Detection of Virulent

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Detection of Virulent
Yersinia enterocolitica in
Processed Pork Using
Polymerase Chain Reaction

• Jafa Armagost

Dr. William Mackay
Edinboro University of Pennsylvania
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Yersinia enterocolitica

• Enterobacteriaceae
• Gram negative Coccoid bacillus
• Facultative anaerobe
• Psychrotroph
• Motile at 27 C

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Yersinia

• Yersinia pseudotuberculosis
• Yersinia pestis

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Yersiniosis

• Ingestion of Y. enterocolitica from contaminated
  food, usually pork products.
• Y. enterocolitica is also found in Ovine sp,
  Mictotus sp, Equine sp and Bos texanus.

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Transmission of Yersinia enterocolitica in Pigs
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Transmission of Yersinia in Pigs (Slaughterhouses)
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Symptoms

• Fever Abdominal pain Diarrhea

Pseudoappendicitis
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Clinical Significance

• Food-borne pathogen
• 96,000 cases (annually)
• Refrigerated temperatures

Sommers 1999
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What Are Public Health Agencies Doing?

• CDC (Centers for Disease Control)
• Food borne active surveillance network
• Investigates outbreaks
• Inspections of imported foods and milk
• An education campaign

• (no pathogen left behind)

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What Can You Do?

• 71º C (160 º F)
• Pasteurization
• Hygiene
• Cross-contamination
• Sanitation

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Yersinia Virulence

• 5 virulent serotypes cause human illnesses (O3,
  O5, O8, O9, O27)
• gt 200 known avirulent serotypes

virF gene (causes virulence)
70 kb Plasmid
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Procedures

• Colony Morphology
• Crystal Violet Binding Assay
• Plasmid Purification
• Polymerase Chain Reaction

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Strains

• GER (serotype 03)
• Contains the virF gene
• EWM5 (serotype 013)
• Negative control

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Colony Morphology

• Virulent

Avirulent
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Crystal Violet
Binds virulent cells Does not bind avirulent
cells
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Crystal Violet Binding Assay

• Crystal violet (100mg/ml)
• Flood surface of plate
• Occasionally rock plate
• Remove dye from plate

USDA/FSIS Microbiology Laboratory Guidebook
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Results Of Crystal Violet Binding Assay

• Virulent

Avirulent
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Plasmid Prep Protocol(Triton-Lysis)

• Cells resuspended (isotonic buffer)
• Lysozyme degrades cell wall
• Non-ionic detergent lyses cell
• Heat denatures carbohydrates
• Phenol chloroform removes proteins
• Alcohol precipitation concentrate nucleic acids
• Treatment with RNase

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Plasmid Prep Results

• ? HinD III /EcoR I
• GER (serotype O3)
• EWM5 (serotype 013)
• ? HinD III /EcoR I

1 2 3 4
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Polymerase Chain Reaction
PCR is used to detect the virF gene. The virF
gene primer pairs
5-TCATGGCAGAACAGCAGTCAG-3
590 base
pairs
5-ACTCATCTTACCATTAAGAAG-3

• To amplify positions 430 to 1020 from the
  virulence fragment.

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Protocol for PCR
Heat Denaturation 94 C 1 min
Primer Annealing 54 C 45 sec
Extension 72 C 2 min
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PCR

• 1) ? HinD III / EcoR I
• 2) 590 bp virF gene fragment
• 3) Negative control
• 4) ? HinD III /EcoR I

1 2 3 4
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Future Procedures

• Take processed pork from local supermarket

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Future Procedures
Enrichment

• 10ml of of Modified Trypticase Soy Broth
• 5 min 25 C
• 12C for 24 hrs under constant shaking
• Irgasan (4µg/ml)
• 12C for 48 hrs

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Future Procedures

• Colony Morphology

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Future Procedures

• Crystal Violet Binding Assay

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Future Procedures

• Plasmid Purification

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Future Procedures

• Polymerase Chain Reaction

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Summary

• Compared morphology between virulent and
  avirulent strains
• Confirmed virulence by
• Crystal Violet Binding Assay
• Plasmid Purification
• Polymerase Chain Reaction

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Grants
Tri-Beta Honor Society Pennsylvania Academy
of Science
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Acknowledgements

• Dr. Christopher Sommers
• (USDA)
• Summer Slater
• (University of Alabama)