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In reference to the following dissertation:
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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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
KKNCWLEDXGWENTS
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 CF CONTENTS
PAGE
A KNO< C LEDGBENTS ..................................................... ii
LIST CF TABLES....................................................... v
LIST CF FIGURES .....................................................vi
ABSTRCT. ...................................................... vii
INTRODUCTION ....................... .................................
The Genus Salmonella............................................. 3
Disease ............. .......................... ............4
Prevention ................................... ..........4
Infective Dose ................................. .. ...... 5
Incidence........ ........ ................................. 6
Ecology ..................... ................ .10
Microflora of Seafoods............................. ...... 13
Relationship of Salmonella to Indicator Organisms................15
Survival of Salmonella During Storage.......................... 16
MTERIALS AND nETHDDS............................................... 18
Materials.............. ..... ......... ....... .. .. ............ 18
Sampling Plan.......................... ... ......................18
Samples....o. ........ ....... ......... ... ........... ...... ......19
Salmonella Analysis.................. ......................... 19
VPN 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......o............................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
SULMRY AND 3NCLLU6IONS ............................................. 48
iii
PAGE
BIBLIOGRAPHY ........................................................51
BIOGRAPHICAL S
LIST OF TABLES
TABLE PAGE
1. MIDFLCR RA CF CYSTERS AND BLLE CRABS............................. 14
2. BIOCHYMIICAL TESTS PERFCRED FTR ISCLATICN CF SALMONELLA .......... 21
3. BASIS FCR DISCARDING ISOLATES....................................22
4. SALM3NELLAE RECOVERED FRCM FOLR SEAFIODS.. .....................26
5. NMST ROBABLE NULBER F SAILNNELLAE PRESENT IN OfSTERS...........31
6. M3ST PROBABLE NUMBER CF SALIDNELLAE
PRESENT IN FRESH--WATER CLAMS ................................. 33
7. SALM NELLA SEROTYPES PREVIOUSLY REPORTED FICM SEAFODS........... 34
8. SERCLOGICAL IDENTIFICATION CF ISCLATES
FRCM FKR SEAICODS AND SEDINENT................................36
9. THE TEN M3ST FREQUENTLY REPORTED SALM3NELLAE
SERDTYPES FRCM HVMAN AND NONHLMAN SOXCES, 1979 ..................37
10. AEROBIC PLATE (DLNT, TOTAL CCLIFCRM, FECAL CCLIFCRM, AND
SAMIDNELLAE RECOVERED FCM IFOLR SEAFOODS......................... 40
LIST OF FIGURES
FIGURE PAGE
1. REPORTED SALWVNELLA ISOLATIONS FRCM HLMNS
BY WEEK IN THE LNITED STATES, 1974-1980..........................7
2. REF RTED SALMNELLAISCLATIONS HiCM HUINS
BY PE IN THE UNITED STATES, 1980..............................9
3. MODE OF TRANSMISSION CF SAIMDNELLOSIS ...........................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
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 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
viii
INTRODUCTION
Seafoods accounted for 8.7% of the reported foodborne disease
outbreaks in the United States in 1979 (10). With the dockside value of
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 found
in seafoods (e.g., nonagglutinating Vibrio cholerae and Clostridiun
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%
of all confirmed outbreaks. The prevalence of the disease can only be
2
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
the seafoods harvested in Florida, the seafoods were harvested from two
approved areas in Florida; the west coast location was at the mouth of
.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
e.g. S. typhi and S. paratyphi
2. Salmonellae pathogenic to animals only
e.g. S. pullorum and S. abortus equi
3. Salmonellae pathogenic to man and animals
It is this last classification which contains the vast majority of ser-
otypes. In man, these species are the major cause of food infections
(6). The genus is further classified into groups A through I based on
their O (somatic) antigens. The serotypes (or species) within each of
these groups are based on the H (flagellar) antigens.
Salmonellae (50) are gram negative, generally motile, generally
produce abundant amounts of hydrogen sulfide, and generally do not
ferment lactose or sucrose. They are capable of growth between 7 and
45 C with optimal growth at 35 to 37 C. The pH range for growth is 4.1
to 9.0 with an optimum of pH 6.5 to 7.5. The lowest water activity at
which growth occurs is 0.94, which is equivalent to a 9% NaCI solution.
The D value, which is the amount of time at a particular temperature
needed to reduce the number of organisms by one log10 cycle, is given
as 0.4 minutes at 140 F for egg products. The D values can vary greatly
4.
among strains and can.be influenced strongly by pH, sugar or salt
content, and the physiological state of the cells. The genus has shown
an ability to survive for relatively long periods of time in chilled,
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, which 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
salmonellae. The symptoms usually include fever, cramps, diarrhea, and
vomiting, which are caused by invasion of the muscosal layer of both
the small and large intestines. The gastroenteritis is self-limiting
with a duration of three to four days and the degree of severity of the
disease is variable, depending on the infecting dose, serotype, and the
health of the individual. Treatment includes administration of fluids
and electrolytes. Occasionally septicemia will develop, resulting in a
localized infection of the gall bladder, spleen, lungs, or urinary
tract (50). In 0.5 to 2% of the salmonellosis patients a carrier state
develops which may last for years (24). This is often characterized by
a chronic infection of the gall bladder.
Prevention
Prevention of salmonellosis is mainly accomplished by the adher-
ence to public health standards. Proper sewage disposal, pasteurization
5
of milk, maintenance .of unpolluted water supplies, and exclusion of
carriers 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
various 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
salmonellae per gram, and ice cream had an infective dose of 2
salmonellae per gram. It can be seen from the above epidemiological
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
United States since the beginning of surveillance in 1950 (11) and has
continued to increase in recent years as shown in Figure 1 (11). Some
of this increase must be attributed to more effective monitoring and
reporting of the disease, although the extent of this contribution can-
not be established. In cases per 100,000 population, however, a slight
decrease was seen in 1980, from 15.06 in 1979 to 14.88 in 1980 with
33,715 cases of salmonellosis reported in 1980. It is estimated that
approximately 1% of the total salmonellosis cases are reported (13),
indicating the total cases in 1980 to be well over 3 million. In Figure
1, the seasonal fluctuation in the incidence is apparent, with the sum-
mer months having higher incidences. This may be the result of the in-
crease in thermally mishandling of foods which occurs more readily in
2 -
4 700
.-
0
'L 600
w soo
CO 500
400
>J
300
200
< 100
rA ; lU J 4's 0111O r sono rur 77".Tnl=" .-'. "O'J' IT-C "s oJ rUnMAM oJ J SCN AJJ
1974 1975- 1976 1977 1978 1979 1980
SEach po'nt r.prespt ts the w~vpklv averaqr number)* r.l ,olatps for tho month
FIGURE 1
REPORTED SALMONELLA ISOLATIONS FROM HUMANS
BY WEEK IN THE UNITED STATES, 1974-1980
8
the sumner months. Salmonella is now the most cormnon etiological agent
of foodborne disease in the United States.
The reported isolations of Salmonella from humans by age in the
United States -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 the
number of deaths attributed to the disease. Deaths due to salmonellosis
reported in the United States in the nine year period from 1970 to 1979
totaled 645 with the highest number of deaths in one year being in 1970
and 1971 with 81 deaths in each year. The lowest number of deaths was
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 other
microbiological genera. This is in contrast to the 1976 figures, which
show Staphylococcus aureus responsible for more outbreaks and cases
(9). In 1976, there were 356 salmonellosis cases reported involving 23
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
aureus reported (9). These figures illustrate the importance of Salmo-
nella as a foodborne pathogen in another area of the developed world.
70-
300
60-
--- MALE 200-
S-- .- FEMALE 2
50- 100-
<1' 2 4 5 6 7 8 9 10 11
. 40- AGE (MONTHS)
I- I
0
30-
20-
10
0-4 5-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80
AGE GROUP (YEARS)
'PER 100,000 POPULATION
FIGURE 2
REPORTED SALMONELLA ISOLATIONS FROM HUMANS
BY AGE IN THE UNITED STATES, 1980
Ecology
Salmonella is ubiquitous in the environment, being one of the. most
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 to
both animals and man. The mode of transmission in 500 salmonellosis
outbreaks from 1966 to 1975 is given in Figure 3 (57). The most common
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 plants,
which are often contaminated with salmonellae, are the main ingredients
of animal feeds. Another reason for the ubiquity of Salmonella is the
apparent capacity of the genus to survive for prolonged periods in the
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 in
the recovery of two serotypes, S. agona and S. hadar. Before 1970, S.
agona had been reported in man only twice. One of the first isolations
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
sources. In 1976 it ranked third among the most frequently isolated
INCLUDES OVER 50 VEHICLES WHICH INDIVIDUALLY
CAUSED LESS THAN 3% OF OUTBREAKS
FIGURE 3
MODE OF TRANSMISSION OF SALMONELLOSIS
CONTAMINATED AI
AND CFRTILIZER
ILTRY INI CTPD ON
FEW INFECTED ON
NV/AL FEED
FARM
MANY INFECTED IN LAIRAGE,POULTRY
PROCESSING PLANTS AND ABATTOIRS
V
BULK EGG PRODUCTSl
IFOWL AND MEATI
CO sJ 5J mED
MAN AND WIS 4
4r f
LOCAL
UNPASTEURPJZED
MIK PRODUCTS
<
DOMESTIC ANIMALS
FIMGRE 4
ANIMAL-TO-MAN TRANSMISSION OF SALMONELLOSIS
I
B
13
serotypes from human sources. S. hadar has been accidentally imported
by a turkey breeder from the United Kingdom. A rapid increase in.the
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. The
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 the
marine environment. It is realized that turtles are a major carrier of
Salmonella and therefore the Food and Drug Administration has
prohibited interstate shipment of turtles. Nine serotypes were isolated
from water in aquaria housing turtles (40). In another study, seagulls
were found to contribute a new serotype to a water environment (52).
Reptiles, also present in the aquatic environment, have been found to
be contaminated with salmonellae while living in Swiss zoos. Thirty
percent of the reptiles in the Basel zoo, 43% of those in the Bern zoo,
and 30% of reptiles in the Zurich zoo were positive for salmonellae
(54). Salmonellae have also been isolated from oysters (3, 60), clams
(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, many
overt pathogens and opportunistic pathogens have been reported to be
present in seafoods. Table I gives a brief summary of the microbes that
have been isolated from oysters and blue crabs. These organisms have
not been reported in mullet or clams.
TABLE 1
MICRDFLCRA OF THE SEAFOODS STUDIED
ORGANISM SEAFOOD LOCATION
Vibrio parahaemolyticus oyster Mississippi Sound
blue crab Chincoteague Bay
oyster laboratory
oyster Long Island Sound
blue crab unknown
Pseudomonas spp. blue crab unknown
oyster Long Island Sound
oyster laboratory
oyster Galveston Bay
Flavobacterium-Cytophaga oyster Long Island Sound
blue crab unknown
oyster Galveston Bay
oyster laboratory
Achromobacter-Alcaligenes oyster Long Island Sound
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 Bay
Clostridiun botulinum type F blue crab York River, VA
REFERENCE
38
35
46
47
59
59
47
46
67
47
59
67
46
47
46
67
59
46
67
59
67
67
67
67
29
69
15
Relationship of Salmonellae to Indicator Groups
The use of specific groups of microorganisms to indicate the pos-
sible presence of pathogenic organisms had its beginning near the turn
of the twentieth century (30). The purpose of these indicator organ-
isms was to detect the presence of sewage contamination in the potable
water supply. Twenty years later, indicators were found to be of use in
the shellfish industry, where bacteriological standards for harvest
waters were being developed. Advantages of utilyzing these indicator
organisms included the relative ease and short duration of the analy-
ses. In 1946, the first quantitative guideline for coliform counts in
water from harvesting areas was established, which placed a limit of
seventy organisms per 100 rrL water. This was modified slightly in 1965,
and in 1974 the fecal coliform limit of fourteen organisms per 100 rrL
water was established. Presently either limit is accepted by the Food
and Drug Administration (30).
Problems have arisen with the use of indicator organisms, since
there is no single group which contains all the desirable characteris-
tics of an indicator. One of the first problems arose in 1961 when a
study by Tennant and Reid (64) found that 26.7% of the coliform organ-
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 fecal
coliforms to the presence of pathogenic organisms (18, 41, 55, 58) as
well as the poor correlation of pollution to pathogenic organisms (14,
55). Attempts have been made (2, 3, 55) to determine the best indicator
group to use in the seafood industry; these studies have yielded con-
trasting results. In general, however, fecal coliforms have been found
to be slightly better indicators than total coliforms (2, 3, 41, 60).
16
Other emerging microbial problems have also complicated interpre-
tation of the results of indicator organisms. No correlation has been
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 advantageous
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 analy-
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 microorganisms.
This is reflected by the FDA's limit of 230 fecal coliforms per 100 rL
of shellfish, which is sixteen times greater than the limit allowed
for waters. It is generally agreed that shellfish have the ability to
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
ammrals 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
It is of interest to the seafood industry to consider the surviv-
ability of Salmonella species during commercial handling and 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 meat (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 Carnission 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.
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 Carnission 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.
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 Carnission 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.
19
Samples
Thirty samples of each seafood (oysters, clams, mullet, and crabs)
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
plastic bags containing lactose broth in a 1:10 proportion. The bags
were heat-sealed, the samples shaken, and the bags incubated. This
19
Samples
Thirty samples of each seafood (oysters, clams, mullet, and crabs)
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
plastic bags containing lactose broth in a 1:10 proportion. The bags
were heat-sealed, the samples shaken, and the bags incubated. This
20
method was used because of the larger size of the mullet and the desire
to analyze the entire fish. All samples were incubated in blender.jars
or bags at 35 C for 24 + 2 hours. Selective enrichment using both
tetrathionate broth and selenite cystine broth followed using a 1-mL
aliquot into 10 rL 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 green
agar (EG), and bismuth sulfite agar (BS). These plates were incubated
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 use
of both selective and nonselective media prior to biochemical analysis.
Biochemical tests performed and media utilized are shown in Table
2. Cultures were discarded if they produced the biochemical reactions
listed in Table 3. All other cultures were subjected to serological
identification using polyvalent antisera (A-I, V.). Positive polyvalent
cultures were specifically identified by the laboratories of the
Florida Department of Health and Rehabilitative Services located in
Jacksonville.
MPN Studies
A quantitative study of the presence of salmonellae in oysters and
fresh-water clams (Polymesoda caroliniana) from the west coast harvest
location was performed using five 10-nL samples of the same dilution
incubated in 90 rL lactose broth (51). The analyses were performed in
TABLE 2
BIOCHEMICAL TESTS PERICRVED FCR ISOLATION CF SALMONELLA
TEST
Presence of urease enzyme
Ability to ferment dulcitol
Ability to ferment lactose
Ability to ferment sucrose
Ability to utilize malonate
as a sole carbon source
Ability to utilize citrate
as a sole carbon source
Ability to convert tryptophan
to indole
Ability to grow in the presence
of potassium cyanide
Production of acid end products
Production of acetylmethyl-
carbinol
Presence of lysine decarboxylase
NED ILM EPLOYED
Urea broth
Phenol red dulcitol broth
Brom cresol purple lactose broth
Brom cresol purple sucrose broth
Malonate broth
Simnon's citte agar slant
Tryptone broth with Kbvac's reagent
KCN broth
VRVP medium with methyl red
VRVP with alpha-naphthol and KOH
Lysine decarboxylase broth
22
TABLE 3
BASIS FCR DISCARDING ISOLATES
1. Urease present
2. Lactose fermentation, unless
a. mralonate 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
23
duplicate and the most probable number technique was used to quantitate
the results. Composite samples of 100 grams for oysters and 200 grams
for clams were prepared to increase the number of individual mollusks
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 with
the MPN study previously mentioned. Oysters and fresh-water clams
(Polymesoda caroliniana) from the west coast harvesting location were
analyzed for salmonellae on days 0, 5, and 10. To simulate commercial
storage conditions, storage was in an insulated cooler with the lid
open inside a walk-in cooler which maintained a temperature of 3.3 to
7.3 C. Because burlap bags are used commercially for storing oysters,
air flow was determined to be advantageous.
Sediment Samples
Five sediment samples taken from a sand bar at the rmuth 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 utilized
to quantitate the mesophilic aerobic bacteria (APC). This analysis was
performed in duplicate using Butterfield's phosphate buffer (66) as the
diluent. Composited samples of fifty grams or more were used for the
oysters and east coast clams while the mullet, crabs, and west coast
clams were larger and therefore not composited. The colonies on the
23
duplicate and the most probable number technique was used to quantitate
the results. Composite samples of 100 grams for oysters and 200 grams
for clams were prepared to increase the number of individual mollusks
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 with
the MPN study previously mentioned. Oysters and fresh-water clams
(Polymesoda caroliniana) from the west coast harvesting location were
analyzed for salmonellae on days 0, 5, and 10. To simulate commercial
storage conditions, storage was in an insulated cooler with the lid
open inside a walk-in cooler which maintained a temperature of 3.3 to
7.3 C. Because burlap bags are used commercially for storing oysters,
air flow was determined to be advantageous.
Sediment Samples
Five sediment samples taken from a sand bar at the rmuth 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 utilized
to quantitate the mesophilic aerobic bacteria (APC). This analysis was
performed in duplicate using Butterfield's phosphate buffer (66) as the
diluent. Composited samples of fifty grams or more were used for the
oysters and east coast clams while the mullet, crabs, and west coast
clams were larger and therefore not composited. The colonies on the
23
duplicate and the most probable number technique was used to quantitate
the results. Composite samples of 100 grams for oysters and 200 grams
for clams were prepared to increase the number of individual mollusks
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 with
the MPN study previously mentioned. Oysters and fresh-water clams
(Polymesoda caroliniana) from the west coast harvesting location were
analyzed for salmonellae on days 0, 5, and 10. To simulate commercial
storage conditions, storage was in an insulated cooler with the lid
open inside a walk-in cooler which maintained a temperature of 3.3 to
7.3 C. Because burlap bags are used commercially for storing oysters,
air flow was determined to be advantageous.
Sediment Samples
Five sediment samples taken from a sand bar at the rmuth 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 utilized
to quantitate the mesophilic aerobic bacteria (APC). This analysis was
performed in duplicate using Butterfield's phosphate buffer (66) as the
diluent. Composited samples of fifty grams or more were used for the
oysters and east coast clams while the mullet, crabs, and west coast
clams were larger and therefore not composited. The colonies on the
24
plates were counted with the aid of a Quebec Colony Counter (American
Optical Co., Buffalo, NY) after incubation at 25 C for five days.
Coliform Analysis
Coliform and fecal coliform analyses were performed simultaneously
with the aerobic plate count analysis. These analyses were performed as
stated in the Compendium of Methods for the Microbiological Examination
of Foods (1) using the most probable number (MPN) technique. One rrL of
each dilution was transferred into either three or five 10-nL 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 mL 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 a
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 or
5-tube MPN table for gassing BGB tubes and the fecal coliform count was
quantitated using the gassing BC tubes and the MPN tables.
Statistical Analysis
To determine if a correlation was present between salmonellae and
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
results given are the r2 values of the linear regressions.
results given are the r values of the linear regressions.
24
plates were counted with the aid of a Quebec Colony Counter (American
Optical Co., Buffalo, NY) after incubation at 25 C for five days.
Coliform Analysis
Coliform and fecal coliform analyses were performed simultaneously
with the aerobic plate count analysis. These analyses were performed as
stated in the Compendium of Methods for the Microbiological Examination
of Foods (1) using the most probable number (MPN) technique. One rrL of
each dilution was transferred into either three or five 10-nL 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 mL 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 a
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 or
5-tube MPN table for gassing BGB tubes and the fecal coliform count was
quantitated using the gassing BC tubes and the MPN tables.
Statistical Analysis
To determine if a correlation was present between salmonellae and
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
results given are the r2 values of the linear regressions.
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 and
the seafood studied. A mean of 11.1% of the oyster homogenate samples
and 2.3% of the clam homogenate samples were found to contain
salmonellae in a study conducted by Andrews et al. (2, 3). In another
study (60), the percentage of positive samples was 11.4% for oysters.
It must be noted that these percentages were all taken from composite
samples, whereas this research was conducted using individual animals
as separate samples. Table 4 details the salmonellae recovered from the
four seafoods involved in this study. The results vary between seafoods
and sampling locations, as well as from previously stated findings of
other investigators.
Ten percent of the west coast oysters that were analyzed contained
Salmonella and 6.7% of the east coast oysters also contained
Salmonella, with the combined average of 8.3% of the oysters containing
salmonellae. These percentages are within the general range of those
found in the previous studies (3, 60). The incidence of Salmonella in
clams, however, was much higher than those previously reported (2),
with the west coast incidence of 43.3% and the east coast incidence of
13.3%. Although there is a marked difference between the samples from
each coast, many factors may account for this difference, among which
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 and
the seafood studied. A mean of 11.1% of the oyster homogenate samples
and 2.3% of the clam homogenate samples were found to contain
salmonellae in a study conducted by Andrews et al. (2, 3). In another
study (60), the percentage of positive samples was 11.4% for oysters.
It must be noted that these percentages were all taken from composite
samples, whereas this research was conducted using individual animals
as separate samples. Table 4 details the salmonellae recovered from the
four seafoods involved in this study. The results vary between seafoods
and sampling locations, as well as from previously stated findings of
other investigators.
Ten percent of the west coast oysters that were analyzed contained
Salmonella and 6.7% of the east coast oysters also contained
Salmonella, with the combined average of 8.3% of the oysters containing
salmonellae. These percentages are within the general range of those
found in the previous studies (3, 60). The incidence of Salmonella in
clams, however, was much higher than those previously reported (2),
with the west coast incidence of 43.3% and the east coast incidence of
13.3%. Although there is a marked difference between the samples from
each coast, many factors may account for this difference, among which
SALIONELLAE
SEAFtOD LOCATION OF
HPRVEST
Oysters West Coast
East Coast
Combined
Clams West Coast
East Coast
Combined
Mullet West Coast
East Coast
Conbined
Crabs West Coast
East Coast
caught
purchased
Combined
TABLE 4
RECOVERED FROM FOR~ SEAFDODS
IATE
SWPLES FOS./
# SAVMLED
July 82
August 81
October 81
August 81
November 81
January 82
February 82
March 82
3/30
2/30
5/60
13/30
4/30
17/60
0/30
0/30
0/60
11/30
9/30
3/12
6/18
20/60
42/240 17.5
PERCENT
10.0
6.7
8.3
43.3
13.3
28.3
0.0
0.0
0.0
36.7
30.0
25.0
33.3
33.3
TOTAL
are the differences in size of the clams and the apparent lower
salinity at the west coast location due to the movement of fresh water
discharged from the mouth of the river. The incidence of Salmonella in
crabs was also high, with 36.7% of the west coast crabs containing
Salmonella and Salmonella isolated from 30.0% of the east coast crabs.
Eighteen of the east coast crabs used for analysis were not caught in
the Intracoastal Waterway, but were purchased live in the vicinity of
the harvest location. These showed little difference in percent
recovery of salmonellae from the twelve east coast crabs harvested in
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-swirming fish. These factors will be discussed in detail in the
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 outcome of
the salmonellae analyses in this study. First, because refrigeration is
known to injure many microorganisms, no attempt was made to cool the
samples. The short period between harvest and analysis maximumn time of
four hours) minimized any opportunity for extensive growth of the
microorganisms present. The aerobic plate counts were generally low,
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
anticipated, individual animals were used to draw a larger number of
smaller samples, thereby increasing the probability of recovering any
28
salmonellae present. Lastly, a preenrichment procedure was used, which
is recommended mainly for processed foods or foods with low levels of
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 15
of 120 (12.5%) from the east coast location. This may be partially
explained by the differences in the surrounding environment of the two
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 in
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 and
animals in the surrounding area.
To investigate the possibility of Salmonella being present in the
entire estuarine environment rather than only in the seafoods studied,
five sediment samples were analyzed for Salmonella from a sand bar
approximately fifty meters from the harvest location of the west coast
oysters. The incidence of salmonellae in three of the five sediment
samples taken from a sand bar in the vicinity of the harvest location
of the clams and oysters on the West Coast indicates salmonellae may be
widely distributed in the environment. In this particular area, the
29
high number of water birds and animals that populate the area may
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-swimning
fish studied, with the other seafoods being "bottom dwellers." Stream
bottom sediment was found to have a higher recovery rate of salmonellae
than surface waters (26), which would indicate that "bottom dwellers"
would be more likely to contain salmonellae. Additionally, mullusks are
known to be filter feeders, which results in the accumulation of
microorganisms from their environment (65). No such report is known
for fin fish. Another possibility for the lack of salmonellae found in
the mullet is the temperature of storage and of incubation. The length
of time the mullet were on ice is unknown, which may have been
detrimental to salmonellae, if present. In addition, the total volume
of fish and broth during the preenrichment section of the mullet
analysis was so great that several hours were required for all thirty
samples to reach the incubation temperature of 35 C. An additional
possibility for the lack of salmonellae found in the mullet is the
sampling method used. The plastic bag procedure was adopted to provide
analysis of the total surface area of the fish. This method can be
substantiated by the research involving sampling methods for detection
30
of salmonellae in raw chicken carcasses. D'Aoust et al. (19) found the
whole carcass rinse method to be superior to the thaw water analysis or
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. Camnercially, the gastrointestinal tract is
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 the
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 coast
oysters using the nmst 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 and
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
meal of raw oysters would, therefore, be minimal. With the infective
dose being dependent on the consumer, food handling and preparation,
,^~ i. .-.,
TABLE 5
NOST F BABLE NUMBER OF SA.M)NELLAE PRESENT IN OYSTERS
DAYS CF SAVPLE MPN PER
SITRPGE 100 g
0 A 0.0
B 2.2
5 A 2.2
B 2.2
10 A 0.0
B 2.2
32
time factors, and the nature of the food, it would appear that this
concentration of salmonellae would not frequently cause gastroenteritis
in nan. 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).
These 207 serotypes represent approximately 12% of the more than 1800
known Salmonella serotypes and variants. The ten most frequently
TABLE 6
MOST PfOBABLE NLUEER CF SALDNELLAE PRESENT
IAYS CF
STCRAPE
0
5
10
SAMPLE
A
B
A
B
A
IN FRESH-WATER CAMS
NPN PER
100 g
5.1
9.2
5.1
2.2
9.2
16.0
/
TABLE 7
SERILOGICAL IDENTIFICATION OF SLATES FIRCM RLR SEAIOCDS AND SEDIMENT
NUM3ER SPIPLES CONTAINING SERITYPE
SPECIES CLAVS OYSTERS CRABS LYESODA SEDIMENT
VEST EAST 'ESTa EAST\ WEST EAST VEST IEST
S. agona 5 1 3
S. allandale 1 1
S. anatuin 1 1
S. bareilly 2 2
S. braenderup 2 1 5 1
S. inverness 1 5 1 3
S. java 3 1
S. muenchen 8 1 3 6
S. redlands 2
S. tallahassee 1 7 1
S. thompson I
Edwardsiella tarda 2 1
b West coast oysters are those from the MPN study as vwll as the the individual animal study.
Polymesoda isolates are those from the fresh-water clam IPN study.
35
recovered serotypes accounted for over 72% of the isolates reported in
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 from
west coast sediment. All other serotypes recovered in the seafoods each
accounted for less than 1% of the reported isolates of Salmonella in
1979. Two serotypes, S. redlands and S. allandale, were not reported to
have been recovered from human or nonhuman 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 in
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). Edwardsiella,
exhibits disease patterns and biochemical characteristics similar to
Salmonella. E. tarda is a motile, hydrogen sulfide producing, lactose
negative, and indole positive organism (16) that has been isolated from
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
species (68). Because of these similarities, indole positive isolates
were analyzed for E. tarda.
TABLE 8
THE TEN MDST FREQUENTLY REPORTED SALMtNELLA SERDTYPES RCMA HLMN AND NONHLMAN SOUCES*, 1979
HLMN
Serotype
typhirmuriun**
enteritidis
heidelberg
newport
infants
agona
saint-paul
typhi
montevideo
oranienburg
Percent
32.6
8.5
8.0
6.2
4.6
3.5
2.8
2.1
2.0
1.9
Rank
1
2
3
4
5
6
7
8
9
N3NHLMN
Serotype
typhimurirLu**
agona
derby
infants
panama
heidelberg
oranienberg
montevideo
cholerae-suis
v. kunzendorf
weltevreden
worthington
reported to CDC
** includes var. copenhagen
Source: Salmonella Surveillance Annual Sumnary (12)
Rank
1
2
3
4
5
6
7
8
9
10
Percent
22.2
12.4
6.1
5.1
4.7
3.5
2.8
2.8
2.6
1.7
1.7
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
Isolation Methodology
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 upon
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 37
C and 45 C. The results of this analysis revealed that 30% of the crabs
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 from
oysters (Salmonellae were recovered from 33/84 (39%) of the aliquots at
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
of the BAM method, which revealed that as few as eight to ten
salmonellae per 100 grams of artificially contaminated shellfish could
consistently be recovered, which indicated an acceptable level of
39
sensitivity. By the use of the fluorescent antibody (FA) test to
analyze water, Cherry et al. (14) were able to detect salmonellae in
60% nmre samples than when using the culture method employing elevated
temperatures. Because of the lack of a method which is optimal, exact
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 aerobic
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 there
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 of
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 recovery
of salmonellae, however, was 43%. Although not quite as marked, east
coast clams exhibited the same behavior, with the aerobic plate count
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 al.
(2) found total coliform and fecal coliform counts in clam meat to be
higher than those found in this study. Total coliform counts ranged
from 13 to 1720 organisms per gram and fecal 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
41
from 4.9 to 330 organisms per gram when salmonellae were detected in
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 that
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 rL with the fecal
coliform range from 33 to 2300 organisms per 100 mL. When studying 539
oysters, however, Andrews and coworkers (3) found one salmonellae-
positive oyster which was harvested from water having a total coliform
NPN of 11 organisms per 100 mL water and a fecal coliform level of less
than 1.8 organisms per 100 nL 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 salmonellae
had fecal coliform levels within the approved range of a maximum of 14
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 100
rL seawater or in the seafood meat may not insure the absence of
pathogenic microorganisms in mollusks harvested from these waters.
Therefore, the absence of pathogenic organisms from approved waters
based on current analysis methods cannot be assumed.
42
Salmonella as a Contaminant or an Autochthon
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 seems 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)
concluded that the possibility should now be entertained that these
bacteria may exist as free-living organisms multiplying under natural
43
conditions. This would indicate that salmonellae are autochthonous,
being indigenous in the estuarine environment.
Although there is some question as to the use of indicator
organisms to determine the safety of foods, this data, although
limited, showed that in several cases, such as the west coast clams,
determining the presence of fecal coliforms was not a good indication
of the presence of Salmonella in these seafoods. Due to the absence of
fecal coliforms, this may be another indication of the possible au-
tochthonous nature of Salmonella.
The United States Department of Agriculture has had to deal with
whether salmonellae in foods are "added" substances or whether they are
a natural part of domestic animals and fish. For example, a current
court decision ban importation of Indian shrimp, if it contains
salmonellae, because salmonellae are thought to be added substances,
thereby causing the product to be adulterated (4). In this case, the
salmonellae were thought to be present because of poor sanitary
practices in the processing plant. Salmonellae can thus be classified
as added, which changes the definition of adulteration to any substance
which "may render the food injurious to health." If the substance is
not added, the product is then considered adulterated only if the
substance would "ordinarily render the food injurious to health."
Salmonellae is not "ordinarily injurious" since its dangers can be
averted through proper cooking and storage. In this way, salmonellae do
not adulterate the food if they are naturally occurring (56). In 1980,
2.7% of the Gulf Coast shrimp in a study were found to be contaminated
with Salmonella (18), but since this contamination is thought to be
inherent in the product, Gulf Coast shrimp is not confiscated. This
44
discrepancy in rulings concerning Salmonella indicates the complexity
of the situation.
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
with opposition.
45
Significance of the Findings v
Because salmonellae were found in three of the four 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
salmonellae.
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,
would also be inhibitory.
In spite of the deleterious effects of heat treatment, pH of the
stomach, and the consumption of alcohol, salmonellae are able to
survive in many instances. In the period of 1963 to 1973 in the United
States, five comnon-source epidemics of salmonellosis (one involving
twelve separate outbreaks) were attributed to fish and shellfish (7).
Therefore, the presence of these salmonellae, even in low numbers, must
not be ignored.
Relatively low numbers of salmonellae were found in oysters and
fresh-water clams. This would indicate that the salmonellae are not in
large enough quantity to cause severe symptoms. However, milder
symptoms may occur and would go unreported more often than severe
symptoms. In addition, as the salmonellae encounter various host
defense mechanisms, a portion of the original population will be
killed. If the total number of salmonellae were low to begin with,
these defense mechanisms might destroy the entire population. This
would account for the low numbers of reported cases of salmonellosis
due to consumption of contaminated seafood products.
Another interesting factor in the findings of this research is the
serotypes that were isolated. As stated earlier, these were varied and
many were not the common serotypes isolated from human and nonhuman
sources (Table 7). These rarer serotypes may be seldom isolated from
47
humans because the virulence may differ between serotypes, with the
more virulent serotypes being isolated more often because of their.
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 nay
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 CaNCLUSIONS
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 (r = 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
four seafoods studied would indicate that either Salmonella as a
contaminant is present due to a relatively large and consistent amount
49
of environmental pollution or that Salmonella may be an autochthonous
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 single
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 method
used for analyzing salmonellae will cause differences in the results
obtained. The lack of refrigeration, minimal competing flora, the use
of a preenrichment medium, and the analysis of individual animals may
have increased the recovery of salmonellae in the study. More sensitive
methods of recovery, such as the use of elevated temperatures, may also
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 from
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 are
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
coanonly isolated from cases of human salmonellosis. Many of these
products are cooked, as are red meats and poultry which also often
contain salmonellae, and the cooking process destroys the salmonellae.
50
If the seafoods are not cooked, they are usually eaten as appetizers.
The pH of the stomach is lower at this period, which will be
detrimental to the salmonellae. Lastly, variation in susceptibility
anong different individuals within the population is well known, and
therefore the low numbers of salmonellae recovered from these seafoods
may not affect many people. All these factors combined may account for
the fact that these salmonellae-containing seafood have not been often
involved in human salmonellosis under normal conditions of harvesting,
processing, storage and consumption.
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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.
ohn A. Kobur eC, Chaian
I yfessor of Wod Science
and Human Nutrition
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.
3 as/L. Obl ger
Professor ofvFood Scien e
and Human Nutrition
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.
Samuel R. Farrah
Associate Professor of Micro-
biology and Cell Science
This thesis was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Master of Science.
December 1982
Dean, 6/lege of Agriculto
Dean for Graduate Studies and
Research
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