dr. butch baker - pedv lab update
DESCRIPTION
PEDv Lab Update - Butch Baker, DVM MS, Interim Director of the Iowa Pork Industry Center, ISU Extension and Outreach, Iowa State University, from the 2014 NIAA Annual Conference titled 'The Precautionary Principle: How Agriculture Will Thrive', March 31 - April 2, 2014, Omaha, NE, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2014_niaa_how_animal_agriculture_will_thriveTRANSCRIPT
Veterinary Diagnostic LaboratoryIowa State University
Emerging Coronaviruses in US Swine“Diagnostic Lab Update”
RB Baker, R Main, & K Mueller
Department of Veterinary Diagnostic and Production Animal Medicine,Veterinary Diagnostic Lab, Iowa State University, Ames, IA 50011
Veterinary Diagnostic LaboratoryIowa State University
Faculty, Staff, & Students of ISU VDL
≈22 Faculty, 100 Technical Staff, & 20 Graduate Students/Residents
Veterinary Diagnostic LaboratoryIowa State University
Serving Food Animal Agriculture Comprehensive Diagnostic Service, Teaching, and Discovery
(Iowa’s only Full-Service & Fully Accredited Veterinary Diagnostic Laboratory)
Processing 55,000+ cases/year from livestock producersApplying world-class technology to real-world problems
Veterinary Diagnostic LaboratoryIowa State University
≈ 75% Swine, 10% Bovine, 10% Poultry, + 5% Everything Else
Veterinary Diagnostic LaboratoryIowa State University
Where the Pigs Are Located in US
≈ 500,000 pigs/week imported to IA to be grown
Total Positive PEDv Cases (NAHLN)Seems to very closely resemble the US pig density map reflecting PEDV’s broad dissemination across the US
Veterinary Diagnostic LaboratoryIowa State University
Porcine Coronaviruses in US Swine
TGE: Transmissible Gastroenteritis
PEDV: Porcine Endemic Diarrhea Virus
PDCoV: Porcine Deltacoronavirus
* Field reports suggest it to be a pathogen
* Koch’s postulates yet to be fulfilled-----------------------------------------------------------------------------------------------------------------------------
PRCV = Porcine Respiratory Coronavirus
* Seems to be more of a friend than a foe
Porcine enteric disease picture in the US has rapidly become more complex
Veterinary Diagnostic LaboratoryIowa State University
Porcine Coronavirus Diagnostic Specimens(TGE, PEDV, & most recently PDCoV)
• Intestine (Fresh & Fixed)
• Feces
• Fecal Swabs
• Oral Fluids
• Serum (Antibody)
Antemortem diagnostic tools are increasingly used (especially Oral fluids & Molecular Dx) to monitor the heath status of US swine in real-time.
Veterinary Diagnostic LaboratoryIowa State University
Porcine Coronavirus Serology(Antibody Testing)
• PEDV – IFA (labor intensive, subjective, but it has been fairly robust)
• PEDV-Elisa (only routine @ UMN that I’m aware)
– Duration of detectable antibody response needs to be understood with PEDV
– Others In Development (ISU, SDSU, et al.)
– Commercially Available PEDV Elisa’s → Very Poor
• TGE-PRCV Differential Elisa
• PDCoV = No Assays Available
Veterinary Diagnostic LaboratoryIowa State University
PEDV IFAPrimary PEDV Antibody Detection Assay Used Today @ ISU VDL
In-House Experimental PEDV Elisa Coming Soon
• Indirect Fluorescence Antibody Test (IFA)– Serology / Antibody Based Assay
– Screening assay for antibody at 1:40 dilution (positive/negative)
– 4-fold dilution (40, 80, 160, 320)
– Limitations• Limited correlation between IFA titer and protective immunity
• IFA antibody duration is short
• Difficult to correlate IFA titer with success of feedback
Dr. Kyoung-Jin Yoon, ISU-VDLDr. John Johnson, ISU-VDL
Veterinary Diagnostic LaboratoryIowa State University
Yoon, Madson, Magstad, Labois, et al – experimentally infected with PEDV.
Veterinary Diagnostic LaboratoryIowa State University
PEDV & TGEV Diagnostics
• Immunohistochemistry (IHC)– Valuable tool, but much less sensitive than PCR
– Detection varies based on timing of infection
• Virus Isolation (VI)– Difficult and time consuming
– Success rate very low
Late PEDv infection (~36 hrs PI): Severe villus atrophy & loss of
absorptive epithelium
(Source: Schwartz, Madson, Magstad et al.)(Source: Dr. Jianqiang Zhang, ISU-VDL)
Late PEDv infection (~36 hrs PI):Few infected cells remain (brown
stain) & absorptive cells destroyed PDCoV = Presently No IHC or Success with VI
Veterinary Diagnostic LaboratoryIowa State University
Porcine Coronavirus Molecular Diagnostic Assays @ ISU VDLRed = New Assays in Past 12-months
• TGE – PCR
• PEDV – PCR
• PEDV Differential – PCR– Original US strain vs. more recently identified variant
• PDCoV – PCR
• Pan-Corona – PCR (Discovery – Yoon et al)
• PEDV Sequencing– S1 Gene (Routine)
– Whole Genome (Research)
• PDCoV Sequencing– (Research to date)
↑ Sensitive, Semi-quantitative, Same-day testing, Used Extensively
Veterinary Diagnostic LaboratoryIowa State University
0
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# P
EDv
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Positive PEDv Cases per Week by Farm Type(Data Collated by USDA NAHLN)
Unknown
Grow-Finish
Sow
**black lines represent the total number of positive cases for that week
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# P
EDv
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Cumulative Positive PEDv Cases by Farm Type
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Grow-Finish
Sow
Veterinary Diagnostic LaboratoryIowa State University
PEDV: DiagnosticsPolymerase chain reaction (PCR)
• Target: Nucleoprotein (ISU) or Matrix– Targets the conserved portion of the genome
• PCR tests are very sensitive and specific– Detect small amounts of target (virus) in the sample
– Does not require much fecal content for detection
• Same day testing
• Sample types = Intestine, Feces, Fecal Swabs, Oral Fluids
• Tests require interpretation
– Based on clinical presentation, sample type, location, etc.
– Is the test outcome what you expected?
3’ORF1a
ORF1bS ORF3 M
N
frameshift
E
L AAA
Dr. Jianqiang Zhang, ISU-VDL
Cycle threshold values: Range: 8-12 in acutely
affected, neonatal pigletsLots of PEDV!
Veterinary Diagnostic LaboratoryIowa State University
PEDV: Diagnostics
PEDV Sequencing
• Spike protein sequence
– Variable portion of genome
– Determine genetic relatedness of PED viruses
– Evaluate genetic evolution
• Sequencing at ISU-VDL
– S1 gene: 2.2 kb• Highly variable portion of spike gene being used for routine sequencing for epidemiological purposes.
Dr. Jianqiang Zhang, ISU-VDL
Two Distinctly Different Groups of PEDVs Circulating in US
• January 2014 – ISU VDL S1 Sequencing
– 15 PEDV positive cases
– 10 cases shared 99.1-100% nucleotide identities to PEDV strains identified in US swine since April 2013
– 5 cases shared 99.6-100% nucleotide identities to each other but only 93.4-94.6% nucleotide identities to the originally identified US strain
Veterinary Diagnostic LaboratoryIowa State University
PEDV Variant: Epidemiology
• January 2014 – ISU VDL retrospective testing – 183 random PEDV PCR positive fecal samples from ISU VDL cases
submitted May to September 2013
– 6 of 183 cases were positive “PEDV variant”
– Five of the 6 PEDV variant positive cases were submitted in May 2013 and the other in early June
• In retrospect, both PEDV strains have been present and circulating in US since April/May 2013.– Earliest PEDV positive case identified in the US was submitted to the
ISU VDL on April 15, 2013.
Veterinary Diagnostic LaboratoryIowa State University
PDCoV• PDCoV not PEDV or TGEV or PRCV
– Delta vs. Alpha Coronaviruses
– Doesn’t appear to be any cross-protection
• Vomiting and diarrhea (Field/Diagnostic Reports)
– Similar clinical signs and lesions to PEDV/TGE
– Reportedly more transient &/or less severe
– Koch’s postulates yet to be fulfilled
• ISU-VDL Available Assays
– PDCV PCR is currently offered
– PDCV sequencing becoming available
• Limited retrospective testing on PDCoV completed to date
Alphacoronavirus: PEDV
Deltacoronavirus: PDCV
Veterinary Diagnostic LaboratoryIowa State University
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# P
DC
oV
Po
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Date by Week
Positive PDCoV Cases @ ISU VDL per Week by Farm Type
Unknown
Grow-Finish
Sow
2-17-14Began PDCV PCR
Testing at ISU VDL
Veterinary Diagnostic LaboratoryIowa State University
0
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# P
DC
oV
Po
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Date by Week
Cumulative Positive PDCoV Cases by Farm Type
Unknown
Grow-Finish
Sow
2-17-14Began PDCV PCR
Testing at ISU VDL
Veterinary Diagnostic LaboratoryIowa State University
ISU VDL PDCoV Positive Swine Cases
Week Received @ VDL
Total # PCR Positive Cases
Farm State
IA IL IN MI MN MO NC NE OH OK PA ? 12/22/13 1 1
12/29/13
1/5/14
1/12/14
1/19/14 2 1 1
1/26/14
2/2/14 2 1 1
2/9/14 6 4 1 1
2/16/14 10 6 1 1 1 1
2/23/14 12 2 1 5 1 1 1 1
3/2/14 14 1 5 1 3 1 1 1
3/9/14 17 8 1 1 1 3 1 1 1
3/16/14 8 3 1 3 1
Total 71 26 10 7 1 3 9 4 3 3 1 3 1
**totals include biological samples only--no environmental or feed samples are included in the this data
Veterinary Diagnostic LaboratoryIowa State University
Emerging Coronaviruses in US Swine“Diagnostic Lab Update”
Lots New, Lots Going On!
Thank YouQuestions ?