Title: Foodborne Disease Role of Pulsedfield Gel Electrophoresis and PulseNet
1Foodborne DiseaseRole of Pulsed-field Gel
Electrophoresis and PulseNet
- Dave Warshauer, Ph.D D(ABMM)
- Deputy Director, Communicable Diseases
- Wisconsin State Laboratory of Hygiene
2The Public Health Burden of Foodborne Disease in
the US
- Mead et al 1999 - Emerg Infect Dis Vol. 5
- 76 million cases per year
- 325,000 hospitalizations
- 5000 deaths
- Cost 8-10 billion/year
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5Other Factors Influencing Emergence of Foodborne
Disease
- Inexperienced food workers
- Average turnover rate about 350 / year
- International transport of contaminated foods
- Introduction of foodborne pathogens to new
geographic areas
From M. T. Osterholm, 2002 ICAAC Presentation
6Trends in Foodborne DiseasePercent of Food
Spending by Site
Food Review 1623-7, 1993
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8Emerging Foodborne Pathogens
Parasites Cryptosporidium parvum Cyclospora
cayetanensis Viruses Noroviruses Prions Spongifo
rm encephalopathies
- Bacteria
- Shiga toxin-producing E. coli (STEC)
- Salmonella Typhimurium DT104
- Salmonella Enteritidis
- Listeria monocytogenes
- Campylobacter jejuni
- Campylobacter fetus
- Yersinia enterocolitica
- Vibrio cholerae O139
- Vibrio vulnificus
- Vibrio parahemolyticus
-
9WSLH WEPS Program
10WEPS Program
- Wisconsin Enteric Pathogen Surveillance Program
- Request submission of all bacterial enteric
pathogens to WSLH as soon as possible for PFGE
analysis and antimicrobial resistance monitoring
(for select organisms) - Vital to sustain our foodborne disease
surveillance
11What is PFGE?
- Molecular subtyping method used to generate the
DNA fingerprint of a bacteria - Other subtyping methods
- Phage typing
- Serotyping
- Biotyping
- Various molecular methods
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14PFGE Method
Step
- Liquid suspension of bacteria
- Encase in agarose plug
- Lyse with detergent and protease
- Wash away detergent and protease
- Digest with restriction enzyme
- Separate restriction fragments of genome DNA on
PFGE apparatus
15Specimen Slice
Comb
Gel
Casting Stand
16Pump
CHEF Mapper
Cooler
Electrophoresis Chamber
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18Gel Doc Imaging System
19PFGE Analysis of E.coli O157H7 isolates
M Milwaukee outbreak O Other outbreak ?
marker/internal reference
M
M
M
M
O
O
O
?
?
?
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23What is PulseNet?
- A national network of public health laboratories
- State health departments
- Local health departments
- Federal agencies (CDC, USDA, FDA)
- Perform standardized molecular typing of
foodborne disease-causing bacteria by PFGE - Sharing DNA fingerprints electronically
- Enhancing foodborne disease surveillance
24PulseNet
- Inception in 1996
- CDC-based APHL administration
- Molecular surveillance network for foodborne
bacterial pathogens - Primary subtyping method is PFGE
- Organisms subtyped include E. coli O157 and
non-O157 STEC, Salmonella, Shigella, Listeria and
Campylobacter
25PulseNet- Contd
- PulseNet International
- - Canada - Middle East
- - Europe - Asia Pacific
- - Latin America
26How Does PulseNet Enhances Surveillance?
- Detects outbreaks by identifying cases with
indistinguishable PFGE patterns - Links apparently unrelated outbreaks
- Speeds outbreak investigations through focusing
efforts on cases with indistinguishable PFGE
patterns - Monitors for illnesses associated with a product
retail samples ? recalls
27single food supplier
outbreak in New York
outbreak in California
outbreak in Wisconsin
same PFGE pattern
-Hypothesis for disease transmission -Test food
samples with PFGE
28Reporting Timeline
292006 Multi-State Spinach E. coli O157 H7
Outbreak
30Spinach Outbreak Timeline
- Sept. 5----Cluster of 5 E.coli O157H7 cases
- Livestock at Manitowoc county fair suspected
- Sept. 7----Report to State Medical Officer of 5
adults with HUS who had undergone plasma exchange - E. coli O157H7 confirmed in 3 patients
- Sept. 8----WSLH posts PFGE pattern of 8 isolates
on PulseNet - 7 had indistinguishable patterns, 1 a small
variation
31PFGE Gel - E. coli O157H7
32Spinach Outbreak (2)
- Sept. 7-9-----12 more E.coli O157H7 received at
WSLH - Sept 11----DPH questionnaires indicate high
percentage of females and link with spinach
consumption - Sept 12----Oregon investigation suggests 6
patients with E. coli O157H7 linked to spinach - Sept 14----CDC confirms 50 cases in 8 states
- Wisconsin, New Mexico, Utah, Oregon
- One death----77 yr old Wisconsin woman
- Sept 14----Conference call with CDC, state HDs,
and FDA - FDA issues advisory for consumers not to eat
bagged spinach
33Spinach Outbreak (3)
- Sept. 15----National Selection Foods of San Juan
Bautista, CA recalls all products containing
spinach (use-by dates from Aug 17 Oct 1) - Sept. 19----New Mexico confirms E. coli from
patients open bag of spinach - Identical PFGE pattern to outbreak strain
- Other states later identify outbreak strain in
bags of spinach - 13 bags from 8 states including 2 from Wisconsin
- Non-O157 isolate from New Mexico spinach
- Sept. 20----2 yr old Utah boy dies
- Oct. 5----Elderly Nebraska woman dies
34National Cases
35Outbreak Statistics
- 204 cases
- 26 states
- 1 Canadian province
- Approximately 50 hospitalized (104)
- Hemolytic uremic syndrome (HUS) (31)
- Higher than usual
- 39 of children
- 10 of adults
- 3 Deaths
36Spinach Outbreak Strain
- E. coli O157H7 identified as EXHX01.0124 strain
- Accounts for lt1 of strains reported each year
- Produces only Type 2 shiga toxin
- Usually more virulent than strains that produce
only Type 1 or both toxins - Characterized as Stx2d-activatable----activated
by human and mouse intestinal mucus
37What was the Source?
- Extensive environmental sampling
- Water
- Processing plant
- Cattle feces
- Wild pig feces
- Investigation by FDA and CDC suggests wild pigs
or contaminated irrigation water as culprits
38How do we Prevent Future Outbreaks
- Preventive measures
- At the field
- Irrigation water
- Proximity to cattle, pig, and other animal
ranches - At the processing plant
- Decontamination steps
- Distribution
- Maintaining appropriate temperatures
- Consumer education
39Conclusions I
- PFGE is extremely valuable as part of
epidemiologic and environmental investigations - Helps separate outbreak from sporadic cases
- Identifies cases that are likely to be linked
- Helps trace source of contamination
- Most of the time, lab and epidemiology correlate
- PFGE is only part of the picture results must be
correlated with epidemiology
40Conclusions II
- PulseNet
- Provides a means for laboratorians to rapidly
exchange cata - Allows active participation of laboratorians in
evolving outbreak investigations - Provides effective two-way communication among
laboratories and CDC
41The Latest Victim