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The incidence of Salmonella in four fish and shellfish species harvested in Florida

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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 Bibliography
 Biographical sketch
University of Florida PETE FLAG IFAS PALMM


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Author: Margaret Bury Fraiser


Title: The incidence of salmonella in four fish and shellfish species harvested in Florida


Publication Date: 1982


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MISSING IMAGE

Material Information

Title:
The incidence of Salmonella in four fish and shellfish species harvested in Florida
Physical Description:
viii, 57 leaves : ill. ; 28 cm.
Language:
English
Creator:
Fraiser, Margaret Bury, 1959- ( Dissertant )
Koburger, John A. ( Thesis advisor )
Oblinger, James L. ( Reviewer )
Farrah, Samuel R. ( Reviewer )
Fry, Jack L. ( Degree grantor )
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:
Copyright Date:
1982

Subjects

Subjects / Keywords:
Salmonella   ( lcsh )
Seafood -- Microbiology   ( lcsh )
Fishes -- Microbiology -- Florida   ( lcsh )
Shellfish -- Microbiology -- Florida   ( lcsh )
Food Science and Human Nutrition thesis M.S
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )
Spatial Coverage:
United States -- Florida -- Crescent Beach

Notes

Abstract:
Members of the genus Salmonella are important etiological agents in foodborne disease outbreaks in the united States and have often been recovered from seafoods. In order to gain information on the distribution of these organisms in seafoods harvested in Florida, the incidence of Salmonella was studied in four seafoods: clams (Mercenaria mercenaria), oysters (Crassostrea virginica), mullet (Mugil cephalus), and blue crabs (Callinectes sapidus). These were harvested from two locations in Florida; the west coast location was at the mouth of the Suwannee River and the east coast location was in the Intracoastal waterway at Crescent Beach. In addition, fresh-water clams (Polymesoda caroliniana) and sediment obtained at the west coast location were analyzed for Salmonella. Determination of salmonellae was performed using the standard procedures of the United States Food and Drug Administration for the analysis of salmonellae in food products. To determine the degree of contamination, a quantitative study of Salmonella in west coast oysters and fresh-water clams was performed. In addition, a storage study to investigate the survivability of Salmonella under commercial storage conditions was also conducted. Results indicated that salmonellae were present in oysters, clams, and blue crabs in percentages of 8.3%, 28.3&, and 33.3% of the samples analyzed, respectively. Salmonellae were not recovered from any mullet samples. Sediment samples taken in the vicinity of the west coast harvest location also contained salmonellae. the stereotypes recovered were those which are less frequently reported to the Centers for Disease Control as agents in human salmonellosis, which may reflect their lesser virulence. Quantitation of salmonellae in oysters and fresh-water clams using the most probable number technique yielded low numbers, ranging from 2.2 to 16.0 salmonellae per 100 grams sample. Storage tests showed that Salmonella were capable of surviving in oysters and fresh-water clams at refrigerated temperatures for ten days. Aerobic plate counts, total coliform estimates and fecal coliform estimates showed no apparent correlation with the incidence of salmonellae.This study indicates that salmonellae may be autochthonous members of the microflora of these seafoods based on the wide distribution, the low numbers recovered, the variety of the serotypes recovered and the lack of correlation with fecal coliform analyses. Under normal handling, processing, and storage conditions, consumption of these seafood products may not cause salmonellosis in the majority of the human United States population due to the low numbers of salmonellae isolated, the destruction of salmonellae during the cooking process, the unfavorable pH of the stomach, and/or the presence of less virulent serotypes.
Thesis:
Thesis (M.S.)--University of Florida, 1982.
Bibliography:
Includes bibliographic references (leaves 51-56).
General Note:
Typescript.
General Note:
Vita.
Funding:
Electronic resources created as part of a prototype UF Institutional Repository and Faculty Papers project by the University of Florida.
Statement of Responsibility:
by Margaret Bury Fraiser.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved, Board of Trustees of the University of Florida
Resource Identifier:
aleph - 000319030
oclc - 09263082
notis - ABU5880
System ID:
UF00074944:00001

MISSING IMAGE

Material Information

Title:
The incidence of Salmonella in four fish and shellfish species harvested in Florida
Physical Description:
viii, 57 leaves : ill. ; 28 cm.
Language:
English
Creator:
Fraiser, Margaret Bury, 1959- ( Dissertant )
Koburger, John A. ( Thesis advisor )
Oblinger, James L. ( Reviewer )
Farrah, Samuel R. ( Reviewer )
Fry, Jack L. ( Degree grantor )
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:
Copyright Date:
1982

Subjects

Subjects / Keywords:
Salmonella   ( lcsh )
Seafood -- Microbiology   ( lcsh )
Fishes -- Microbiology -- Florida   ( lcsh )
Shellfish -- Microbiology -- Florida   ( lcsh )
Food Science and Human Nutrition thesis M.S
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )
Spatial Coverage:
United States -- Florida -- Crescent Beach

Notes

Abstract:
Members of the genus Salmonella are important etiological agents in foodborne disease outbreaks in the united States and have often been recovered from seafoods. In order to gain information on the distribution of these organisms in seafoods harvested in Florida, the incidence of Salmonella was studied in four seafoods: clams (Mercenaria mercenaria), oysters (Crassostrea virginica), mullet (Mugil cephalus), and blue crabs (Callinectes sapidus). These were harvested from two locations in Florida; the west coast location was at the mouth of the Suwannee River and the east coast location was in the Intracoastal waterway at Crescent Beach. In addition, fresh-water clams (Polymesoda caroliniana) and sediment obtained at the west coast location were analyzed for Salmonella. Determination of salmonellae was performed using the standard procedures of the United States Food and Drug Administration for the analysis of salmonellae in food products. To determine the degree of contamination, a quantitative study of Salmonella in west coast oysters and fresh-water clams was performed. In addition, a storage study to investigate the survivability of Salmonella under commercial storage conditions was also conducted. Results indicated that salmonellae were present in oysters, clams, and blue crabs in percentages of 8.3%, 28.3&, and 33.3% of the samples analyzed, respectively. Salmonellae were not recovered from any mullet samples. Sediment samples taken in the vicinity of the west coast harvest location also contained salmonellae. the stereotypes recovered were those which are less frequently reported to the Centers for Disease Control as agents in human salmonellosis, which may reflect their lesser virulence. Quantitation of salmonellae in oysters and fresh-water clams using the most probable number technique yielded low numbers, ranging from 2.2 to 16.0 salmonellae per 100 grams sample. Storage tests showed that Salmonella were capable of surviving in oysters and fresh-water clams at refrigerated temperatures for ten days. Aerobic plate counts, total coliform estimates and fecal coliform estimates showed no apparent correlation with the incidence of salmonellae.This study indicates that salmonellae may be autochthonous members of the microflora of these seafoods based on the wide distribution, the low numbers recovered, the variety of the serotypes recovered and the lack of correlation with fecal coliform analyses. Under normal handling, processing, and storage conditions, consumption of these seafood products may not cause salmonellosis in the majority of the human United States population due to the low numbers of salmonellae isolated, the destruction of salmonellae during the cooking process, the unfavorable pH of the stomach, and/or the presence of less virulent serotypes.
Thesis:
Thesis (M.S.)--University of Florida, 1982.
Bibliography:
Includes bibliographic references (leaves 51-56).
General Note:
Typescript.
General Note:
Vita.
Funding:
Electronic resources created as part of a prototype UF Institutional Repository and Faculty Papers project by the University of Florida.
Statement of Responsibility:
by Margaret Bury Fraiser.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved, Board of Trustees of the University of Florida
Resource Identifier:
aleph - 000319030
oclc - 09263082
notis - ABU5880
System ID:
UF00074944:00001

Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
    Abstract
        Page vii
        Page viii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
    Materials and methods
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
    Results and discussion
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
    Summary and conclusions
        Page 48
        Page 49
        Page 50
    Bibliography
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
    Biographical sketch
        Page 57
        Page 58
Full Text












THE INCIDENCE OF SALMONELLA IN FOUR FISH
AND SHELLFISH SPECIES HARVESTED IN FLORIDA










BY
MARGARET BURY FRAISER






















A THESIS PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE


UNIVERSITY OF FLORIDA

1982














KKNCWLEDGBEVNTS

The author wishes to thank Dr. John A. Koburger, her advisor, for

his guidance and encouragement in this project. She is thankful for his

knowledge of the subject and patience in teaching her. Appreciation is

also extended to Dr. James L. Oblinger and Dr. Samuel R. Farrah for

their advice and assistance during the research and thesis preparation.

Appreciation is expressed to the National Fisheries Institute for

financial support during this and other related projects. Thanks are

also given to Mary Miller for her willingness to listen and to share

materials.

Finally, the author thanks her parents for their encouragement and

financial support throughout her education and her husband for his

love, support, meal preparation, and word processor knowledge.














TABLE OF GINTNTENTS

PAGE

A K
LIST CF TABLES....................................................... v

LIST OF FIGLRES .....................................................vi

ABSTRCT. ...................................................... vii

INTRODLCTION ............................................. .......... 1
The Genus Salmonella............................................3
Disease ..................................... ....... 4
Prevention...................................... ......
Infective Dose ...........................................5
Incidence.......................... ..................... 6
Ecology ........................................10
Microflora of Seafoods.................... 1......... ...... 13
Relationship of Salmonella to Indicator Organisms...............15
Survival of Salmonella During Storage...........................16

MSTERIALS AND ETHSDS...............................................18
Materials.................................. .... ......... ...... 18
Sampling Plan....... ...... ................ .......... ...... .. 18
Samples ....... .. ............................................ 19
Salmonella Analysis......................... ........... ....... 19
nPN Studies ..................................... ............... 20
Storage Studies............ ............................ ........ 23
Sediment Samples..................................................23
Aerobic Plate Count....................................... ..... 23
Coliform Analysis .......... ...... .... .................. ...........24
Statistical Analysis.................... .. .......... ..... .......24

RESULTS AND DISCUSSION..............................................25
Salmonella Analysis Results........................................ 25
Isolation of Salmonellae from Various Seafoods ...........25
MPN Studies....................o...........................30
Storage Studies............. ..... ...... ..................... 32
Serotypes.............................. .................. 32
Isolation Methodology.....................................38
Relationship to Total Coliform, Fecal Coliform, and
Aerobic Plate Count.................. .......................39
Salmonella as a Contaminant or an Autochthon....................42
Significance of the Findings....................................45








PAGE
BIBLIOGRAPHY ........................................................51

BIOGRAPHICAL S












LIST OF TABLES

TABLE PAGE

I. MIGCFLCR COF C(STERS AND BLLE CRABS............................. 14

2. BIOCHEMICAL TESTS PERFCRvED FOR ISCLATICN CF SALMONELL A....... 21

3. BASIS FCR DISCARDING ISCLATES....................................22

4. SALM3NELLAE REXnVERED FRCM FOLR SEAFODS .... ................... 26















31 L.. FI.JLLU L3


I I .lK-F--I

1. REFPRTED SAITvNELLA ISOLATIONS RCM HLMVNS
BY WEEK IN THE .NITED STATES, 1974-1980................. .......7

2. REFCRTED SAILMNELLAISCLATIONS iCM HUAMNS
BY PE IN THE UNITED STATES, 1980..............................9

3. MODE OF TRANSMISSION CF SAIDNELLOSIS ...........................11

4. ANIMML-TO-MAN TRANSMISSION OF SALM3NELLOSIS.....................12













Abstract of Thesis Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science


THE INCIDENCE OF SALMONELLA IN FOR FISH
AND SHELLFISH SPECIES HARVESTED IN FLCRIDA

By

Margaret Bury Fraiser

December 1982

Chairman: John A. Koburger
Major Department: Food Science and Human Nutrition

Members of the genus Salmonella are important etiological agents in

foodborne disease outbreaks in the United States and have often been

recovered from seafoods. In order to gain information on the distribu-

tion of these organisms in seafoods harvested in Florida, the incidence

of Salmonella was studied in four seafoods: clams (Mercenaria mercenar-

ia), oysters (Crassostrea virginica), mullet (Mugil cephalus), and blue

crabs (Callinectes sapidus). These were harvested from two locations in

Florida; the west coast location was at the mouth of the Suwannee River

and the east coast location was in the Intracoastal Waterway at Crescent

Beach. In addition, fresh-water clams (Polymesoda caroliniana) and sedi-

ment obtained at the west coast location were analyzed for Salmonella.

Determination of salmonellae was performed using the standard pro-

cedures of the United States Food and Drug Administration for the analy-

sis of salmonellae in food products. To determine the degree of contami-

nation, a quantitative study of Salmonella in west coast oysters and

fresh-water clams was performed. In addition, a storage study to








IIIVtC. L I Lile U[L V VCIUl i Ly UJ. O"JAIIL I IIC Zi ULIUCL LI.AJIII C -.ICI 3LUIagC:

conditions was also conducted.

Results indicated that salmonellae were present in oysters, clams,

and blue crabs in percentages of 8.3%, 28.3%, and 33.3% of the samples

analyzed, respectively. Salmonellae were not recovered from any mullet

samples. Sediment samples taken in the vicinity of the west coast har-

vest location also contained salmonellae. The serotypes recovered were

those which are less frequently reported to the Centers for Disease Con-

trol as agents in human salmonellosis, which may reflect their lesser

virulence. Quantitation of salmonellae in oysters and fresh-water clams

using the most probable number technique yielded low numbers, ranging

from 2.2 to 16.0 salmonellae per 100 grams sample. Storage tests showed

that Salmonella were capable of surviving in oysters and fresh-water

clams at refrigerated temperatures for ten days. Aerobic plate counts,

total coliform estimates and fecal coliform estimates showed no apparent

correlation with the incidence of salmonellae.

This study indicates that salmonellae may be autochthonous members

of the microflora of these seafoods based on the wide distribution, the

low numbers recovered, the variety of the serotypes recovered and the

lack of correlation with fecal coliform analyses. Under normal handling,

processing, and storage conditions, consumption of these seafood prod-

ucts may not cause salmonellosis in the majority of the human United

States population due to the low numbers of salmonellae isolated, the

destruction of salmonellae during the cooking process, the unfavorable

pH of the stomach, and/or the presence of less virulent serotypes.




Chai an













INTRODUCTION

Seafoods accounted for 8.7% of the reported foodborne disease

outbreaks in the United States in 1979 (10). With the dockside value ol

seafoods landed in Florida at approximately 173 million dollars (48),

there is both an economic and an etiological need to study the presence

of pathogens within these products. Historically, Salmonella typhi was

the major pathogen isolated from seafoods. For instance, in 1925, 150

deaths and thousands of cases of typhoid fever were due to the presence

of salmonellae in oysters (25). Other pathogens are now commonly founc

in seafoods (e.g., nonagglutinating Vibrio cholerae and Clostridium

botulinum), in addition to opportunistic pathogens ( e.g., Vibrio

parahaemolyticus, enteroviruses, Pseudomonas aeroginosa, and Aeromonas

hydrophila) which are being recognized with greater frequency. Because

of the increase in reporting of foodbone disease associated with

seafood, stricter microbiological guidelines are being considered for

harvesting areas and seafood products, placing pressure on both the

seafood industry and the federal government. In an attempt to better

understand this problem, additional data concerning the presence and

distribution of pathogens in seafoods would be helpful in dealing with

this problem.

Members of the genus Salmonella are the primary etiological agents

in foodborne disease outbreaks in the United States (10). In 1979 the

genus Salmonella produced 40% of all confirmed foodborne cases and 29%








estimated because the reported infections are estimated to be only a

fraction of the total number. It is postulated that about 1% of the

salmonellosis cases are reported (13); in 1980 there were 33,715 cases

reported, making the total estimated cases in the United States well

over 3 million. This low rate of reporting is probably a result of a

low fatality rate and an illness which is frequently mild and

self-limiting. In addition, salmonellosis can not be accurately

diagnosed on clinical grounds alone, leaving many cases unreported,

particularly those which are not associated with a recognized

food-poisoning outbreak. Although salmonellosis may appear to be a

relatively harmless disease, it is responsible for substantial costs in

the form of medical care, hospitalization, and lost income due to

absence from work. The total cost of salmonellosis in 1969 was

estimated to be at least $300 million (49). Salmonellosis is also

potentially fatal to the old, the very young, and the infirm. Because

of the ease of transmission between both humans and animals, the

control of salmonellosis is complex.

The purpose of this study was to investigate the incidence of Sal-

monella in four fresh seafoods commercially harvested in Florida: oys-

ters (Crassostrea virginica), clams (Mercenaria mercenaria), striped

mullet (Mugil cephalus), and blue crabs (Callinectes sapidus). Salmo-

nella was chosen as a representative of the pathogenic microflora be-

cause it is an established overt pathogen, it is associated with the

estuarine environment, and standarized methodology for its detection is

available. In an attempt to obtain a more representative sampling of
+h1 cn fnnrlcT^4 1r -A+-A ;-n rIr-;!- +Ak- hA- -oAnle I.-ra k-r-a-.4A *mnh +Ir -






.3

the Suwannee River and the east coast location was in the Intracoastal

Waterway at Crescent Beach.

The Genus Salmonella

The genus Salmonella, a member of the family Enterobacteriaceae,

now contains over 1800 serotypes with the rrst prominent member of the

genus being Salmonella typhi, the causal agent of typhoid fever. The

genus can be divided into three classifications, based on their patho-

genicity for various animals:

1. Salmonellae pathogenic to man only














frozen, or dried foods and feeds. Salmonellae have also been found to

persist for prolonged periods at ambient temperatures in dried nonfat

milk, egg products, and rendered animal byproducts (50).

Disease

There are four typical disease syndromes associated with the genus

Salmonella, vAhich are enteric fever, septicemia, gastroenteritis, and a

carrier state. Only one of these, gastroenteritis, is commonly caused

by ingestion of contaminated foods, although gastroenteritis can occa-

sionally progress into the other syndromes. The gastroenteritis, called

salmonellosis, will begin to occur 8 to 48 hours after ingestion of the

salmonella. The svrrntoms usually include fever. cramos. diarrhea. and










barrierss as food handlers are common measures exercised in the

developed world. On an individual level, personal hygiene is helpful in

prevention of salmonellosis.

Infective Dose

The infective dose has been disputed since 1951, when McCullough

and Eisele reported doses in the range of 105 to 109 bacteria were

needed for human infection (43, 44, 45). McCullough and Eisele also

reported differences in the infective doses between serotypes and

strains; this was thought to be due to a difference in the virulence of

variouss serotypes (43, 44, 45). Because this is the only research re-

ported using human subjects, these figures have been used repeatedly

and are still in use today. Epidemiologic studies from salmonellosis

outbreaks, however, have shown doses causing the disease to be much

smaller. An outbreak in 1973 involving chocolate revealed a total con-

tamination of fewer than 100 salmonellae for a primary case of

infection (17). In an outbreak involving ground beef, there were only

6-23 viable salmonellae per 100 grams upon epidemiological analysis of

the frozen sample (23). Experience shows, however, that there is a re-

duction in the number of viable organisms after freezing, which would

increase the number of viable salmonellae in the meat at the time of

ingestion as much as 100-fold (71). In the case of ground beef, it is

unlikely that a person who contracted salmonellosis from this product

would have eaten 100 grams of raw meat, although the amount may have

been larger if the contaminated meat was cooked rare, which may not

kill salmonellae. In other cases, frozen eggs were found to have an

infective dose of 0.6 salmonellae per gram, cereal products had 7 to 14










salmoneiiae per gram. It can oe seen Irom me aoove epiaemioiogical

studies that the tolerance to salmonellae varies with the food product

and the person involved. Because of this, regulatory agencies forbid

the sale of foods which contain any salmonellae. Exceptions to this are

red meats and poultry, which often have high percentages (30%) of

contamination (57). These products, being integral parts of the food

chain, are difficult to rid of salmonellae. Because of this and the

fact that these products will commonly be cooked, destroying the

salmonellae, the United States Department of Agriculture has made an

exception, allowing salmonellae to be present in red meats and poultry

sold in the United States if the salmonellae are considered inherent in

the product.

Incidence

The incidence of salmonellosis has been steadily increasing in the





























1000


900


C 800
Z
0
< 700
-J

L 600
0




UJ
C 500



w
400


300


200


< 100


arIima rsono'ru J s O0no.' ruI i "nl0 . a ..-'"O'J'' S I'C a s J s- rMAMJ J ASCNDOJFMAMJJ A SO O
J if M1;A J OeO 1;M AMJ JJ 0
1974 1975- 1976 1977 1978 1979 1980

SEach point r.presp.ts the wve.klv averaqr numbr)*n r.l ,olalps for thn month








FIGURE 1
REPORTED SALMONELLA ISOLATIONS FROM HUMANS
BY WEEK IN THE UNITED STATES, 1974-1980










of foodborne disease in the United States.

The reported isolations of Salmonella from humans by age in the

United States -l-s 1980 is shown in Figure 2 (11). The very high

incidence in children less than one year old can be attributed to sev

eral factors, including low immunity, higher incidence of examination

and the increased probability of transmission by the fecal/oral route

Another increase in the incidence can be seen in the elderly; this in

crease is due to aging which causes a decrease in resistance to such

organisms.

Another indication of the severity of salmonellae is shown in th

number of deaths attributed to the disease. Deaths due to salmonellos

reported in the United States in the nine year period from 1970 to 19

totaled 645 with the highest number of deaths in one year being in 19

and 1971 with 81 deaths in each year. The lowest number of deaths wa

in 1974 with 59 deaths being reported.

In Canada, it is of interest to note that in 1977 the foodborne

illnesses due to Salmonella species exceeded those caused by all othe

microbiological genera. This is in contrast to the 1976 figures, whi

show Staphylococcus aureus responsible for more outbreaks and cases

(9). In 1976, there were 356 salmonellosis cases reported involving

outbreaks. In that same year, there were 520 cases involving 25

outbreaks reported due to Staphylococcus aureus. In 1977, however,

there were 763 cases of salmonellosis reported involving 32 outbreaks

with 305 cases involving 22 outbreaks pertaining to Staphylocuccus





















70T-


300-

60-
MALE 200
-. --- FEMALE 20

50- _\ O 100-


S<1 I 2 4 5 67 8 9 10 11
S 40- AGE (MONTHS)


0
30




20-




10


o ---- --- -- --- --- ---------- -

0-4 5-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 t 0
AGE GROUP (YEARS)

*PER 100,000 POPULATION





FIGURE 2
REPORTED SALMONELLA ISOLATIONS FROM HUMANS
BY AGE IN THE UNITED STATES, 1980









Salmonella is ubiquitous in the environment, being one of the. n

widely distributed overt pathogens in the world. The.main reason for

this ubiquity is the easy transmissibility of the organism. Transmis

sion of salmonellae can be through a variety of means (5): animal to

animal, animal to man, man to man, man to animal, or a common source

both animals and man. The mode of transmission in 500 salmonellosis

outbreaks from 1966 to 1975 is given in Figure 3 (57). The most corm

of these transmissions was the animal to man, in which red meats and

poultry play a large part as illustrated in Figure 4 (5). A circular

pattern can be seen by noting that the byproducts of processing plant

which are often contaminated with salmonellae, are the main ingredien

of animal feeds. Another reason for the ubiquity of Salmonella is th

apparent capacity of the genus to survive for prolonged periods in th

estuarine environment. For example, Salmonella in oysters have been

reported to have survived in artificial brackish water for forty-nine

days (33).

The Centers for Disease Control (CDC) now believes that much of

the human salmonellosis is directly or indirectly related to Salmon-

ella-contaminated animal feeds (56). Epidemiolgical evidence that

supports this position is growing, as seen particularly by the rise i

the recovery of two serotypes, S. agona and S. hadar. Before 1970, 5

agona had been reported in man only twice. One of the first isolation

of the serotype in the United States was due to contaminated Peruvian

fishmeal. After this, S. agona was isolated more frequently from

non-human sources, followed by an increase in isolations from human




















DAIRY PETS
PRODUCTS

S3%
4%
INEGGS
6%

10% \\ UNKNOWN
28%
PEnSON-TO-PERSO


MEAT
13%
MISCELLANEOUS
POULTRY 19%
17%






INCLUDES OVER 50 VEHICLES WHICH INDIVIDUALLY
CAUSED LESS THAN 3% OF OUTBREAKS







FIGURE 3
MODE OF TRANSMISSION OF SALMONELLOSIS













CONTAMINArT'D ANIMAL FEED

AND FERTILIZER





POULTRY PIGS' CATTLE L
FE'W INFECTED ON FARM
CROWDED TRANSPORT




MANY INFECTED IN LAIRAGE,POULTRY
PROCESSING PLANTS AND ABATTOIRS






LOCAL
_____ ____Y __UNPASTEURIZED
BULK EGG PRODUCTS IFOWL AND MEAT PRODUctS
CO~S' 5UM ED BY

MAN AND WIS DOMESTIC ANIMALS

CARR ] cE









by a turkey breeder from the United Kingdom. A rapid increase in tt

early 1970s was seen in the LK much like that of S. agona in this

country due to recycling of waste products from processing plants.

increase in the LK has lead to an increase in the United States,

although the numbers of isolates have been smaller.

There has been little study of the incidence of salmonellae in

marine environment. It is realized that turtles are a major carried

Salmonella and therefore the Food and Drug Administration has

prohibited interstate shipment of turtles. Nine serotypes were isoli

from water in aquaria housing turtles (40). In another study, seagt

were found to contribute a new serotype to a water environment (52).

Reptiles, also present in the aquatic environment, have been found 1

be contaminated with salmonellae while living in Swiss zoos. Thirty

percent of the reptiles in the Basel zoo, 43% of those in the Bern

and 30% of reptiles in the Zurich zoo were positive for salmonellae

(54). Salmonellae have also been isolated from oysters (3, 60), clz

(2), shrimp (18), cockles (34), and lalakupong (34). In addition,

salmonellae have been isolated from marine waters (2, 3, 27, 36, 60:

and sediment (26).

Microflora of Seafoods

In addition to the Salmonella serotypes already mentioned, man]

overt pathogens and opportunistic pathogens have been reported to be

present in seafoods. Table I gives a brief summary of the microbes 1

have been isolated from oysters and blue crabs. These organisms ha\










TABLE 1
MICRDFLCRA OF THE SEAFOODS STUDIED
CRGANI M SEAFOOD LOCATION
Vibrio parahaemolyticus oyster Mississippi Soul
blue crab Chincoteague Ba:
oyster laboratory
oyster Long Island Soul
blue crab unknown
Pseudomonas spp. blue crab unknown
oyster Long Island Soul
oyster laboratory
oyster Galveston Bay
Flavobacteriun-Cytophaga oyster Long Island Soui
blue crab unknown
oyster Galveston Bay
oyster laboratory
Achromobacter-Alcaligenes oyster Long Island Soul
oyster laboratory
oyster Galveston Bay
Bacillus blue crab unknown
oyster laboratory
oyster Galveston Bay
Acinetobacter blue crab unknown
oyster Galveston Bay
Aeromonas oyster Galveston Bay
Moraxella oyster Galveston Bay
Coryneforms oyster Galveston Bay
Vibrio cholerae oyster Apalachicola Ba
Clostridiun botulinum type F blue crab York River, VA









The use of specific groups of microorganisms to indicate the po

sible presence of pathogenic organisms had its beginning near the tu

of the twentieth century (30). The purpose of these indicator orga

isms was to detect the presence of sewage contamination in the potab

water supply. Twenty years later, indicators were found to be of use

the shellfish industry, where bacteriological standards for harvest

waters were being developed. Advantages of utilyzing these indicate

organisms included the relative ease and short duration of the analy

ses. In 1946, the first quantitative guideline for coliform counts

water from harvesting areas was established, which placed a limit of

seventy organisms per 100 rrL water. This was modified slightly in 19

and in 1974 the fecal coliform limit of fourteen organisms per 100 r

water was established. Presently either limit is accepted by the Fo

and Drug Administration (30).

Problems have arisen with the use of indicator organisms, since

there is no single group which contains all the desirable character

tics of an indicator. One of the first problems arose in 1961 when

study by Tennant and Reid (64) found that 26.7% of the coliform orga

isms did not ferment lactose with acid and gas in 48 hours at 35.5 C

Many studies have shown the poor correlation of coliforms and/or fec

coliforms to the presence of pathogenic organisms (18, 41, 55, 58) a

well as the poor correlation of pollution to pathogenic organisms (1

55). Attempts have been made (2, 3, 55) to determine the best indica

group to use in the seafood industry; these studies have yielded con
.1.--:, , .I .*- - -^-^ -- -- -- 1 2^, r -__- 1---- L -_ r,










station of the results of indicator organisms. No correlation has -beei

seen between fecal coliforms and Vibrio cholerae (15, 28, 35, 37), an

emerging problem with shellfish. In addition, Hood (28) found that

Vibrio cholerae was able to survive for longer periods in estuarine

waters than fecal coliforms. For increased safety, it is advantageou:

for indicator organisms to survive longer than the anticipated patho-

gen. Also, the increased incidence of enteroviruses in oysters has

caused Ellender et al. in 1980 (21) to suggest the use of a viral ana

sis of shellfish as an adjunct to bacteriological analyses so that

shellfish safety is verified.

Another problem concerning the microbial regulation of harvesting

waters is the question of whether shellfish concentrate microorganism

This is reflected by the FDA's limit of 230 fecal coliforms per 100 m

of shellfish, which is sixteen times greater than the limit allowed

for waters. It is generally agreed that shellfish have the ability ti

concentrate organisms from overlaying waters (8, 21, 31); however, it

is not agreed whether this is of major sanitary importance (31, 58).

Therefore, water and sediment analyses must be interpreted with

caution. In addition, Presnell and Miescier (53) found that wild

mammals and birds were the most likely sources of coliforms and fecal

coliforms in water and sediment. They expressed the need to ascertain

the presence of warm-blooded wildlife in areas adjacent to shellfish-

growing areas.

Survival of Salmonellae During Storage






17

conditions. Although no intensive studies have been conducted

pertaining to this subject, several studies have given indications-of

the survivability of salmonellae within seafoods and nonseafoods. In

nonseafood products, salmonellae survived less than ten days at 5 to

7 C in ground rabbit neat (62), and in a study involving "soul foods",

S. typhimurium survived for five days at 10 C in all foods studied,

which included collard greens, field peas, sweet potatoes, and semi-

processed pig offals (61). In seafoods, Kelly and Arcisz (39) found

that S. typhosa remained viable within the bodies or shell liquors of

oysters long enough to cause illness when oysters are eaten within the

"usual period" elapsing between the time they are removed from the

infected water and the time they are consumed. Salmonellae were

isolated from the shell liquor of oysters after refrigeration at 5 C

for forty-nine days. In a study evaluating depuration techniques (33),

S. typhimurium was found to persist in oysters for the entire

forty-nine days of the experiment. This was noteworthy in that

depuration procedures currently in use for commercial oysters are based

on the removal of fecal coliforms within 48 hours and may be deceptive

and 'ineffective in removing some human waterborne disease organisms.

In a storage study of oysters conducted at 20 to 25 C, S.. typhimurium

and S. senftenberg were detected until the sixteenth day of storage

(65). Collectively, these results support the hypothesis that

salmonellae can survive normal handling procedures practiced in the

seafood industry today.














MATERIALS AND METHODS

Materials

All microbiologcial culture media were obtained dehydrated from

Difco Laboratories (Detriot, MI) or Baltimore Biological Laboratories

(Cockeysville, MD). Media were prepared according to directions and

were at ambient temperature before inoculation, with the exception of

pour plates. Salmonella antisera were obtained from Fisher Scientific

Co. (Pittsburgh, PA) or Difco Laboratories (Detroit, MI).

All glassware was sterilized at 121 C for thirty minutes using wet

heat or at 170 C for one hour using dry heat.

Sampling Plan

The number of samples of each animal from each coast required for

analysis was dependent on a variety of factors. Because of the

pathogenicity of Salmonella and the zero tolerance in foods, the

sampling plan needed to be relatively stringent. If the food is to be

consumed raw, as clams and oysters frequently are, Salmonella is

considered a direct hazard in foods and therefore the sample size

recommended by the International Camnission on Microbiological

Specifications for Foods should be at least thirty for investigative

purposes (32). Based on this information, thirty animals of each

species and from each coast were analyzed, for a total of 240

individual samples.










11iirly sbdiipies ux 0 c1 n sCCLU Iu bd uysiers, kict>, IIUIICLj
were harvested between August 1981 and August 1982, from both a west

coast location and an east coast location within the state of Florida.

The west coast location was at the mouth of the Suwannee River and the

east coast location was in the Intracoastal Waterway 500 meters south

of the bridge on State Road 206 in Crescent Beach. Crabs were harvested

by trapping, oysters by tonging, and clams by digging. The mullet were

fresh whole commercial samples purchased in the vicinity of the

sampling locations (Suwannee, FL and St. Augustine, FL), and had been

iced prior to transportation to the laboratory. Eighteen of the thirty

east coast crabs were purchased live in St. Augustine and were not iced

during transportation. The samples were transported to the laboratory

at the University of Florida (Gainesville) in sanitized insulated

coolers with analysis begun within four hours of harvest or purchase.

No attempt was made to cool the samples (with the exception of the

mullet) because of the short time span involved between collection and

analysis.

Salmonella Analysis

The method for isolation of Salmonella generally followed the

Bacteriological Analytical Manual (B4M) (66) as follows. Individual

shellfish were blended for two minutes at 8000 rpm in a 1:10 dilution

using lactose broth. Crab weighing over 100 grams were blended with

lactose broth and brought to a final volume of 900 grams total volume.

Mullet samples were not blended, but placed whole into individual
,-1,^: r ^-- -- ---^:--*_ --- I ~- 1 r- -L L. 1 -r~t -- --^ *- :, r^ L. -









to analyze the entire fish. All samples were incubated in blender.ja

or bags at 35 C for 24 + 2 hours. Selective enrichment using both

tetrathionate broth and selenite cystine broth followed using a 1-rrL

aliquot into 10 rrL selective enrichment and incubated at 35 C for 24

2 hours. Samples from each tube were streaked onto three selective

plating media: xylose lysine desoxycholate agar (XLD), brilliant gree

agar (EG), and bismuth sulfite agar (BS). These plates were incubate

for 24 + 2 hours at 35 C. Two typical colonies from each medium, if

present, were transferred to triple sugar iron agar (TSI) slants and

lysine iron agar (LIA) slants, which were incubated at 35 C. All

isolates with positive LIA reactions were retained for biochemical

analysis, as stated in the BPM (66). Isolates were purified by the u

of both selective and nonselective media prior to biochemical analysis

Biochemical tests performed and media utilized are shown in Tabl

2. Cultures were discarded if they produced the biochemical reaction

listed in Table 3. All other cultures were subjected to serological

identification using polyvalent antisera (A-I, V.). Positive polyvale

cultures were specifically identified by the laboratories of the

Florida Department of Health and Rehabilitative Services located in

Jacksonville.

vPN Studies

A quantitative study of the presence of salmonellae in oysters a

fresh-water clams (Polymesoda caroliniana) from the west coast harves

location was performed using five 10-rL samples of the same dilution

























TABLE 2
BIOCHEMICAL TESTS PERIFORED FCR ISOLATION CF SALMONELLA

TEST vEDI LM EPLOYED
Presence of urease enzyme Urea broth
Ability to ferment dulcitol Phenol red dulcitol broth
Ability to ferment lactose Brom cresol purple lactose broth
Ability to ferment sucrose Brom cresol purple sucrose broth
Ability to utilize malonate Malonate broth
as a sole carbon source
Ability to utilize citrate Simron's citte agar slant
as a sole carbon source
Ability to convert tryptophan Tryptone broth with Kovac's reagent
to indole
Ability to grow in the presence KCN broth
of potassium cyanide
Production of acid end products vRVP medium with methyl red
Production of acetylmethyl- WRVP with alpha-naphthol and KNH
carbinol
Presence of lysine decarboxylase Lysine decarboxylase broth



























TABLE 3
BASIS FCR DISCARDING ISOLATES

1. Urease present

2. Lactose fermentation, unless
a. malonate test is positive
b. acid slant on TSI

3. Sucrose fermentation, unless acid slant on TSI

4. Growth in KCN, VP positive, and NR negative










the results. Camposite samples of 100 grams for oysters and 200 grz

for clams were prepared to increase the number of individual mollusk

sampled during each analysis.

Storage Studies

A storage study was conducted in June 1982, to observe the

survival of salmonellae in oysters and fresh-water clams under

refrigerated conditions. This study was performed in conjunction wi

the MIPN study previously mentioned. Oysters and fresh-water clams

(Polymesoda caroliniana) from the west coast harvesting location wer

analyzed for salmonellae on days 0, 5, and 10. To simulate comnerci

storage conditions, storage was in an insulated cooler with the lid

open inside a walk-in cooler which maintained a temperature of 3.3 t

7.3 C. Because burlap bags are used commercially for storing oyster

air flow was determined to be advantageous.

Sediment Samples

Five sediment samples taken from a sand bar at the rrmouth of the

Suwannee River were analyzed for Salmonella. This bar was chosen

because of the large number of waterbirds commonly found in the

vicinity.

Aerobic Plate Count

The pour plate method using standard plate count agar was utili

to quantitate the mesophilic aerobic bacteria (APC). This analysis

performed in duplicate using Butterfield's phosphate buffer (66) as

diluent. Composited samples of fifty grams or more were used for th










Optical Co., Buffalo, NY) after incubation at 25 C for five days.

Coliform Analysis

Coliform and fecal coliform analyses were performed simultaneous.

with the aerobic plate count analysis. These analyses were performed i

stated in the Compendium of Methods for the Microbiological Examinati<

of Foods (1) using the most probable number (MPN) technique. One rrL (

each dilution was transferred into either three or five 10-ntL tubes

containing lauryl tryptose broth and incubated at 35 C for 48 + 2

hours. As soon as gas was detected by the use of Durham tubes, a

loopful of broth was transferred to tubes containing 10 nL of either

brilliant green bile 2% broth (BGB) or EC medium. The BGB tubes were

incubated at 35 C for 48 + 2 hours and the EC medium was incubated in

constant temperature bath (Blue M, Blue.Island, IL) at 44.5 C for 48

2 hours. The number of coliforms were quantitated using the 3-tube oi

5-tube MPN table for gassing BGB tubes and the fecal coliform count w,

quantitated using the gassing BE tubes and the vPN tables.

Statistical Analysis

To determine if a correlation was present between salmonellae an<

total coliform, fecal coliform, or aerobic plate count values, three

linear regression were made of the percentage salmonellae recovered

versus total coliform, fecal coliform, and aerobic plate count. The
2
results given are the r values of the linear regressions.














RESULTS AND DISCUSSION

Salmonella Analysis Results

Isolation of Salmonellae From Various Seafoods

Previous studies have shown that the number of seafood.samples

found to contain salmonellae varies with respect to sampling method

the seafood studied. A mean of 11.1% of the oyster homogenate samp.

and 2.3% of the clam homogenate samples were found to contain

salmonellae in a study conducted by Andrews et al. (2, 3). In anotl

study (60), the percentage of positive samples was 11.4% for oyster!

It must be noted that these percentages were all taken from corposil

samples, whereas this research was conducted using individual anima.

as separate samples. Table 4 details the salmonellae recovered from

four seafoods involved in this study. The results vary between seafc

and sampling locations, as well as from previously stated findings <

other investigators.

Ten percent of the west coast oysters that were analyzed contain

Salmonella and 6.7% of the east coast oysters also contained

Salmonella, with the combined average of 8.3% of the oysters contair

salmonellae. These percentages are within the general range of those

found in the previous studies (3, 60). The incidence of Salmonella

clams, however, was much higher than those previously reported (2),

with the west coast incidence of 43.3% and the east coast incidence
























TABLE 4
SALAMNELLAE RECOVERED FRCM FOLR SEAFKODS

SEAFOOD LOCATION OF DTE S*PLES FOS./ PERCENT
HARVEST # SVIPLED

Oysters West Coast July 82 3/30 10.0
East Coast August 81 2/30 6.7
Combined 5/60 8.3

Clams West Coast October 81 13/30 43.3
East Coast August 81 4/30 13.3
Combined 17/60 28.3

Mullet West Coast November 81 0/30 0.0
East Coast January 82 0/30 0.0
Conbined 0/60 0.0

Crabs West Coast February 82 11/30 36.7
East Coast March 82 9/30 30.0
caught 3/12 25.0
purchased 6/18 33.3
Ca-nbined 20/60 33.3

TOTAL 42/240 17.5









salinity at the west coast location due to the movement of fresh wal

discharged from the mouth of the river. The incidence of Salmonelli

crabs was also high, with 36.7% of the west coast crabs containing

Salmonella and Salmonella isolated from 30.0% of the east coast cral

Eighteen of the east coast crabs used for analysis were not caught

the Intracoastal Waterway, but were purchased live in the vicinity <

the harvest location. These showed little difference in percent

recovery of salmonellae from the twelve east coast crabs harvested

the Intracoastal Waterway. Mullet samples were all negative for

Salmonella; however, this may be a result of prior icing, incubation

temperatures, sampling methodology, and/or the fact that mullet are

free-swirrming fish. These factors will be discussed in detail in tl

following pages. If Salmonella is a contaminant as opposed to an

indigenous member of the estuarine environment, these variations in

percent incidence would be expected due to sporatic contamination.

There are several factors which may have influenced the outconx

the salmonellae analyses in this study. First, because refrigerator

known to injure many microorganisms, no attempt was made to cool th<

samples. The short period between harvest and analysis (maximum tinr

four hours) minimized any opportunity for extensive growth of the

microorganisms present. The aerobic plate counts were generally lo\

indicating that competing microorganisms probably had not grown and

were not in such high quantity as to markedly inhibit the growth of

salmonellae. Thirdly, because low numbers of salmonellae were










is recommended mainly for processed foods or foods with low levels ol

contamination. It is possible that increased recovery of any injured

salmonellae occurred because of the use of a preenrichment medium,

which allowed the organisms to overcome any physiological stress.

A difference was seen in the number of salmonellae-positive

samples isolated from each coast. Salmonellae were isolated from a

total of 27 of 120 (22.5%) samples from the west coast location and I

of 120 (12.5%) from the east coast location. This may be partially

explained by the differences in the surrounding environment of the tv

areas, which differed in human population, wildlife, and sewage

disposal techniques. The west coast location is not highly populated

with only a limited number of riverfront vacation homes and septic

tanks for sewage disposal. There were many water birds and animals i

the estuary of the Suwannee River because of the limited human

population. The east coast location, on the other hand, is much more

populated as well as industrialized with sewage treatment plants which

empty into the Intracoastal Waterway. There were fewer water birds a

animals in the surrounding area.










contribute to this incidence. These birds and animals were not as

prevalent at the east coast location, which could substantiate the

higher numbers of seafood being contaminated with salmonellae from the

West Coast. The importance of animal and waterbird transmission is

illustrated by research which indicates the seagull's capacity to

transmit a new serotype of salmonellae into an environment (52).

Differences were also noted between the species studied, with the

major distinction being that no salmonellae were isolated from the

mullet. This may have been due to mullet being the only free-swinrning










whole carcass rinse method to be superior to the thaw water analysis o

the skin method. By using this method, the gastrointestinal tract was

not punctured, and hence the contents of the tract were not accessible

to the preenrichment broth. Caonercially, the gastrointestinal tract i

not usually punctured during cleaning the fish. The gastrointestinal

tract of the other seafoods studied were blended with the other parts

of the animals. This difference in sampling methoddology between the

mullet and the other seafoods studied may have had some bearing on the

outcome of the analysis.

It should be noted that due to an incubator malfunction during th

preenrichment and selective enrichment segments of the analysis for

salmonellae in the east coast crabs, the incubation temperature

fluctuated between 37 and 45 C. Because salmonellae were recovered,

these results are presented.

WPN Studies

Quantitating the number of salmonellae per 100 grams of west coas

oysters using the most probable number (MPN) technique showed that

salmonellae were present in low numbers (Table 5). The highest number

obtained was 2.2 salmonellae per 100 grams of oysters with an upper

confidence limit of 12.6 and a lower confidence limit of 0.1 organisms

per 100 grams. Because of the small size of the oysters (between 3 an

15 grams each), it would be possible for one organism to be present in

every 300 oysters as a lower limit and two organisms present in each

oyster as an upper limit. The number of salmonellae consumed at one



























TABLE 5
NOST F BABLE NUMBER OF SA.M)NELLAE PRESENT IN OYSTERS

DAYS CF SVPLE MPN PER
SITRAPE 100 g

0 A 0.0
B 2.2

5 A 2.2
B 2.2

10 A 0.0
B 2.2










concentration of salmonellae would not frequently cause gastroenteritis

in man. From a commercial standpoint, however, this concentration is

important because of the United States Food and Drug Adminis- tration's

ruling that no salmonellae are allowed in foods.

Quantitation of the number of salmonellae per 100 grams of west

coast fresh-water clams (Polymesoda caroliniana) yielded higher numbers

than those found in the oysters (Table 6), with the highest number

being 16.0 salmonellae per 100 grams of clam with a lower confidence

limit of 3.3 and an upper confidence limit of 52.9 organisms per 100

grams of clam. These numbers are still relatively low, indicating that

few salmonellae would be present in a single serving.

Storage Studies

In order to predict the survivability of the salmonellae under

commercial handling and storage conditions, a ten-day storage study was

performed in conjunction with the NPN study of west coast oysters and

fresh-water clams (Tables 5 and 6). It is apparent from these studies

that salmonellae are capable of survival at 5 C for ten days. This,

along with other studies (33, 39, 65), indicate that salmonellae can

survive typical handling and storage procedures presently practiced in

the seafood industry.

Serotypes

Salmonellae found in this study were of a variety of serotypes

(Table 7). The Centers for Disease Control (CDC) reported a total of

207 different Salmonella serotypes isolated from human sources (12).



























TABLE 6
OMST PROBABLE NULER CF SALM3NELLAE PRESENT IN FRESH-WATER CLAMS

DAYS CF SAMPLE NPN PER
STCRAGE 100 g

0 A 5.1
B 9.2

5 A 5.1
B 2.2

10 A 9.2
B 16.0











/

/
SECLZXuICAL IDENTIFICATION CF iSCLATES fflCM RXR SEARXU

NLM3ER SPIPLES CONTAINING SEIDTYPE
SPECIES CLAMS OYSTERS CRABS PILYNESO!
\VEST EAST 'VESTa EAST WST EAST VEST

S. agona 5 1
S. allandale 1 1
S. anatun 1 I 1
S. bareilly 2 2
S. braenderup 2 1 5 1
S. inverness 1 5 1 3
S. java 3 1
S. rmenchen 8 1 3 6
S. redlands 2
S. tallahassee 1 7 1
S. thormpson I

Edwardsiella tarda 2 1

b West coast oysters are those from the MPN study as vell as the th4
Polymesoda isolates are those from the fresh-water clam WPN study,










1979, which are listed in Table 8 (12). This illustrates the vast

differences in the frequency in which the various serotypes cause

gastroenteritis. The only serotype recovered in the seafoods studied

which is also found in this list, as well as the list of the ten most

frequently reported serotypes from animal sources (Table 8), is S.

agona, which was recovered from a sampling of east coast crabs and fr

west coast sediment. All other serotypes recovered in the seafoods ea

accounted for less than 1% of the reported isolates of Salmonella in

1979. Two serotypes, S. redlands and S. allandale, were not reported

have been recovered from human or nonhunan sources in 1979. Table 9

details Salmonella serotypes isolated from seafoods in other studies.

It can be seen that five of these serotypes were among the ten most

frequently reported serotypes from either nonhuman or human sources i

1979.

Edwardsiella tarda, another member of the family Enterobac-

teriaceae, was found in west coast clams, both fresh-water clams

(Polymesoda) and salt-water clams (Mercenaria) (Table 7). Edwardsiell

exhibits disease patterns and biochemical characteristics similar to

Salmonella. E. tarda is a motile, hydrogen sulfide producing, lactos

negative, and indole positive organism (16) that has been isolated fr

patients having acute gastroenteritis, enteric fever, septicemia,

meningitis, wound infections, and surgical incisions (16, 68). The

ecology of E. tarda is also similar to Salmonella, having a wide

geographic distribution and the ability to infect numerous animal
C.--- ; / \ Q:__..__ /t --C -:_: 1 --: 4..: -- *.-.J +; *., ;, 1--+-
















TABLE 8
THE TEN M3ST FREQUENTLY REPORTED SALCtDNELLA SEOTIYPES FRCM HLMWA A

HLMN
Rank Serotype Percent Rank
1 typhimurium** 32.6 1 ty
2 enteritidis 8.5 2 ag
3 heidelberg 8.0 3 de
4 newport 6.2 4 in
5 infants 4.6 5 pa
6 agona 3.5 6 he
7 saint-paul 2.8 7 or
8 typhi 2.1 8 rm
9 montevideo 2.0 9 ch
10 oranienburg 1.9 v
10 we
WO

reported to CDC
** includes var. copenhagen
Source: Salmonella Surveillance Annual Sunmary























TABLE 9
SALMONELLA SEIOTYPES PREVIOUSLY REPORTED FRCM SEARFODS
SERDLYPE CLAMS OYSTERS OYSTERS SHRIMP
S. anatun X
S. blockley X
S. braenderup X
S. cerro X
S. derby X X
S. heidelberg X
S. hilversam X
S. infants X
S. lexington X
S. manhattan X
S. newport X
S. paratyphi B X
S. senftenberg .X
S. tennessee X
S. thompson X X
S. typhimurium X X
S. virchow X

Reference 2 3 61 18









Although this study did not involve methodology, it is of

importance to discuss the effectiveness of recovering all the

salmonellae present in the seafoods analyzed. Successful isolation of

salmonellae is a complex multifactorial procedure and is dependent upc

the food studied, the medium used, and the laboratory personnel

performing the analysis. For the purpose of this study, the procedure

recommended in the BPM (66) was the most practical in that a standard

was needed. Higher recovery may have been possible by the use of an

alternative method, such as elevated temperatures or the use of more

selective media.

The problem with the isolation method was first identified during

an analysis of east coast crabs. In March 1982, due to incubator

failure, the preeenrichment and selective enrichment sections of the

analysis were incubated at a higher temperature, fluctuating between 3

C and 45 C. The results of this analysis revealed that 30% of the crat

contained salmonellae. Two months later when the analysis was repeated

no salmonellae were recovered. In August 1982, when the analysis was

performed for the third time, salmonellae again were not recovered.

This confirms the finding of Miller and Koburger (unpublished data)

that elevated temperatures provide higher recovery of salmonellae front

oysters (Salmonellae were recovered from 33/84 (39%) of the aliquots Z

41 C and 43 C as opposed to 11/42 (26%) when using 35 C.). While

studying clams and oysters, Andrews et al. (2) tested the sensitivity

nf thp RPM mpthne- ujhirh rivpalprf that as few as eight to ten









analyze water, Cherry et al. (14) were able to detect salmonellae in

60% more samples than when using the culture method employing elevate

temperatures. Because of the lack of a method which is optimal, exa(

numbers and comparison of recovery between studies should be

interpreted cautiously.

Relationship to Total Coliforms, Fecal Coliforms,

and Aerobic Plate Count

A relationship between total coliforms, fecal coliforms or aerol

plate count and the recovery of salmonellae was not apparent, with r

values of 0.15, 0.02, and 0.11, respectively (Table 10). Although the

are relatively few data in this study, these results emphasize the

inadequacy of relying solely upon an indicator system for determining(

the safety of a seafood. An extreme case can be seen in the results

the analyses of west coast clams. The total coliform level of 5

organisms per gram was low, the aerobic plate count of 260 organisms

per gram was far below the guideline of 500,000 organisms per gram

(42), and the fecal coliform count was zero. The percentage recover]

of salmonellae, however, was 43%. Although not quite as marked, easi

coast clams exhibited the same behavior, with the aerobic plate coun-

at 1100 organisms per gram, coliforms at 2 organisms per gram, and

fecal coliforms at 2 organisms per gram. Thirteen percent of the

samples analyzed contained salmonellae from this group. Andrews et i

(2) found total coliform and fecal coliform counts in clam meat to be

higher than those found in this study. Total coliform counts ranged



















AEROBIC PLATE G(UNT, TOTAL (LIFCRM, FECAL


TABLE 10
CGLIFCRIM, AND SAUvN ELLAE RECOVERED FI~M FRXR SEAFOODS


TOTAL
C(LIFIRMS*
MPN/g


FECAL
CDLIFC~TS*
NPN/g


SAUMDNELLAE
samples pos./ Percentage
# sampled


West coast oysters
East coast oysters

West coast clams
East coast clams

West coast mullet
East coast mullet

West coast crabs
East coast crabs
caught
purchased


July 82
August 81

October 81
August 81

November 81
January 82

February 82
March 82


* these numbers represent means of duplicate analyses


SEAFOOD


ATE


APC*
CFU/g


1100 Est
800 Est

260
1100 Est

4.4 X 105
5.1 X 10
5
7.5 X 105
1.3 X 108
7.9 X 104
2.6 X 10


78
306

4700
28
0
56


3/30
2/30

13/30
4/30

0/30
0/30


11/30
9/30
S 3/12
6/18


0.2
27

4
28
0
56


10.0
6.7

43.3
13.3


36.7
30.0
25.0
33.3










clams.

Many studies have compared the incidence of salmonellae in

seafoods with the total coliform and fecal coliform counts of the

harvest waters. When studying 214 clams, Andrews et al. (2) found th;

it was not until the total coliform PN of the waters exceeded 200

organisms per 100 rL that salmonellae were recovered from the clams.

The range of total coliform NPN of water where salmonellae were

recovered was from 490 to 11,000 organisms per 100 mL with the fecal

coliform range from 33 to 2300 organisms per 100 mL. When studying 5:

oysters, however, Andrews and coworkers (3) found one salmonellae-

positive oyster which was harvested from water having a total coliforr

NPN of 11 organisms per 100 mL water and a fecal coliform level of le!

than 1.8 organisms per 100 mL water. Of their oyster samples found

positive for salmonellae, 7.5% had total coliform counts within the

approved range of a maximum of 70 organisms per 100 rL water. In

addition, a total of 2.4% of the oysters found positive for salmonella

had fecal coliform levels within the approved range of a maximum of li

organisms per 100 mL overlying water.

A measureable difference can be seen in these studies of the

correlation of coliforms to salmonellae, which clearly indicates more

study should be directed in this area. All that is now known is that

low numbers or even the absence of coliforms or fecal coliforms in 10(

iL seawater or in the seafood meat may not insure the absence of

pathogenic microorganisms in mollusks harvested from these waters.










Although salmonellae are known to be ubiquitous in the

environment, their presence has been consistently thought to be

associated with fecal contamination. Being a human and animal ent ric

pathogen, this assumption seens reasonable. It is thought that

salmonellae cannot survive out of the human or animal body for extended

periods of time, and that the natural environment would therefore not

be a reservoir for the organism. Several studies related to water

quality have questioned these concepts. In 1967, Fair and Morrison

(22) were able to isolate salmonellae from a stream of high quality

where the total coliform count was 30 organisms per 100 nL water.

Throughout the entirety of the stream, there were no known additions of

human excreta. Salmonellae were isolated from all seven sampling

stations and the source of contamination was thought to be due to wild

and domestic animals. This article initiated the theory that naturally

occurring potable surface waters do not exist. A 1971 study agreed with

this theory when salmonellae were easily recovered within 350 ft. of

the origin of a supposedly "unpolluted" mountain stream where the only

reasonable source of pollution appeared to be terrestrial or aquatic

wildlife (14). Hendricks and Morrison (27) showed that salmonellae and

other enteric bacteria can not only maintain their populations in

polluted or unpolluted river water at temperatures as low as 10 C, but

also were capable of multiplication. This fact may help to explain the

ease with which salmonellae have been recovered from various streams.

Based on these studies as well as his own studies, Cherry et al. (14)





organisms to determine the safety of food!

limited, showed that in several cases, su(

determining the presence of fecal coliform

of the presence of Salmonella in these sez

fecal coliforms, this may be another indi(

tochthonous nature of Salmonella.

The United States Department of Agric

whether salmonellae in foods are "added" ,

a natural part of domestic animals and fi.

court decision ban importation of Indian !

salmonellae, because salmonellae are thou

thereby caulsing the product to he adiilter;


practices in the processing plant. Salmonellae can


I


....... ./ vw,


Y


----- -- -- -------


--


ist clams,

indication

e. absence of

sible au-




) deal with

other they are

a current

tains

ibstances,

; case, the


"`^












In the United States, salmonellae in red meats and poultry have

been thought to be inevitable. The National Research Council stated in

1975 (49) that complete elimination of salmonellae from domestic

animals was not feasible at that time, but that the development of

infection-free breeding stock, elimination of contaminants from feed,

improved conditions for holding animals before slaughter, and more

careful slaughtering practices are measures that can reduce the hazard.

Three Scandinavian countries, Denmark, Finland, and Sweden, have

attempted to raise salmonellae-free domestic meat animals by the use of

salmonellae-free feed ingredients and strict destruction or quarantine

of those animals contaminated with salmonellae (56). Various surveys

have indicated that these countries have a low incidence of Salmonella

in poultry, with the percent contamination ranging from 1% to 7%. The

United States percent contamination is estimated at approximately 30%

(70). Unfortunately, the impact of these programs has in no way

eliminated human salmonellosis. Reported cases per 100,000 population

are still high, with 10 cases per 100,000 people in Denmark, 44 in

Finland, 43 in Sweden, and 14 in the United States. These figures may

be distorted since they are reported cases and not true incidences.

Although the incidence may be reduced by the employment of these

precautionary measures, salmonellosis cannot be completely eradicated

at this time. Improved methods for the eradication of salmonellae would

be costly to the population on the whole, and therefore would be met










Because salmonellae were found in three of the tour seafoods

studied, it is necessary to evaluate the significance of these

findings. In some cases, the number of samples in which salmonellae

were recovered was quite high, which causes one to question why

regulatory agencies have not placed restrictions on these products,

and, more importantly, why there is not a higher incidence of

salmonellosis caused by seafood products.

Most of these products will be cooked before eating. Members of

the genus Salmonella are killed during ordinary cooking times and

temperatures in foods having a high moisture content. In most

instances, foods that are to be heat processed can be made completely

safe with respect to salmonellae contamination without impairing the

quality of the food (7). The specific times and temperatures required

to kill salmonellae depend upon the number of bacteria present, the pH

of the food, the species of Salmonella, and the water activity of the

food. If the product is not cooked, this first defense fails. Such is

the case when eating raw clams and oysters.

The probability of salmonellae surviving the digestion process is

complicated by the time of consumption. Because the pH of the stomach

is lowest just after the meal begins, more bacteria are killed at this

time. An inversely proportional relationship between the amount of

gastric acid and the number of bacteria in the stomach has been found

(63). Because raw oysters and clams are most often consumed as

appetizers, the pH of the stomach would inhibit the majority of the






46

A second factor involved in the consumption of raw oysters and

clams is the fact that alcohol often accompanies these appetizers..

Beer and wine have low pH's, which would be deleterious to the

salmonellae before the stomach is reached as well as aid the stomach in

producing acid conditions. The alcohol itself, in large quantities,












capacity to survive the host defenses and produce more severe symptoms.

There is also evidence that virulent species of Salmonella multiply

intracellularly whereas avirulent species do not (16). This would also

increase the incidence of particular species over others. Addition-

ally, after studying the lesser virulence of Salmonella isolates from

streams in New York State, Dondero et al. (20) concluded that it is not

inconceivable that the Salmonella genus in the environment may

frequently contain nonpathogenic strains.

The wide variety of serotypes isolated from these seafoods may

also indicate that there is no single source of contamination. Eleven

serotypes were recovered from these products, with many of the samples

containing more than one serotype. As many as three serotypes were

recovered from an individual animal. The low numbers of salmonellae

recovered indicates that the contamination is minimal, and the number

of serotypes would indicate that the contamination is from a variety of

sources.














SUVMvRY AND G NCLUSIONS

Analysis for the presence of Salmonella in four seafoods harvested

in the state of Florida was performed. Samples from both the east and

west coasts of Florida were used as being representative of the sea-

foods harvested in Florida. Aerobic plate counts, total coliform

estimates, fecal coliform estimates, storage studies, and quantitative

studies accompanied these analyses.

Results indicated that salmonellae were present in oysters, clams

and crabs in percentages of 8.3%, 28.3%, and 33.3% of the samples

analyzed, respectively. Salmonellae were not recovered from the mullet

samples. Sediment samples taken in the vicinity of the west coast

harvest location also contained salmonellae. Quantitation of the

salmonellae by the use of the MPN technique yielded between 2.2 and

16.0 salmonellae per 100 grams of oysters and fresh-water clams.

Storage tests showed that salmonellae are capable of surviving in

oysters and fresh-water clams at refrigerated temperatures (3-7 C) for

at least ten days. Aerobic plate counts, total coliform estimates, and

fecal coliform estimates did not correlate (r2 = 0.11, 0.02, and 0.15,

respectively) with the incidence of salmonellae.

Based on the results of this study, the following conclusions can

be drawn:

1. The incidence and distribution of Salmonella in three of the










member of the microflora of the estuarine environment. This is based

on the following observations: Higher numbers of salmonellae were

recovered from the west coast location, which is less populated with

humans and more populated with water birds and animals. With the

variety of serotypes recovered, it is probable that there was no sing

source of contamination. Sediment analysis revealed that salmonellae

may be present in the environment as well as in the seafoods studied.

2. The method of treatment of the samples and the isolation meth,

used for analyzing salmonellae will cause differences in the results

obtained. The lack of refrigeration, minimal competing flora, the us,

of a preenrichment medium, and the analysis of individual animals may

have increased the recovery of salmonellae in the study. More sensiti

methods of recovery, such as the use of elevated temperatures, may al

have increased recovery in this category of foods.

3. Under commercial storage and handling conditions, Salmonella

will be able to survive in oysters at least ten days. Temperatures fr

5 to 10 C do not kill salmonellae, although the salmonellae do not

appear to multiply readily at these temperatures.

4. Many factors may influence the lack of confirmed reports of

seafoods transmitting salmonellae to humans. The serotypes isolated a

not those that are known to cause the majority of the human

salmonellosis cases reported in the United States. The virulence of

these serotypes, therefore, may not be as great as those serotypes

commonly isolated from cases of human salmonellosis. Many of these










The pH of the stomach is lower at this period, which will be

detrimental to the salmonellae. Lastly, variation in susceptibility

among different individuals within the population is well known, and

therefore the low numbers of salmonellae recovered from these seafoc

may not affect many people. All these factors combined may account

the fact that these salmonellae-containing seafood have not been oft

involved in huran salmonellosis under normal conditions of harvesting

processing, storage and consurrption.














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BIORAPHICAL SKETCH

Margaret Bury Fraiser was born on April 29, 1959, in Yreka,

California. She graduated from A & M Consolidated High School, College

Station, Texas, in May, 1977. She attended Sterling College in

Sterling, Kansas, and Universitaet Salzburg in Salzburg, Austria, and

received a Bachelor of Science degree in youth leadership from Sterling

College in December, 1980. She enrolled as a graduate student in the

Food Science and Human Nutrition Department at the University of

Florida in January, 1981. She expects to receive a Master of Science

degree in food science and human nutrition with a minor in microbiology

in December, 1982.











I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a thesis for the degree of Master of
Science.


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