surveillance and disease control approaches for pigs and their application to asf
DESCRIPTION
Presented by Raymond (Bob) Rowland at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011TRANSCRIPT
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Surveillance and disease control approaches for pigs and their
application to ASF
Raymond (Bob) RowlandCollege of Veterinary Medicine,
Kansas State University, Manhattan, KansasJuly 21, 2011, Nairobi, Kenya
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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
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CDRicht
AD:Rowland
Theme 1: Vaccines C: Richt AC:
Garcia-SastreTheme 2: Detection
C: LipkinAC: R. Hesse
Theme 3: Modeling/Epidemiology
C: GrayACs: HabteMariam/Webby/Scoglio
Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)
RVFV: (Richt/Young/Ksiazek/Flick)
FMDV: (Estes/Rodriguez/ Golde)
AIV: (Garcia-Sastre/ Richt)
Vaccine Platform: (Garcia-Sastre/Rowland/Ma/
Rodriguez)
Translational Partners:(LAH/BI-Vetmedica/Merial/ GenVec Inc./Bioprotection
Systems)
RVFV/FMDV/AIV: (Lipkin/Palacios/Wilson)
Novel Pathogens:(Lipkin/Briese)Field Devices:
(Higgins/Culbertson/Anderson/Hesse)
Translational Partners:(Orion Biosciences
Inc./INT/BI-Vetmedica/Akonni Inc,
Synbiotics)
RVFV: (Linthicum/Habtemariam)
FMDV: (Perez/HabteMariam/Scott)
AIV: (Stallknecht/Gray/Webby)
Others (Data collection, GIS, etc.):
(Scoglio/Erickson/Anderson/Schroeder/Damon)
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CDRicht
AD:Rowland
Admin. Core with Admin. Core Dir. (ACD)/Emergency Response
Coordinator and Internal/External Committees
Theme 1: Vaccines C: Richt AC:
Garcia-SastreTheme 2: Detection
C: LipkinAC: R. Hesse
Theme 3: Modeling/Epidemiology
C: GrayACs: HabteMariam/Webby/Scoglio
Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD)
RVFV: (Richt/Young/Ksiazek/Flick)
FMDV: (Estes/Rodriguez/ Golde)
AIV: (Garcia-Sastre/ Richt)
Vaccine Platform: (Garcia-Sastre/Rowland/Ma/
Rodriguez)
Translational Partners:(LAH/BI-Vetmedica/Merial/ GenVec Inc./Bioprotection
Systems)
RVFV/FMDV/AIV: (Lipkin/Palacios/Wilson)
Novel Pathogens:(Lipkin/Briese)Field Devices:
(Higgins/Culbertson/Anderson/Hesse)
Translational Partners:(Orion Biosciences
Inc./INT/BI-Vetmedica/Akonni Inc,
Synbiotics)
International Collaborations(Africa, Asia, Europe)
RVFV: (Linthicum/Habtemariam)
FMDV: (Perez/HabteMariam/Scott)
AIV: (Stallknecht/Gray/Webby)
Others (Data collection, GIS, etc.):
(Scoglio/Erickson/Anderson/Schroeder/Damon)
National Collaborations with:Agriculture Industry
Federal, State, and Local AgenciesGovernmental Groups
Non-governmental groups
Overlay: Outreach/ Training/EducationC: Roth; AC: Montelone
Outreach/Training/Education: (Roth/Stewart/ Montelone)
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Elimination vs. Eradication Eradication• 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.
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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
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PRRS“Reproductive Failure of Unknown Etiology”,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
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Blue ear disease-Kansas 2006
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PRRS is a swine disease cofactorReston ebolavirus (REBOV)
Philippines 2008
Co-Infection of pigs with REBOV and PHFD PRRSV
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PRRSV as a disease cofactor
Days after weaning Mortality in groups of 200 experimentally challenged pigs
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MGP5
N2b
GP2GP3
GP4
heterotrimer
viralenvelope
Glycosylation sitesnucleocapsid homopolymer
heterodimer
5a
Complex surface topology & compositionSites for neutralization remain largely unknown
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Dockland-Vet Res (2010)154: 86
Minor Proteins• GP2• 2b (E)•GP3•GP4•5a
N
M
GP5
Complex surface topology & composition
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Genetic diversity (poor heterologous protection)
Pep
tide
seq
uenc
e va
riabi
lity
Mutations in a single isolate over time
nsp2
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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
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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)
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PRRSV Ecology/EpidemiologyNeumann-2003
• 60% herds infected• $600 million/yr
FAD Threats•Feral pigs (8 million)•Backyard farms
Endemic infection Acute outbreaks
Vaccines as biosecurity tools• Block introduction (antibody)• Break endemicity (T cells and innate responses)•Prevent or lower shedding
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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
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Large Populations
Loss of immunity- appearance of an escape mutant-introduction of a new virus
Continuous Infection
Natural Termination/Elimination/Extinction
Susceptible Infectious Resistant
Susceptible Infectious ResistantSIRS
SIRSmall Populations
Population size matters
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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 production
How much does $20 million buy in research?
• Developing infectious disease models that are predictive• Implementing regional approaches to PRRSV elimination
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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!)
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Sow Herd Filtration StudyScott Dee, University of Minnesota
Pipetsone, MN
Attic installation of filter boxes
Advances in biosecurity
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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
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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
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Minnesota(Bob Morrison, Montse Torremorell)
164,000 pigs-83 sites
InfectedUnknownFreeNurserySowsFinish
2004
2006
2010
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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
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pA2pA3pA4pA5pAM
2a
2b3
4 56
7
5’ UTR
pA1a1b
pAN
pAGFP GFP
PRRSV as dual vaccine vector
N
M
GP5
GFP
Nsp2-fusion protein
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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
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Analysis of virus and antibodies in Oral Fluids Jeff Zimmerman, Iowa State
Non-invasive and easy to collectCollect dailyPopulation sampleModification of existing tests (PCR and Ab)Sensitivity
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Circulation of three infectious agentsPRRSV, SIV, PCV2
Oral fluid testing for routine surveillance of infectious diseases in swine populations
Jeff Zimmerman, IA State
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Measure SurfaceTag Florescence Green laser
Red laserMeasure Internal Dye Florescence
AntigenHost antibody (analyte)
Luminex- Microsphere Immunoassay
Each sphere is coated with a different antigen“multiplexing to assess quantity and quality of immunity”
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Microimmunoassay (MIA) Luminex• Substitute for standard ELISA• Can detect multiple analytes (antigens)
including native and denatured proteins, peptides
• Interrogate host antibody responseDIVA
Targets of neutralizing antibodyImmunopathogenic responses
• Sensitivity/Specificity• Multiple variations
• 2010 MAGPIX instrument (magnetic beads)LED detectionLower cost-instrument and sample prep96 well formatSimple sample prep
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Serum IgG and IgM responses (Luminex)PRRSV N protein
n=16 pigs
0 5 10 15 20 250
1
2
3
4
5
6
7
2000
4000
6000
8000
10000
12000
0
MFI LogPRRSV
Day after infection
RNAIgG
IgM
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Oral fluid PRRSV IgG and IgM responses(Luminex, PRRSV protein)
0
2000
4000
6000
8000
10000
12000
14000
1 3 5 7 9 11 13 15 17 19 21 23 25
IgM- anti-PRRSV N mean for 12 pensIgG- anti-PRRSV N mean for 12 pens
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Pig movementPoint-of-care tests
Transportwithin a region
Transportout of a region
TransportInto a region
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Host geneticsPRRS 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 markersthat predict outcomes following infection
3. Conduct “ultra-deep” phenotyping to identify gene pathways and novel biomarkers related to virus replication and disease
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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
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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)
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“Deep” phenotyping
Weight gain over time Log PRRSV RNA over time
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Viral Load(AUG)
Quantified as area under the curve from day 0 to 21
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Subpopulations with unique phenotypic properties
PRRS tolerant
J.P. SteibelMich. St. U.
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GWAS-Illumina Porcine 60k Beadchip
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Genomic Association Model
y = Xb + ziα ii=1
k
∑ δ i +ε
Where y = phenotypic observations for VL or WGX = incidence matrix relating fixed effects to phenotypesb = vector of fixed effects of experiment(pen) and
experiment*parityzi = vector of genotypes at SNP i , coded 0/1/2αi = substitution effect of SNP iδi = 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
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Bayes B analysispi = 0.99
Prop
ortio
n of
Gen
etic
Var
ianc
e
Prop
ortio
n of
Gen
etic
Var
ianc
e
5-SNP window ordered by chromosome5-SNP window ordered by chromosome
Viral Load (AUG) Weight Gain (ADWG)
SSC 4SSC 4
SSC X
SSC 17
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Jack Dekkers and Nick Boddicker
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Integrated approach (no single solution)
• Good vaccines• Good diagnostics• Good understanding of ecology epidemiology• Good surveillance approaches• Good understanding of social psychology • Good pig
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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