a 4.4 AC11a 3.8 GY71 1.2 GY183 12.8 i16b R Marker NamecM Dist V J13c 8.7 GY51 25.0 F15b 18.6 rSSRY19 12.1 rG4 5.4 rK11c 7.5 rAC5 7.6 rR13b Marker NamecM Dist V rP1a 10.9 rGY156 21.2 GY134 9.9 rGY220 GY63 6.0 rSSRY19 16.0 rAC9a Marker NamecM Dist R GY100 31.4 GY71 25.9 rGY48 12.3 AE2 24.8 U1 Marker NamecM Dist rGY163 16.4 rGA-127 15.2 CDY106 27.8 rSSRY49 12.4 rGY119 K Dist cM Marker Name DistMarker cMName rGY117 17.3 rSSRY175 1.5 rSSRY85 6.9 GY82 8.3 CDY106 14.0 W16b 14.1 AC9b 10.0 GY119 19.0 rI2 K SSRY174 rSSRY82 Cassava Map 2004: 202 SSRs, 307 RFLPs, 100 RAPDs, 3 isozymes

15 RFLP Marker Segregation in an F 1 Cross of Non-inbred Parents

16 Three different CMD Resistance genes have been mapped and Can now be pyramided using molecular markers DistMarker cMName rGY115 7.9 rGY9 15.6 rGY1 16.1 rSSRY28 11.3 Ai19 CMD2 R Gene Tagging of Multiple Sources of CMD Resistance Genes D Y66 18.5 rI18b 20.5 rJ1a 20.0 rGY57 21.0 rGY25 21.2 SSRY9 23.9 SSRY3 16.2 SSRY23 CMD1 Fregene et al. 2000 Akano et al. 2001 M SSRY182 SSRY102 24.4 SSRY230 16.6 Ns905 9.1 GY39 11.2 SSRY299 NS170 SSRY182 SSRY102 24.4 SSRY230 16.6 Ns905 9.1 GY39 11.2 SSRY299 SSRY102 24.4 SSRY230 16.6 Ns905 9.1 GY39 11.2 SSRY299 CMD3 Hurtado et al. 2007

17 High resolution map around CMD2 Candidate BAC Sequence Contigs of BAC clones around the CMD2 locus Positional Cloning of CMD2

18 Susceptible to CMD High Starch TMS30555 Female Parent 1288 F 1 s Resistant to CMD (CMD2 gene) X Male Parent TME3

19 HindIII partial digestion Purification and cloning 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 by Robotics Mega base pair-sized DNA in Agarose Plugs Cassava BAC Libraries 97kb 6.9kb 48.5kb

20 BAC Clone Fingerprinting Digestion with Hind III

21 Construction of a Contig Around CMD2 CMD2 NS158 RME--1 RME--2 SSRY-28 BAC 33 Contig construction and genetic mapping of the ends of a BAC cone (BAC-33b) 4cM 7cM

22 Bringing the Benefits of Modern Science to Farmers Fields Molecular markers can help reduce breeding populations Proof of concept: Farmer participation demonstrate quickly superiority of new germplasm Social scientists/mol biologists

23 Two scientists Elizabeth Balyejusa: molecular markers –PhD –Grant awards –MBL and NARO Yona Baguma: modified starch content –PhD –Heading Biotechnology Division, NARO

24 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) Improvement granted by the Bean Improvement Cooperative (BIC) to Matthew Blair.

25 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 EBalyejusa Kizito E, Rönnberg-Wästljung AC, Egwang T, Gullberg U, Fregene M, Westerbergh A.Rönnberg-Wästljung AC Egwang TGullberg UFregene MWesterbergh A

26 Genetic diversity and variety composition of cassava on small-scale farms in Uganda: an interdisciplinary study using genetic markers and farmer interviews. Balyejusa Kizito EBalyejusa Kizito E, Chiwona-Karltun L, Egwang T, Fregene M, Westerbergh A.Chiwona-Karltun L Egwang TFregene MWesterbergh A Genetica. 2007 Jul;130(3):301-18. Epub 2006 Nov 3.

27 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..

28 Malaria

29 Malaria kills 110 Ugandan children <5 years old annually

30 Pregnant women (1 st pregnancies) bear the brunt: Poor pregnancy outcomes Maternal anemia Maternal deaths

31 Malaria control Drug treatment: RESISTANCE Insecticide treated bed nets: RESISTANCE Vector control IRS: RESISTANCE; LOGISTICS Vaccines: future ray of hope

32 Targeted training Malaria drug resistance monitoring Malaria vaccine studies

33 Malaria vaccines Activity phase Vaccine Type DevelopmentClinical trialsTotal PreErythrocytic 13427 Asexual 37946 Transmission- Blocking 213 Combination 628 Total 581674

34 Merozoite RBC membrane SERA Localization of SERA (serine repeat antigen) in P. falciparum infected RBC by immuno-gold staining

35 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

36 After processing Before processing NSH Recombinant proteins Serine repeats (35) 23 191-225 IIII II 17382 193-225 P47P50P18 SE47 SE36 989 P6 1 P50A

37 01234 0 2 4 6 8 10 12 14 01234 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 FeverAnti-SERA IgG and blood parasitemia Parasitemia (1000 μl blood) No Fever < 37.5 Fever > 37.5 Anti-SE47' IgG3 ELISA OD Anti-E50A IgG1 ELISA OD Anti-SE47Anti-SE50AAnti-SE47Anti-SE50A

38 Mean ODs for IgG subclass responses to SE47 and SE50A as a function of age in Apac District, Northern Uganda IgG 1IgG 2 IgG 3 IgG 4 11- 14 Anti-SE47' IgG Anti-SE50A IgG 0 - 12 - 5 15 - 20 6 - 8 21 - 30 9 - 10 31 - 40 > 40 11- 14 0 - 12 - 5 15 - 20 6 - 8 21 - 30 9 - 10 31 - 40 > 40 Age group ELISA OD

39 Parasite growth inhibition by human serum 75 50 25 0 Honduras 1 FCR3 Parasite Growth inhibition (%) IgG3 ELISA titer against SE47 10 5 10 2 10 3 10 4 K1 Patient Serum Parasite Growth Inhibition Assay after 24 h. Parasite culture

40 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 0 Parasite Growth Inhibition (%) 0 10 2030 40 8

41 SM MM Median SE36 titre compared with level of pigmented leucocytes IQR 7,000 6,5001,500

42 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

43

44 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%

45 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%

46 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%

47 P. vivax: chloroquine prophylactic or treatment failure P. vivax prophylactic or treatment failure

48 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

49 Mutation Sites in the PfCRT Transmembrane Protein K76 N75 M74 C72 H97 A220 N326 Q271 R371 I356

50 Molecular Diagnosis of Chloroquine Resistance in the Field

51 PCR detection of pfcrt K76T polymorphisms P1 P2 P3 P4-w P4-m D1 D2 Apo1 DIAGNOSTIC PCRPCR1: P1+P2 PCR2: P3+P4-w or P4-m RESTRICTIONPCR1: P1+P2 PCR2: D1+D2 Digestion with Apo1

52 Restriction digestion analysis of pfcrt K76T M 1 2 3 4 5 Dd2 3D7 C M 300bp

53 Distinguishing Recrudescent from reinfection with Microsatellite ta99 6 9 19 33 35 53 Dd2 Hb3 CM

54 Pteridine Dihydrofolate Tetrahydrofolate pABA dihydropteorate synthase (DHPS) Thimidine, Methionine synthesis dihydrofolate reductase (DHFR) Pyrimethamine (PM) Sulfadoxine (SD) GTP Mode of action of antifolates

55 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

56 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

57

58 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: Dip BSc drug resistance –Best AMANET student paper 2006 Prossy Namuwulya –Short term training Plasmodium transfection: LUMC

59 Gender equality/equity issues addressed? Not entirely

60 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

61 CBT in Malaria and Cassava Research addresses 6/8 MDGs Science and Technology for Sustainable Development in Africa

62 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

63 Thank you


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