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Biosciences research at the International Livestock Research Institute (ILRI)

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Presented by Steve Kemp and Vish Nene at a University of Nairobi seminar, Nairobi, 5 June 2013

Presented by Steve Kemp and Vish Nene at a University of Nairobi seminar, Nairobi, 5 June 2013

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  • 1. Biosciences  research  at    Interna.onal  Livestock    Research  Ins.tute  (ILRI)  A  seminar  given  by  Steve  Kemp  and  Vish  Nene  at  University  of  Nairobi  5th  June  2013  
  • 2. 2  
  • 3. 3  
  • 4. 4  Source:  FAOSTAT,  2010  data  
  • 5. Four  out  of  the  five  highest  value  global  commodi.es  are  livestock  5  Source:  FAOSTAT,  2010  data  
  • 6. %  growth  in  demand  for  livestock  products  2000  -­‐  2030  6  FAO,  2012  
  • 7. ILRI  Mission  and  Strategy    §  ILRI envisions a world where all people have access to enoughfood and livelihood options to fulfill their potential.§  ILRI’s mission is to improve food and nutritional security and toreduce poverty in developing countries through research forefficient, safe and sustainable use of livestock— ensuring betterlives through livestock§  ILRI works in partnerships and alliances with other organizations,national and international, in livestock research, training andinformation. ILRI works in all tropical developing regions of Africaand Asia.§  ILRI is a member of the CGIAR Consortium that conducts foodand environmental research to help alleviate poverty and increasefood security while protecting the natural resource base.
  • 8. Strategic  objec.ves  §  ILRI  and  its  partners  will  develop,  test,  adapt  and  promote  science-­‐based  prac%ces  that—being  sustainable  and  scalable—achieve  beXer  lives  through  livestock.  Ø  ILRI  and  its  partners  will  provide  compelling  scien%fic  evidence  in  ways  that  persuade  decision-­‐makers—from  farms  to  boardrooms  and  parliaments—that  smarter  policies  and  bigger  livestock  investments  can  deliver  significant  socio-­‐economic,  health  and  environmental  dividends  to  both  poor  na.ons  and  households.  Ø  ILRI  and  its  partners  will  work  to  increase  capacity  amongst  ILRI’s  key  stakeholders  and  the  ins.tute  itself  so  that  they  can  make  beXer  use  of  livestock  science  and  investments  for  beXer  lives  through  livestock.  
  • 9. ILRI’s  competencies  Integrated sciences BiosciencesGender and equity VaccinesResilience GenomicsValue chains and innovation BreedingZoonotics and food safety BecAFeeds Genomics and gene deliveryLivestock and environment (bothdirections)Feed biosciencesPolicy, investment and trade Poultry geneticsAnimal health deliveryPayment for ecosystem servicesConservation of indigenous animalgenetic resourcesRuminants and monogastrics
  • 10. ILRI’s  research  teams  10  Integrated sciences BiosciencesAnimal science for sustainableproductivityBecA-ILRI hubFood safety and zoonoses Vaccine platformLivestock systems and theenvironmentAnimal bioscienceLivelihoods, gender and impact Feed and forage biosciencePolicy, trade, value chains Bioscience facilities
  • 11. ILRI  Resources  •  Staff:  700.  •  Budget:  $60  million.    •  30+  scien.fic  disciplines.    •  130  senior  scien.sts  from  39  countries.  •  56%  of  interna.onally  recruited  staff  are  from  22  developing  countries.  •  34%  of  interna.onally  recruited  staff  are  women.    •  Large  campuses  in  Kenya  and  Ethiopia.    •  70%  of  research  in  sub-­‐Saharan  Africa.  
  • 12. ILRI  Offices  Mali  Nigeria  Mozambique  Kenya  Ethiopia  India  Sri  Lanka  China  Laos  Vietnam  Thailand  Nairobi: HeadquartersAddis Ababa: principal campusIn 2012, offices opened in:Kampala, UgandaHarare, ZimbabweOffice in Bamako, Malirelocated toOuagadougou, Burkina FasoDakar, Senegal
  • 13. Biosciences  eastern  and  central  Africa  –  ILRI  Hub  § a  strategic  partnership  between  ILRI  and  NEPAD.  § a  biosciences  plahorm  that  makes  the  best  lab  facili.es  available  to  the  African  scien.fic  community.  § building  African  scien.fic  capacity.  § iden.fying  agricultural  solu.ons  based  on  modern  biotechnology.  § hosted  at  ILRI’s  headquarters,  Nairobi,  Kenya.      
  • 14. § Biosciences  infrastructure    
  • 15. § Biorepository    •  Sampling is a very time-consuming andexpensive exercise.•  We have an ethical and scientificresponsibility to make the best use of thateffort and money!
  • 16. § Biorepository    
  • 17. § Sequencing  and  bioinforma.cs    The Bioinformatics platformhas 88 compute cores, 31TBof network-attachedGlusterFS storage and backup systems.• 454 GSFLX– 500 Mbases in 7 hour run– $10/Mb– 500bp read lengths– Homo-polymer problem• Illumina MiSeq– 1.5-2Gbases in 27 hour run– $0.15/Mb– <150bp read lengths
  • 18. § Sequencing  and  bioinforma.cs    
  • 19. § Trypanosomias  research.  § Vaccine  research    
  • 20. African Trypanosomiasis•  Caused by extracellular protozoanparasites – Trypanosoma•  Transmitted between mammals by Tsetseflies (Glossina sp.)•  Prevalent in 36 countries of sub-SaharaAfrica.In cattle•  A chronic debilitating and fatal disease.•  A major constraint on livestock andagricultural production in Africa.•  Costs US$ 1 billion annually.In human (Human Sleeping Sickness)•  Fatal•  60,000 people die every year•  Both wild and domestic animals are themajor reservoir of the parasites for humaninfection.
  • 21. Trypanosomias  research  Trypanosomes cause fataldisease in humans and livestock.T. congolense,T. vivaxT brucei rhodesienseT brucei gambiense
  • 22. Control and Treatment of African TrypanosomiasisVector Control (Tsetse Fly)•  Using toxic insecticide•  Not sustainable•  Negative impacts on environmentVaccine•  Tryps periodically change the major surfaceantigen – variant surface glycoprotein (VSG) andevade the host immune system.•  More than 2 decades, there is no effectivevaccine developed.Drug•  Drug toxicity and resistance•  Expensive
  • 23. BovinsBovins et GlossinesGlossinesCattleTsetseCattle and tsetseOrigins of N’Dama and Boran cattleN’DamaBoran
  • 24. Contribution of 10 genes from Boran and N’Damacattle to reduction in degree of trypanosomosisBoran (relatively susceptible)The N’Dama and Boran each contribute trypanotolerance alleles at 5of the 10 most significant QTL, indicating that a synthetic breed couldhave even higher tolerance than the N’Dama.N’Dama (tolerant)-15-10-5051015-15-10-5051015
  • 25. Studying the tolerant/susceptible phenotype hasproblems:• Separating cause from effect• Separating relevant from irrelevant.• Dominance of the ‘what is happening to thisweeks trendy gene/protein/cytokine?’approach.
  • 26. An EST Library screen identifiesARHGAP15282H->P mutation in the Bta2(anaemia) QTLØ Screened EST libraries made from fourtissues from N’Dama and Boran for SNPwithin shortlisted genes.
  • 27. NDama (n = 35) Boran (n = 28)282P-Allele 0.990 0.125282H-Allele 0.010 0.875Gene frequencyH → P mutation at AA282Alignment of N’Dama ARHGAP15 withhomologuesCow NDama KFITRRPSLKTLQEKGLIKDQIFGSPLHTLCEREKSTVPRFVKQCIEAVEK !Cow Boran KFITRRPSLKTLQEKGLIKDQIFGSHLHTLCEREKSTVPRFVKQCIEAVEK !Human KFISRRPSLKTLQEKGLIKDQIFGSHLHTVCEREHSTVPWFVKQCIEAVEK !Pig KFITRRPSLKTLQEKGLIKDQIFGSHLHTVCERENSTVPRFVKQCIEAVEK !Chicken KFISRRPSLKTLQEKGLIKDQIFGSHLHLVCEHENSTVPQFVRQCIKAVER !Salmon KFISRRPSMKTLQEKGIIKDRVFGCHLLALCEREGTTVPKFVRQCVEAVEK !
  • 28. ARHGAP15 is a RAC binding protein and the mutation at theproximal end of the RAC binding domain affects in vitro activityThe tolerant allele would be expected to inhibit RAC1 activity inthe MAPK pathway which plays a key role in regulatinginflammatory responses and could lead to the observeddifferences in expression or amplify downstream expressiondifferences caused by other factors.
  • 29. African Trypanosomes Infectivity• T. congolense• T. vivax• T. brucei brucei• T. brucei rhodesienseT. brucei gambienseCattle Human Baboon (Papio papio)+ - -+ + -Human and baboon resistance is due to innate TrypanosomeLytic Factor (TLF) in serum which is a subclass of high densitylipoprotein (HDL) and can create pores in Tryps lysosomemembrane and kill the trypanosomes by loss of osmoregulation.- + -
  • 30. Can we construct a transgenic cow with resistance toAfrican Trypanosomiasis ?•  Establish a transgenic cattle model with AfricanTrypanosomiasis resistance using nuclear transfer (cloning).•  On the background of a Kenyan indigenous breed – KenyanBoran.•  Introduce the gene – apoL-I from Baboon into Boran, whichis the key trypanolytic component of Baboon’s protectiveTrypanosome Lytic Factor (TLF) against both cattle andhuman-infective trypanosomes.
  • 31. Complete  protec%on  from  human  infec%ve  Trypanosomes  by  baboon  apoL-­‐I  in    transient  transgenic  mice  0 20 40 60 80 100 120 140020406080100Vector (N=6)apoL-I + Hpr (N=5)apoL-I (N=5)**Days post infection• P  =  <  0.01  • Vector  vs.  treatment  Thomson  et  al  PNAS  2009    106:19509-­‐19514    
  • 32. Apol-3Construct withBaboon ApoL-I GenomicSequencePotentialregulatorySequenceMyh 9(myosin heavy chain 9)Chromosome 5Cattle Apol Family Locus(6, 2 like, 4 like, 3)Targeting StrategyApol-6, 2 like, 4 like
  • 33. Project StrategyGenomic locus ofBaboon apoL-I geneVector constructionValidate the construct intransgenic mouseBovine embryonic fibroblasts(BEF) primary cultureTransfection & screeningapoL-I Transgenic BEFsNuclear TransferTransgenic calvesPhenotypingTrypanosome resistanttransgenic Boran bullILRIILRIKenyaBoranRoslinInstituteNew YorkUniversityMichiganStateUniversity
  • 34. NuclearTransfer(Cloning)Electrofusion278 daysBovineEmbryonicfibroblastOocyteOocyte-cell coupletBlastocystCloned calf born
  • 35. Enuclea.on  Polar bodyPolar bodyPolar bodyMIIplateUV+Transmitted lightRemove the PB and surroundingcytoplasm, as little as possibleCheck removal of MII plateunder UV light
  • 36. Cell  Transfer  FibroblastSelect the smallest, round cells withsmooth and shining edgeInject the selected fibroblast into theperi-vitelline space and push the cell intouch with the oocyte cytoplasm.Oocyte-cell couplet
  • 37. Electrofusion  Lineperpendicular tothe electrodeselectrodes
  • 38. Cell line: Kenya Boran, BEFs_E5_286, Male  No. of Oocytes  No. ofReconstructedEmbryos  No. ofBlsts  No. of Blststransferred  No. ofEmbryoTransfer  Pregnancy   Abortion  No.  of  born  calves  1244   723   85   22   16   5   3   2     58.1%   11.8%       31.3%   60.0%   40%  Summary  of  Control  Nuclear  Transfer    
  • 39. Name: TatuDate of Birth:16 July 2012 (Kapiti)Sex: MaleBirth Weight:46 kgDate of Death: 19 July 2012 (74 hrs)Cause of death: Low temperature,low blood glucose …ID: CL001 (Tumaini)Date of Birth: 21 August 2012(ILRI)Sex: MaleBirth Weight: 35 kgCurrent age: 7.5 months, healthyTwo Cloned Calves born through Caesarean Section
  • 40. AtBirth6-MonthCL001 (Tumaini)
  • 41. Identification of cloned calves with microsatellite markersMS Marker ID   Chromosome  Alleles Size    E5(Cell line)  231-F(Tatu)  BH058(Mother)  CL001(Tumaini)  Comment  RM006   7  103.24   103.24     103.23  Calf same as E5  106.96   106.95   106.88   106.93       110.7    BM4440   2       123.69    Calf same as E5No allele as dam  132.21   132.24     132.31  136.54   136.55     136.57       143.41    INRA053   7  90.96   90.92     90.86  Calf same as E5  102.69   102.7   102.7   102.7       110.14    BMS1116   7       141.67    Calf same as E5  143.87   143.77     143.83  146.03   145.93   145.96   145.96  ILST098   2       93.02    Calf same as E5No allele as dam  101.08   101     101.08  104.77   104.73     104.79       110.45    Two born calves are the same as the cell line in 11 microsatellite markers.
  • 42. Future ActivitiesTransfection of Boran BEFs line(Roslin Institute, UK)Establish Apol-I Transgenic Boran by NuclearTransfer with Transgenic CellsPhenotyping (confirm Tryps resistance)•  Apol-I expression pattern•  Killing of Trypanosomes in vitro (serum) and in vivo(challenge)•  Monitor the health conditions with growthIncrease Genetic Diversity•  Establish more transgenic cattle withKenya Boran BEFs lines•  Establish transgenic cattle with otherKenyan indigenous breedsTransgene Delivery•  Develop a breeding programme todisseminate the transgene with farmersRegulatory, legal, safety & public awareness issues
  • 43. Future ActivitiesTumainiA cloned Kenya Boran calfmade by SCNT from a Boranembryo fibroblast cell lineCloned NOT transgenic
  • 44. Current and future animalvaccine research activities atILRIVaccine  Biosciences  Interna.onal  Livestock  Research  Ins.tute  Seminar  at  CAVS,  Kabete  Campus,  5th  June  2013    
  • 45. Importance  of  animal  health  research  in  the  developing  world  Ø Livestock offer a powerful pathway out of poverty for ~750million poor farmers in South Asia and Africa by providingnutritional and economic security.Ø Infectious livestock diseases feature prominently among theconstraints faced by livestock agriculture.•  Endemic diseases•  Epidemic/pandemic diseases•  Trans-boundary diseases•  Emerging and re-emerging diseases•  Zoonotic diseases and food safetyØ For many reasons diseases are neglected problems in affectedcountries, a situation exacerbated by a general lack ofinvestment, vaccine R & D and manufacturing capacity.
  • 46. List  of  current  ILRI  high  priority  diseases  targeted  for  control  Ø African swine fever (ASF) – swine•  African disease threatens the global $150 billion/year pig industryØ Contagious bovine pleuropneumonia (CBPP) – cattle•  Regional losses to CBPP amount to ~ $60 million/yearØ East Coast fever (ECF) – cattle•  Regional losses exceed $300 million/year; kills ~ 1million cattle/yearØ Peste de petits ruminants (PPR) – small ruminants•  Losses in Kenya alone amount to ~ $13 million/yearØ Rift Valley Fever (RVF) – small ruminants, cattle andhuman•  2006/7 outbreak in Kenya cost ~ $30 million•  309 human cases in Kenya, Somalia and Tanzania; 140 deathsVaccines save lives and livestock and contribute to foodsecurity and poverty alleviation
  • 47. Socio-­‐economic  impact  of  East  Coast  fever    in  sub-­‐Saharan  Africa    Ø ECF present in 11 countries; it could spreadto 8 moreØ ~46 million cattle in region; ~28 million at riskØ ~1million deaths/year; losses > 300 $ millionØ Small-holder farmers who would benefit: ~ 20
  • 48. Theileria  parva    life  cycle    R. appendiculatusschizont-infected cellssporozoitespiroplasmsmerogony
  • 49. An  infec.on  and  treatment  vaccine  A live vaccine for the control of ECF(Muguga cocktail)Problems: Liquid nitrogen cold chain, cost, immunologicaltypes
  • 50. Immune  responses  that  contribute  to  immunity  Anti-sporozoiteAnti-schizont
  • 51. An.-­‐sporozoite  immunity:  p67  can  induce  immunity  to  ECF  p67Np67Mp67C21 225226 571572 6519 709reduction in severe ECF by 50% in lab (25% immunity in field)Average
  • 52. A  classical  CD8+  cytotoxic  T  cell  response  to  the  schizont  stage  of  T.  parva  CTLPCTLPT cell receptor (TCR) on CTL recognizesparasite peptide associated with MHC class I molecules
  • 53. Flowchart  of  CTL  an.gen  discovery  ACTGGTACGTAGGGCATCGATCGACATGATAGAGCATATAGCATGACGATGCGATCGACAGTCGACAGCTGACAGCTGAGGGTGACACCAGCTGCCAGCTGGACCACCATTAGGACAGATGACCACACACAAATAGACGATTAGGACCAGATGAGCCACATTTTAGGAGGACACACACCABioinformaticstoolsPredict ~ 5000gene sequences& list candidatevaccine antigensClone genes ofvaccine interestFilter genes viaimmunologicalassaysT. parva genome sequenceARandom cDNAlibraryBCandidate CTL antigensMap CTL epitopes
  • 54. Mapped  parasite  CTL  an.gens/epitopes  CTL epitope Peptide sequence MHC class I gene BoLA sero-typeTp1214-224 VGYPKVKEEML N*01301 A18 (HD6)Tp227-37 SHEELKKLGML T2b~Tp249-59 KSSHGMGKVGK N*01201 A10 (T2a)Tp296-104 FAQSLVCVL T2c~Tp298-106 QSLVCVLMK N*01201 A10 (T2a)Tp4328-336 TGASIQTTL N*00101 A10 (5.1)Tp587-95 SKADVIAKY T5~Tp7206-214 EFISFPISL T7~Tp8379-387 CGAELNHFL N*00101 A10 (5.1)
  • 55. NetMHCpan  –  an  ar.ficial  neural  network  to  predict  CTL  an.gens/epitopes  Center for Biological Sequence Analysis at the Technical University of DenmarkIncorporates correlated effectsMorten Nielsen
  • 56. Use  of  pep.de-­‐MHC  tetramers  in  ECF  CD8+Perforin+Tp1+ cellsCTRCTRBB007BB007
  • 57. Diversity  of  BoLA  MHC  class  I  genes?  Cattle -multiplexRNA isolation from PBMCs454 pyrosequencingRT-PCRFull length cDNAExon 2- Exon 3• Highthroughput• Rarevariants Nicholas Svitek –post-doc
  • 58. Genotypic  diversity  –  a  hallmark  of  T.  parva,  can  compara.ve  genomics  help?  Muguga, Marikebuni, Uganda ~ 64,000SNPsSNP distribution: ~ 65% exons, ~15% introns, ~ 20% inter-genic81/4076 genes under positive selection (includes Tp2)[Henson et al., BMC Genomics 13: 503,2012]Joana da Silva – hybrid capture NGSSequencing more cattle and buffalo derivedparasites
  • 59. An.-­‐schizont  immunity:  trial  of  Tp  an.gens  Graham et al., PNAS, 2006: 30% vaccinated cattle
  • 60. We  need  beXer  methods  to  generate  immune  responses  in  caXle  Anti-sporozoiteAnti-schizontExploring vaccinationsystemsNew adjuvantsViral vectored systemsOld & new antigens
  • 61. A  porholio  of  innova.on  and  vaccine  related  technology  plahorms  Yeast&with&M.#myc&LC&genome&(Delete&puta5ve&&virulence&factors)&Less&virulent&M.#myc&LC&ACTGGTACGTAGGGCATCGATCGACATGATAGAGCATATAGCATGACGATGCGATCGACAGTCGACAGCTGACAGCTGAGGGTGACACCAGCTGCCAGCTGGACCACCATTAGGACAGATGACCACACACAAATAGACGATTAGGACCAGATGAGCCACATTTTAGGAGGACACACACCABioinformaticstoolsPredict genesequences andlist candidatevaccine antigensTest experimental vaccineClone genes ofvaccine interest(100’s of genes)Filter genes viaimmunologicalassaysPathogen genome mining(1000’s of genes)Molecular immunologytools to assess immuneresponses in cattle(10’s genes)BASIC&RESEARCH&Increasing&our&knowledge&base&&“Knowledge*lays*the*founda2on*for*science”***!!  Map&immune&responses&to&infec>on&!  Dissect&pathogen&biology&&&diversity&!  Study&hostDvectorDpathogen&interac>ons&!  Characterize&pathogen&virulence&factors&!  Inves>gate&the&epidemiology&of&disease&!  Iden>fy&vaccine&and&diagnos>c&molecules&&&&&&APPLIED&RESEARCH&Developing&new&vaccines&&&diagnos>cs&&“Vaccines*are*cost8effec2ve*an28disease*inven2ons”*&!  Assess&candidate&subunit&vaccines&!  Assess&aHenuated&pathogen&vaccines&!  Assess&different&vaccina>on&systems&!  Engineer&thermoDstable&vaccine&formula>ons&!  Develop&smarter&easier&to&use&diagnos>c&tests&!  Facilitate&transla>on&of&outputs&to&products&BASIC&RESEARCH&Increasing&our&knowledge&base&&“Knowledge*lays*the*founda2on*for*science”***!!  Map&immune&responses&to&infec>on&!  Dissect&pathogen&biology&&&diversity&!  Study&hostDvectorDpathogen&interac>ons&!  Characterize&pathogen&virulence&factors&!  Inves>gate&the&epidemiology&of&disease&!  Iden>fy&vaccine&and&diagnos>c&molecules&&&&&&APPLIED&RESEARCH&Developing&new&vaccines&&&diagnos>cs&&“Vaccines*are*cost8effec2ve*an28disease*inven2ons”*&!  Assess&candidate&subunit&vaccines&!  Assess&aHenuated&pathogen&vaccines&!  Assess&different&vaccina>on&systems&!  Engineer&thermoDstable&vaccine&formula>ons&!  Develop&smarter&easier&to&use&diagnos>c&tests&!  Facilitate&transla>on&of&outputs&to&products&
  • 62. Acknowledgments  Large number of past and current scientists at ILRI(Evans Taracha et al) and collaborators (LICR, Oxford Uni, Merial)Immuno-informatics approach:John Barlow – University of VermontBill Golde – USDA-ARS (Plum Island)Soren Buus – University of CopenhagenMorten Nielsen - Technical University of DenmarkILRI CRP fundsTIGR and Craig VenterDFIDNSF-BMFG (BREAD program)USAID – Feed the Future via USDA-ARS
  • 63. The presentation has a Creative Commons licence. You are free to re-use or distribute this work, provided credit isgiven to ILRI.ilri.orgBox 30709, Nairobi 00100, KenyaPhone: + 254 20 422 3000Fax: +254 20 422 3001Email: ILRI-Kenya@cgiar.orgBox 5689,Addis Ababa, EthiopiaPhone: +251 11 617 2000Fax: +251 11 617 2001Email: ILRI-Ethiopia@cgiar.orgother officesChina • India • MaliMozambique • Nigeria • TanzaniaThailand • Uganda • VietnamBetter lives through livestockILRI is a member of the CGIAR ConsortiumBeFer  lives  through  livestock  ilri.org
  • 64. The presentation has a Creative Commons licence. You are free to re-use or distribute this work, provided credit isgiven to ILRI.ilri.orgBox 30709, Nairobi 00100, KenyaPhone: + 254 20 422 3000Fax: +254 20 422 3001Email: ILRI-Kenya@cgiar.orgBox 5689,Addis Ababa, EthiopiaPhone: +251 11 617 2000Fax: +251 11 617 2001Email: ILRI-Ethiopia@cgiar.orgother officesChina • India • MaliMozambique • Nigeria • TanzaniaThailand • Uganda • VietnamBetter lives through livestockILRI is a member of the CGIAR ConsortiumBeFer  lives  through  livestock  ilri.org

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