Vibrio parahaemolyticus: Incidence, Growth and Survival in

Chesapeake Bay Oysters

Salina Parveen, Ph.D.Food Science and Technology Program

Department of Agriculture, Food and Resource Sciences

University of Maryland Eastern ShorePrincess Anne, Maryland

Introduction

Vibrio parahaemolyticus (Vp) is a Gram-negative halophillic rod

Grow on or in ordinary media containing 1-8% NaCl

Highly motile in liquid media with polar flagellum

Grow well from 25 to 44°C

(Oliver and Kaper 2001)

Introduction

Optimum temperature for growth is 37°C

Can not proliferate below 10°C

Occurs naturally in marine, coastal and estuarine environments

Isolated from seawaters, sediments and seafoods

(Oliver and Kaper 2001)

A significant cause of bacterial seafood-borne illness

U.S. CDC estimates ~7,880 Vibrio illnesses/year

~2,800 are estimated to be associated with Vp and raw oyster consumption

Common symptom-gastroenteritis with occasional bloody diarrhea

Primary septicemia- individuals with underlying chronic illness(US FDA 2005)

Introduction

Introduction

Pathogenic and non-pathogenic strains

Pathogenic strains of Vp produce a thermostable direct hemolysin (TDH) and thermostable related hemolysin (TRH)

95% of clinical strains produce TDH and TDH was also detected in 47% of oysters

A number of significant Vp illness outbreaks have been reported in the U.S.

Recently, two cases of Vp infections have been reported from Chesapeake Bay oysters

(CDC 1999; Blackstone et al. 2003; Food safety network 2006; Anon. 2008)

Introduction

Several investigators reported the incidence of Vp in the Gulf and Pacific coasts

Seasonal cycle of Vp in sediment and water in the U.S. was first reported by Kaneko and Colwell

Reported that densities of total and pathogenic Vp in shellfish at harvest were directly related to harvest water temperature

No information is available about the incidence of total and pathogenic Vp in Chesapeake Bay oysters

(Kaneko and Colwell, 1973, 1975; Kaysner et al. 1990; DePaola et al. 2003)

Introduction

Recently, Food and Drug Administration (FDA) has published risk assessments for Vp in raw oysters

Identified data gaps that increase the uncertainty of risk assessments

Lack of predictive models for the growth and survival of total and pathogenic Vp in oysters

(Miles et al. 1997; US FDA 2005)

Introduction

Risk assessments have had to make very board extrapolations from bacteriological broth-studies

Effects of temperature and time on the growth and survival of Vp in post-harvest oysters

A single harvest region, limited seasons and to one specific storage temperature

Harvest region, season and the harvest water conditions influence subsequent Vp growth and survival during storage

(Gooch et al. 2002)

Objectives

To determine the incidence of total and pathogenic Vp in oysters and waters in the Chesapeake Bay

To examine the correlation of Vp levels in oysters and waters with environmental parameters

To develop predictive models for the growth and survival of total and pathogenic Vp in oysters as a function of temperature

Objectives

To validate the models with model-independent data, considering season of harvest, geographical harvest area, harvest water salinity and temperature well as Asian oysters

To compare the growth and survival of total and pathogenic Vp in American and Asian oysters

Methodology

Collection of Samples

Oysters (American oyster, Crassostrea virginica) were collected from the Chesapeake Bay, MD and Gulf Coast, AL

Asian Oysters (C. ariakensis) were collected from the Chesapeake Bay, VA

Water temperature, salinity, pH and conductivity were measured, with a model 85 dissolved oxygen conductivity meter

(Cook et al. 2002)

Methodology

Gulf of Mexico

Mississippi Sound

MobileBay

Sampling Sites Cedar Point

Chesapeake Bay

Sampling Sites

Methodology

Chlorophyll content was measured by using YSI chlorophyll sensor

Fecal coliforms by standard methods

Water samples were collected in sterile 1-liter wide mouth containers After harvest, the oysters were shipped to the laboratory

All microbiological analyses were initiated within 24 hours of sample collection

(Cook et al. 2002)

Methodology

Incidence Study

Two separate set of samples of 12 oysters each were analyzed

Growth and Survival Study

Oysters were stored at 5, 10, 15, 20, 25 and 30°C

At selected time intervals, two separate set of

samples of 6 oysters each were analyzed

Methodology Preparation of Samples

12 or 6 oysters (scrubbed, shucked) Added phosphate buffered saline (PBS) and

blended for 90s Serial 10-fold dilutions were prepared in PBS

(DePaola and Kaysner 2004)

Methodology Bacteriological Analysis-A Direct Plating-Colony

Hybridization Original homogenate and 10-fold serial dilutions

spread plated on T1N3

Incubated 16-18 hr at 35C

colony lifts

Hybridization (tlh & tdh), colorimetric detection and

enumeration

(DePaola and Kaysner 2004)

Colonies of Vp

Multiplex Real Time PCR (q-PCR)

Three tubes Most Probable Number (MPN) Method

Enrich oyster homogenate in APW (Alkaline peptone water)

Boil for 10 min.

Analyze by q-PCR (tdh and trh genes)

(Nordstrom et al. 2007)

Methodology

Primary and Secondary Model Development

log10 values and mean and standard deviation were plotted

with Excel spread sheet software

The dynamic model described by Baranyi and Roberts (1994) was used to fit curves to the experimental data and to estimate values for the primary parameters-using DMFit curve-fitting software

lag phase duration (LPD [h]) growth/inactivation rate (GR [log CFU/h]) Maximum population density (MPD [log CFU/g])

Methodology Secondary model was produced using Table Curve 2D

(SPSS Inc., Chicago, IL) with built-in and customized equations

The Ratkowsky square root model was used to model GR

Model performance was measured by bias (Bf) and accuracy (Af) factors

Statistical analysis-Regression analysis

(Baranyi et al. 1999)

Results and Discussion

Incidence of total Vp in oysters at three sites in Chesapeake BayBC:Broad Creek; Chester River:CR; Eastern River (ER)

(Parveen et al. 2008)

0

100

200

300

400

500

600

700

Nov Dec Jan Feb Mar Apr May Jun July Aug Sep Oct

Month

To

tal V

p (

CF

U/g

)

0

5

10

15

20

25

30

35

Wa

ter

tem

pe

ratu

re (°

C)

BC CR EB Water Temperature

Detection of Vp in oyster and water samples by Direct-plating and real-

time PCR (q-PCR)(June to October)

Gene % of positive

oyster samples

% of positive

water samples

Direct plating q-PCR Direct plating

q-PCR

tlh

(n=15)

100 100 0 100

trh

(n=15)

Not Done 40.0 Not done 40.0

tdh

(n=15)

3% 20.2 0 13.3

Results and Discussion

0

0.5

1

1.5

2

2.5

0 100 200 300

Time (h)

log

CF

U/g

0

1

2

3

4

5

6

0 200 400

Time (h)

log

CF

U/g

The inactivation/growth profile of total Vp in shell stock oysters at 5, 15 and 30°C with primary curve fitting (Chesapeake Bay, 2005)

0

1

2

3

4

5

6

0 50 100

Time (h)

log

CF

U/g

5°C 15°C 30°C

Results and Discussion

Primary growth/inactivation parameters for the temperature ranged from 5-30°C. GR- Growth/inactivation rate; LPD- Lag phase duration; MPD-Maximum population density; ND-Not detected; CNBD-Could not

be determined

Storage GR LPD MPDTemperature (log CFU/h) (h) (CFU/g) (°C) 2005 2006 2005 2006 2005 2006

51015202530

-0.002 -0.001 0.054 0.107 0.280 0.264

-0.001-0.002 0.022 0.058 0.177 0.175

NDNDNDND10.664.07

NDNDNDNDNDND

CNBDCNBD 4.55 5.05 6.03 5.40

CNBDCNBD 4.40 6.50 6.69 5.52

Results and Discussion

Predicted and observed growth rate (GR) of total Vp in shell stock oystersCB-Chesapeake Bay

Results and Discussion

Bf=exp

Af=exp

Model Performance-Bias (Bf) and Accuracy (Af) Factors

Bf=1.00Af=1.03

Where m is number of observations, p is predicted, o is observed and μ is growth rate

m

k

op

m

kk

1

)()(

m

m

k

opkk

1

2)()(

Results and Discussion

Predicted and observed growth rate (GR) of total Vp in shell stock oystersCB-Chesapeake Bay; GC-Gulf Coast

Results and Discussion

Validation of Models

Oysters Bf Af

CB-American oysters

CB-Asian oysters

GC-American oysters

Combined

1.02

1.02

1.03

1.02

1.04

1.05

1.06

1.05

Results and Discussion

P>0.05

Effects of harvest temperature and salinity on growth rate

Results and Discussion

P>0.05

P> 0.05Effects of harvest regions/seasons on growth rate

P> 0.05

Results and Discussion

Comparison of growth and survival of total and pathogenic Vp in American oysters

Results and Discussion

Comparison of growth and survival of total and pathogenic Vp in

Asian oysters

Results and Discussion

Comparison of growth and survival of total and pathogenic Vp in

American and Asian oysters

Conclusions

Total Vp was detected in 79% of the samples using direct plating-colony hybridization method at densities ranging from 1.5x101 to 6.0x102 cfu/g

Levels of total Vp at all sites followed a seasonal trend with higher levels in the warmer months

Real time PCR was able to detect significant number of pathogenic Vp in water samples

Conclusions

Total Vp was slowly inactivated at 5°C and 10°C

Maximum GR was observed at 25°C

MPD displayed a peak form at 25°C

No significant LPD was observed The bias (Bf) and accuracy (Af) factors were 1.00

and 1.03, respectively

Conclusions

The Bf and Af factors for the growth rates determined in Gulf Coast oysters during the summer, fall and spring were 1.02 and 1.04; 1.02 and 1.04 and 1.03 and 1.07, respectively

The Bf and Af factors for the growth rates in Asian oysters were 1.02 and 1.05, respectively

These results suggest that the model developed for the growth and survival of total Vp in Chesapeake Bay oysters are valid for, and predictive of, growth occurring in oysters harvested from Gulf Coast over multiple seasons as well as in Asian oysters

Harvest region/season, temperature and salinity had no effects on GR

Conclusions GR of pathogenic Vp were higher than those observed

for total Vp in American and Asian oysters at 10,15, 20, 25, & 30°C

These results also indicate that total and pathogenic Vp multiplied more rapidly in American oysters than Asian oysters

The results of this study will assist risk managers and the seafood industry in designing more effective food safety systems

Further confirmation and mechanism of pathogenic Vp GR require additional study as this may substantially affect risk predictions and impact of controls

Current Studies

Development of predictive models for the growth and survival of pathogenic Vp in oysters

Development of predictive models for the growth and survival of V. vulnificus (Vv) in oysters

Investigation of the effects of storage condition on sensory and textural characteristics of oysters

Future Studies

Secondary models will be incorporated into spreadsheet format and into the USDA Pathogen Modeling Program

(PMP; www.arserrc.gov/mfs/pathogen.htm)

Raw data sets will be archived in ComBase (http://wyndmoor.arserrc.gov/combase/), an international database of predictive microbiology information

Acknowledgements

UMES

Ligia DaSilvaChanelle White

Apsara Hettiarachchi

Meshack Mudoh Jurgen Schwarz Tom Rippen Geoff Rutto

FDA MDE VIMSAngelo DePaola Kathy BrohawnAngelo DePaola Kathy Brohawn S. S. Allen Allen John Bowers John Bowers Bill Beatty Bill BeattyJessica Jones Jessica Jones John McKay John McKay Jeff KrantzJeff Krantz and and

othersothers

Univ. of Tasmania, AustraliaUniv. of Tasmania, AustraliaMark TamplinMark Tamplin

The Maryland Watermen’s Association,The Maryland Watermen’s Association, Inc.Inc.United States Department of Agriculture National Research Initiative grant # 2006-35201-16644

QUESTIONS