Surveillance and disease control approaches for pigs and their       application to ASF          Raymond (Bob) Rowland    ...
USDA Coordinated Agricultural Project                        (PRRS CAP)•   Stakeholder driven (scientists, producers,    v...
Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)                                         CD        ...
Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)                                                   ...
Elimination vs. EradicationEradication•   Based on laws- legislated•   Government supported. Indemnify against losses.•   ...
Arteriviruses (order Nidovirales)Porcine reproductive and respiratory syndrome virus (PRRSV)                (Type 1 and Ty...
PRRS“Reproductive Failure of UnknownEtiology”,Kerry K. Keffaber, 1989, AASP1.    Anorexia during finishing2.    Transient ...
Blue ear disease-Kansas 2006
PRRS is a swine disease cofactor  Reston ebolavirus (REBOV)        Philippines 2008               Co-Infection of pigs wit...
PRRSV as a disease cofactorDays after weaning            Mortality in groups of 200                            experimenta...
Complex surface topology & composition  Sites for neutralization remain largely unknown           heterodimer            h...
Complex surface topology & composition        M                    Minor   N          GP    Proteins              5     • ...
Genetic diversity                               (poor heterologous protection)                                            ...
Subversion of innate immunity by NSP1        (inhibition of the Type 1 IFN response)• Degradation of CREB-binding protein ...
Virus properties that relate                   to control and eradication•   Complex virion composition and surface topolo...
PRRSV Ecology/Epidemiology •               60% herds infected              Neumann-2003             •   $600 million/yr  E...
PRRS vaccines• Modified live virus (MLV) vaccine introduced in 1994-  suitable for infected herds• MLV limitations-virus s...
Population size matters                  Natural Termination/Elimination/Extinction   Small                               ...
Trends (10 year)• Less government involvement in disease control and  eradication (government may not indemnify producers)...
PRRS herd control methodologies•   Syndromics (1990)•   Test and removal (1992)•   Vaccination (1994)•   Depop-repop•   Al...
Sow Herd Filtration StudyScott Dee, University of Minnesota          Pipetsone, MN                 Attic installation of f...
Regional approach to elimination• Herd-level strategies generally fail (virus re-enters from and  unknown source)• For her...
PRRS CAP regional elimination projects•Oral fluid analysis for molecular and     immunological sureveillance•Risk analysis...
Minnesota                     (Bob Morrison, Montse Torremorell)                           164,000 pigs-83 sites2004  Infe...
Technologies to facilitate elimination•Oral fluid analysis for molecular and     immunological surveillance•Risk analysis ...
PRRSV as dual vaccine vector5’ UTR   Nsp2-fusion protein                                           2b                     ...
Advantages of dual vaccine• Targets PRRSV and cofactor infection• Insertion of Ag attenuates or inactivates wild-type viru...
Analysis of virus and antibodies in Oral Fluids                                      Jeff Zimmerman, Iowa State           ...
Circulation of three infectious agents             PRRSV, SIV, PCV2                    Oral fluid testing for routine surv...
Luminex- Microsphere Immunoassay       Measure Surface       Tag Florescence            Green laser                       ...
Microimmunoassay (MIA) Luminex• Substitute for standard ELISA• Can detect multiple analytes (antigens)  including native a...
Serum IgG and IgM responses (Luminex)                 PRRSV N protein                             n=16 pigs               ...
Oral fluid PRRSV IgG and IgM responses        (Luminex, PRRSV protein)   14000   12000   10000    8000    6000    4000    ...
Pig movement    Point-of-care tests                          TransportTransport                 Into a regionout of a regi...
Host genetics   PRRS Host Genetics Consortium (PHGC)    (CoPDs, Bob Rowland and Joan Lunney, ARS)1. Use genotyping and phe...
Nursery Pig Model• 200 pigs (2-3 weeks of age) from different sources in  Canada and the US• 5-10 pigs set aside as refere...
Phenotypic data (deep phenotype)•   Morbidity and mortality•   Viremia, qRT-PCR (ABI) log PRRSV RNA templates/rxn•   Virus...
“Deep” phenotypingWeight gain over time      Log PRRSV RNA over time
Viral Load                         (AUG)Quantified as area under the curve from day 0 to 21
Subpopulations with unique phenotypic properties                                  PRRS tolerant                           ...
GWAS-Illumina Porcine 60k Beadchip
Genomic Association Model                                     k                  y = Xb + ∑ z iα i δ i + ε                ...
Bayes B analysis                                                                      pi = 0.99                           ...
Jack Dekkers and Nick Boddicker
Integrated approach (no single solution)•   Good vaccines•   Good diagnostics•   Good understanding of ecology epidemiolog...
Researchable Issues• Role of PRRSV as a cofactor in ASF pathogenesis and  ecology• Development of tools for the study of A...
Surveillance and disease control approaches for pigs and their application to ASF
Surveillance and disease control approaches for pigs and their application to ASF
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Surveillance and disease control approaches for pigs and their application to ASF

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Presented by Raymond (Bob) Rowland at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011



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Surveillance and disease control approaches for pigs and their application to ASF

  1. 1. Surveillance and disease control approaches for pigs and their application to ASF Raymond (Bob) Rowland College of Veterinary Medicine, Kansas State University, Manhattan, Kansas July 21, 2011, Nairobi, Kenya
  2. 2. USDA Coordinated Agricultural Project (PRRS CAP)• Stakeholder driven (scientists, producers, veterinarians)• “Out-of-the-box” approaches to infectious disease problem solving• Conduct activities that are unique and not routinely supported by existing mechanisms• Activities converge at the control and elimination of virus in the field• Leverage
  3. 3. Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) CD Richt AD: Rowland Theme 1: Vaccines C: Richt AC: Theme 2: Detection Garcia-Sastre C: Lipkin Theme 3: Modeling/ AC: R. Hesse Epidemiology RVFV: (Richt/Young/Ksiazek/Flick) RVFV/FMDV/AIV: C: Gray (Lipkin/Palacios/Wilson) ACs: HabteMariam/Webby/Scoglio FMDV: (Estes/Rodriguez/ Golde) Novel Pathogens: (Lipkin/Briese) RVFV: AIV: (Linthicum/Habtemariam) Field Devices: (Garcia-Sastre/ Richt) FMDV: (Higgins/Culbertson/ (Perez/HabteMariam/Scott) Vaccine Platform: Anderson/Hesse) AIV: (Garcia-Sastre/Rowland/Ma/ Translational Partners: (Stallknecht/Gray/Webby) Rodriguez) (Orion Biosciences Others (Data collection, GIS, Inc./INT/ etc.): Translational Partners: BI-Vetmedica/Akonni Inc, (Scoglio/Erickson/Anderson/ (LAH/BI-Vetmedica/Merial/ Synbiotics) Schroeder/Damon) GenVec Inc./Bioprotection Systems)
  4. 4. Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) Admin. Core with Admin. Core Dir. CD (ACD)/Emergency Response Richt Coordinator and Internal/External International Collaborations Committees AD: (Africa, Asia, Europe) National Collaborations with: Rowland Agriculture Industry Federal, State, and Local Agencies Theme 1: Vaccines Governmental Groups C: Richt AC: Theme 2: Detection Non-governmental groups Garcia-Sastre C: Lipkin Theme 3: Modeling/ AC: R. Hesse Epidemiology RVFV: (Richt/Young/Ksiazek/Flick) RVFV/FMDV/AIV: C: Gray (Lipkin/Palacios/Wilson) ACs: HabteMariam/Webby/Scoglio FMDV: (Estes/Rodriguez/ Golde) Novel Pathogens: (Lipkin/Briese) RVFV: AIV: (Linthicum/Habtemariam) Field Devices: (Garcia-Sastre/ Richt) FMDV: (Higgins/Culbertson/ (Perez/HabteMariam/Scott) Vaccine Platform: Anderson/Hesse) AIV: (Garcia-Sastre/Rowland/Ma/ Translational Partners: (Stallknecht/Gray/Webby) Rodriguez) (Orion Biosciences Others (Data collection, GIS, Inc./INT/ etc.): Translational Partners: BI-Vetmedica/Akonni Inc, (Scoglio/Erickson/Anderson/ (LAH/BI-Vetmedica/Merial/ Synbiotics) Schroeder/Damon) GenVec Inc./Bioprotection Systems) Overlay: Outreach/ Training/Education Outreach/Training/Education: C: Roth; AC: Montelone (Roth/Stewart/ Montelone)
  5. 5. Elimination vs. EradicationEradication• Based on laws- legislated• Government supported. Indemnify against losses.• Draconian. Non-palatable to producers.Elimination• Stakeholder driven- Incentive to maintain profitability.• Varied in scope (herd, region).• Incorporates education and organization (sociology).• Must be “voluntary”, can be chaotic and divisive.
  6. 6. Arteriviruses (order Nidovirales)Porcine reproductive and respiratory syndrome virus (PRRSV) (Type 1 and Type 2 genotypes) Lactate dehydrogenase elevating virus (LDV) Equine arteritis virus (EAV) Simian hemorrhagic fever virus (SHFV)• Enveloped• Positive single-stranded RNA genome (10-15kb)• During replication, produce a nested set of subgenomic mRNAs with a common leader and poly A tail• Macrophage-tropic• Persistent infection and severe and fatal disease
  7. 7. PRRS“Reproductive Failure of UnknownEtiology”,Kerry K. Keffaber, 1989, AASP1. Anorexia during finishing2. Transient increase in respiration rates3. Temperature increase (102-106)4. Mid- to late-term abortions5. Delay in return to heat and infertility6. Pre-weaning mortality7. Respiratory distress
  8. 8. Blue ear disease-Kansas 2006
  9. 9. PRRS is a swine disease cofactor Reston ebolavirus (REBOV) Philippines 2008 Co-Infection of pigs with REBOV and PHFD PRRSV
  10. 10. PRRSV as a disease cofactorDays after weaning Mortality in groups of 200 experimentally challenged pigs
  11. 11. Complex surface topology & composition Sites for neutralization remain largely unknown heterodimer heterotrimer GP5 GP3 M GP2 GP4 viralenvelope 2b 5a N Glycosylation sites nucleocapsid homopolymer
  12. 12. Complex surface topology & composition M Minor N GP Proteins 5 • GP2 • 2b (E) •GP3 •GP4 •5aDockland-Vet Res (2010)154: 86
  13. 13. Genetic diversity (poor heterologous protection) Mutations in a single isolate over timePeptide sequence variability nsp2
  14. 14. Subversion of innate immunity by NSP1 (inhibition of the Type 1 IFN response)• Degradation of CREB-binding protein (CBP) in the nucleus, making it unable to form a complex with p300 and interferon regulatory factor 3 (IRF3; Kim et al., 2010)• Inhibition of dsRNA-induced IRF3 and IFN promoter activities (Kim et al., 2010)• Interaction with PIAS1 (Yoo unpublished)• Inhibition NF-kB function (Song et al., 2010)• Interference of RIG-I signaling (Yoo et al., 2010)• Inhibit nuclear translocation of STAT1 (Chen et al., 2010) nsp1α nsp1β
  15. 15. Virus properties that relate to control and eradication• Complex virion composition and surface topology• Subversion of innate and adaptive immunity• Capacity to generate a large degree of genetic diversity in structural and nonstructural proteins• Macrophage tropism (lymphotropism during subclinical infection)• Capacity to exist as a subclinical infection and cause severe disease• Delayed and reduced Ab neutralizing activity (no heterologous protection)• Co-factor with other infections (ASFV and CSFV)
  16. 16. PRRSV Ecology/Epidemiology • 60% herds infected Neumann-2003 • $600 million/yr Endemic infection Acute outbreaks FAD Threats •Feral pigs (8 million) •Backyard farms Vaccines as biosecurity tools • Block introduction (antibody) • Break endemicity (T cells and innate responses) •Prevent or lower shedding
  17. 17. PRRS vaccines• Modified live virus (MLV) vaccine introduced in 1994- suitable for infected herds• MLV limitations-virus shedding, persistent infection, incomplete immune protection, inability to differentiate infected from vaccinated animals (DIVA), potential for reversion to virulence• Killed vaccines are not effective• Acclimation with wild-type virus as an alternative to vaccination
  18. 18. Population size matters Natural Termination/Elimination/Extinction Small SIRPopulations Susceptible Infectious Resistant Continuous Infection SIRS Susceptible Infectious Resistant LargePopulations Loss of immunity- appearance of an escape mutant-introduction of a new virus
  19. 19. Trends (10 year)• Less government involvement in disease control and eradication (government may not indemnify producers)$20 million spent to support market prices during the pandemic SIV outbreak covered 84 minutes of productionHow much does $20 million buy in research?• Developing infectious disease models that are predictive• Implementing regional approaches to PRRSV elimination
  20. 20. PRRS herd control methodologies• Syndromics (1990)• Test and removal (1992)• Vaccination (1994)• Depop-repop• All in - all out• Acclimation with known viruses• Herd closure (200 days-virus extinction)• Barn filtration (high density areas – expensive!)
  21. 21. Sow Herd Filtration StudyScott Dee, University of Minnesota Pipetsone, MN Attic installation of filter boxes Advances in biosecurity
  22. 22. Regional approach to elimination• Herd-level strategies generally fail (virus re-enters from and unknown source)• For herd-level control and elimination to be sustainable, the effort must be regional• Sufficient tools, technologies, resources and leadership (political will) to initiate and conduct regional scale projects• Regional efforts will identify gaps in knowledge that future research can fill
  23. 23. PRRS CAP regional elimination projects•Oral fluid analysis for molecular and immunological sureveillance•Risk analysis tools (PADRAP)•Risk-based testing and surveillance•Point of care testing (serology and PCR)•Sociology•Economic cost-benefit analysis•Vaccines•Host genetics
  24. 24. Minnesota (Bob Morrison, Montse Torremorell) 164,000 pigs-83 sites2004 Infected Unknown 2006 Free Nursery Sows Finish 2010
  25. 25. Technologies to facilitate elimination•Oral fluid analysis for molecular and immunological surveillance•Risk analysis tools (PADRAP)•Risk-based testing and surveillance•Point of care testing (serology and PCR)•Sociology•Economic cost-benefit analysis•Vaccines•Host genetics
  26. 26. PRRSV as dual vaccine vector5’ UTR Nsp2-fusion protein 2b 1b 3 6 1a 2a 4 5 7 pA GFP GFP pA 2 pA M 3 GFP pA 4 pA N GP5 pA 5 M pA N pA
  27. 27. Advantages of dual vaccine• Targets PRRSV and cofactor infection• Insertion of Ag attenuates or inactivates wild-type virus• Compliance and DIVA markers• In the case of nsp2, antigen expressed as a fusion protein-incorporated in macromolecular complexes (replication complexes) or VLP• PRRSV is resistant to MDA or existing antibody
  28. 28. Analysis of virus and antibodies in Oral Fluids Jeff Zimmerman, Iowa State Non-invasive and easy to collect Collect daily Population sample Modification of existing tests (PCR and Ab) Sensitivity
  29. 29. Circulation of three infectious agents PRRSV, SIV, PCV2 Oral fluid testing for routine surveillance of infectious diseases in swine populations Jeff Zimmerman, IA State
  30. 30. Luminex- Microsphere Immunoassay Measure Surface Tag Florescence Green laser Host antibody (analyte)Measure Internal AntigenDye Florescence Red laser Each sphere is coated with a different antigen “multiplexing to assess quantity and quality of immunity”
  31. 31. Microimmunoassay (MIA) Luminex• Substitute for standard ELISA• Can detect multiple analytes (antigens) including native and denatured proteins, peptides• Interrogate host antibody response DIVA Targets of neutralizing antibody Immunopathogenic responses• Sensitivity/Specificity• Multiple variations• 2010 MAGPIX instrument (magnetic beads) LED detection Lower cost-instrument and sample prep 96 well format Simple sample prep
  32. 32. Serum IgG and IgM responses (Luminex) PRRSV N protein n=16 pigs IgG RNA 7 12000 6 10000 5MFI 8000 Log 4 6000 PRRSV 3 4000 2 2000 IgM 1 0 0 0 5 10 15 20 25 Day after infection
  33. 33. Oral fluid PRRSV IgG and IgM responses (Luminex, PRRSV protein) 14000 12000 10000 8000 6000 4000 2000 0 1 3 5 7 9 11 13 15 17 19 21 23 25 IgM- anti-PRRSV N mean for 12 pens IgG- anti-PRRSV N mean for 12 pens
  34. 34. Pig movement Point-of-care tests TransportTransport Into a regionout of a region Transport within a region
  35. 35. Host genetics PRRS Host Genetics Consortium (PHGC) (CoPDs, Bob Rowland and Joan Lunney, ARS)1. Use genotyping and phenotyping tools to determine if there are host genes that control host response to infection (resistance vs. susceptibility)2. Identify relative importance of different protein markers that predict outcomes following infection3. Conduct “ultra-deep” phenotyping to identify gene pathways and novel biomarkers related to virus replication and disease
  36. 36. Nursery Pig Model• 200 pigs (2-3 weeks of age) from different sources in Canada and the US• 5-10 pigs set aside as reference pigs• 15-16 pigs per pen• A week after arrival pigs challenged with isolate NVSL 97-7985• Collect blood at 0, 3, 7, 10, 14, 21, 28, 35, 42 days dpi (serum, RNA Tempus tubes)• Weigh weekly• Collect tonsils and ears at Day 42
  37. 37. Phenotypic data (deep phenotype)• Morbidity and mortality• Viremia, qRT-PCR (ABI) log PRRSV RNA templates/rxn• Virus Load, area under curve for the 21 days• Weight (weekly)• Total antibody and virus neutralizing activity (42 dpi)• Circulating cytokine levels• Transcriptome analysis of whole blood and tonsil (ultradeep phenotyping)
  38. 38. “Deep” phenotypingWeight gain over time Log PRRSV RNA over time
  39. 39. Viral Load (AUG)Quantified as area under the curve from day 0 to 21
  40. 40. Subpopulations with unique phenotypic properties PRRS tolerant J.P. Steibel Mich. St. U.
  41. 41. GWAS-Illumina Porcine 60k Beadchip
  42. 42. Genomic Association Model k y = Xb + ∑ z iα i δ i + ε i =1Where y = phenotypic observations for VL or WG X = incidence matrix relating fixed effects to phenotypes b = vector of fixed effects of experiment(pen) and experiment*parity zi = vector of genotypes at SNP i , coded 0/1/2 αi = substitution effect of SNP i δi = indicator for whether SNP i was included (δi=1) or excluded (δi=0) in the model for a given iteration of the Monte Carlo Markov Chain The prior probability of δi= 0 was set equal to pi = 0.99
  43. 43. Bayes B analysis pi = 0.99 Viral Load (AUG) Weight Gain (ADWG) SSC 4 SSC XProportion of Genetic Variance Proportion of Genetic Variance SSC 4 SSC 17 5-SNP window ordered by chromosome 5-SNP window ordered by chromosome
  44. 44. Jack Dekkers and Nick Boddicker
  45. 45. Integrated approach (no single solution)• Good vaccines• Good diagnostics• Good understanding of ecology epidemiology• Good surveillance approaches• Good understanding of social psychology• Good pig
  46. 46. Researchable Issues• Role of PRRSV as a cofactor in ASF pathogenesis and ecology• Development of tools for the study of ASF in BSL-2 (pseudotyped virus system)• Multiplex serological assays (Luminex) for investigating ASFV immune response and cofactors• Novel surveillance approaches (oral fluids)• Risk assessment and biosecurity• Role of host genes in ASF disease susceptibility, resistance and persistence• Transcriptome analysis to investigate virus-host interactions during acute and subclinical infection• Education

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