Dr. Butch Baker - PEDv Lab Update

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Veterinary Diagnostic Laboratory Iowa 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

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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_thrive

Transcript of Dr. Butch Baker - PEDv Lab Update

Page 1: Dr. Butch Baker - PEDv Lab Update

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

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Veterinary Diagnostic LaboratoryIowa State University

Faculty, Staff, & Students of ISU VDL

≈22 Faculty, 100 Technical Staff, & 20 Graduate Students/Residents

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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

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Veterinary Diagnostic LaboratoryIowa State University

≈ 75% Swine, 10% Bovine, 10% Poultry, + 5% Everything Else

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Veterinary Diagnostic LaboratoryIowa State University

Where the Pigs Are Located in US

≈ 500,000 pigs/week imported to IA to be grown

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Total Positive PEDv Cases (NAHLN)Seems to very closely resemble the US pig density map reflecting PEDV’s broad dissemination across the US

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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

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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.

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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

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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

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Veterinary Diagnostic LaboratoryIowa State University

Yoon, Madson, Magstad, Labois, et al – experimentally infected with PEDV.

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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

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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

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Veterinary Diagnostic LaboratoryIowa State University

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Positive PEDv Cases per Week by Farm Type(Data Collated by USDA NAHLN)

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**black lines represent the total number of positive cases for that week

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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!

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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.

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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

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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.

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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

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Veterinary Diagnostic LaboratoryIowa State University

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Testing at ISU VDL

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Veterinary Diagnostic LaboratoryIowa State University

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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

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Veterinary Diagnostic LaboratoryIowa State University

Emerging Coronaviruses in US Swine“Diagnostic Lab Update”

Lots New, Lots Going On!

Thank YouQuestions ?