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

Material Information

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

Subjects

Subjects / Keywords:
Clams ( jstor )
Coasts ( jstor )
Crabs ( jstor )
Fecal coliforms ( jstor )
Food ( jstor )
Oysters ( jstor )
Salmonella ( jstor )
Salmonella infections ( jstor )
Seafoods ( jstor )
Shellfish ( jstor )
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Fishes -- Microbiology -- Florida ( lcsh )
Food Science and Human Nutrition thesis M.S
Salmonella ( lcsh )
Seafood -- Microbiology ( lcsh )
Shellfish -- Microbiology -- Florida ( lcsh )
Suwannee River, FL ( local )
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:
Copyright Margaret Bury Fraiser. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
028555649 ( ALEPH )
09263082 ( OCLC )
ABU5880 ( NOTIS )

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










BY
MARGARET BURY RAISER






















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














PCKNJ.EDGEVENTS

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 vrd processor knowledge.













TABLE OF CfNTENTS

PAGE
ACKNC LEO HVE TS . i i

LIST OF TABLES . v

LIST OF FIGURES . vi

ABS C . . . vii

INTR DLCTION .
The Genus Salmonella Storag. 3
Disease . . . . 4 Prevent ion . . . 4 Infective Dose . . 5 Inci dence . . 6 Ecology . . . . . . . .24
Microflora of Seafoods u. . . . . 13 Relationship of Salmonella to Indicator Organismso. 15 Survival of Salmonella During Storage . .16

WTERIALS AND ETpeDS . . . . . . .18
Materials. .o. . . . .o.o. .38
Sampling Plan. . . . . o . .o . .o.18 Samples . P . o . . . . . . 19 Salmonella Analysis.o.oh. 19
/NPN Studies . o. 20 Storage Studies Finding. .o.-o-. . . 2o4
Sediment Samples. . . 23 Aerobic Plate Count . . _ . o. o. . . . . 23 Col iform Analysis .o. . o oo . o. . . . . . . . _24
Statistical Analysis .o. . . . . . 24

RESULTS AND DISCUSSION.-o. . - --25
Salmonella Analysis Results .o. 25
Isolation of Salmonellae from Various Seafoods . _25 NvPN Studies . . . .o .o. . . .30 Storage Studies. . . . . . . . . 32 Sero types. . . . . . .o. 32 I solIa t ion Met hodol1ogy . . o. o38
Relationship to Total Coliform, Fecal Coliform, andAerobic Plate Count. . . . . . . o .o.39
Salmonella as a Contaminant or an Autochthon .o. 42 Significance of the Findings . o. _ 45

SLm*RY AND Go3NCL5 INS . .o.48

iii








BIBLIC APHY . . .51













LIST CF TABLES


TABLE PACE

1. MIICRJCRA OF (CSTERS AND BLLE CRABS . 14 2. BIOCF IEICAL TESTS PERFR\ED FrR ISCLATICN CF SALMONELLA . 21 3. BASIS ICR DISCARDING ISCLATES . 22 4. SALWNELLAE RECXERED F t XR SEAFXODS . 26 5. NOST REMABLE NLBER CF SALMCiELLAE PRESENT IN OYSTERS . 31

6. NOST PRBABLE NUMBER CF SALWNELLAE PRESENT IN FRESH-\wTER CLAMS . 33 7. SALMa.A SEROTYPES IFEVI ISLY REMTED FiO S . 34

8. SERCLOGICAL IDENTIFICATION CF ISCLATES RA X)LR SEACCDS AND SEDIvENT . 36

9. THE TEN &DST FREQLENTLY REF(RTED SALUMELLAE SEIDTYPES FRCM HWAN AND NONHLMNN SXRCES, 1979 . 37 10. AEI IC FATE (XLNT, TOTAL CCLIFCRM, FECAL CCLIFCRi, AND
SALMDNELLAE REVERED FI2 FOLR SEAI(CDS . 40














LIST OF FIGURES


FIGURE PAGE

1. PERTED SALWNELIA ISaLATICNS HRCM HUMVNS
BY WE IN THE .NITED STATES, 1974-1980 . 7

2. RE:RRTED SAILNELLAISCILATIONS RRv HIMNS
BY ,AZ IN THE UNITED STATES, 1980 . 9 3. MME CF TRANSMISSION CF SAIMNELL.OSIS . 11 4. ANIML-TIO-AN TRANSMISSION OF SALM3IELLOSIS . 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 SA.MONELLA IN FOIR 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 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 est coast harvest location also contained salmonellae. The serotypes recovered were those which are less frequently reported to the Centers for Disease Control as agents in humn salmonellosis, which may reflect their lesser virulence. Quantitation of salmonellae in oysters and fresh-water clams using the rmst 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.


Chai


viii













INTICDLCTICN

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.&., nonagglutinating Vibrio cholerae and Clostridium botulinwi), 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 wvuld 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 Salmonella in four fresh seafoods commercially harvested in Florida: oysters (Crassostrea virginica), clams (Mercenaria mercenaria), striped mullet (Mugil cephalus), and blue crabs (Callinectes sapidus). Salmonella was chosen as a representative of the pathogenic microflora because 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 5uwannee 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 rrost 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 pathogenicity 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 serotypes. 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 0 (somatic) antigens. The serotypes (or species) within each of these groups are based on the H (flagellar) antigens.

Salrmonellae (50) are gram negative, generally rmtile, 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 timie 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 occasionally 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 adherence to public health standards. Proper sewage disposal, pasteurization






5

of milk, maintenance _of unpolluted water supplies, and exclusion of carriers as food handlers are conrrxn 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, Wien McCullough and Eisele reported doses in the range of 10 5 to 10 9 bacteria were needed for humann 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 reported 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 contamination 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 reduction in'the number of viable organisms after freezing, which would increase the number of viable salmonellae in the neat at the tine 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, whiich mray 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 me~ats 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 Departme~nt of Agriculture has made an exception, allowing salmonellae to be present in red mreats and poultry sold in the United States if the salmo~nellae are considered inherent in the product.

Incidence

The incidence of salnKonellosis 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 rrvre effective monitoring and reporting of the disease, although the extent of this contribution cannot 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 summr r months having higher incidences. This may be the result of the increase in thermally mishandling of foods which occurs rmre readily in




































Un -UU
z
0
700.
.-J
0
'L 600 .
0

C 500 z 400
>

L 300


200
cc


II



1974 1975' 1976 1977 1978 1979 1960

* Each 13Ont rpresPt.is the wpkiv avevaq numbl,, rt qolalps for fho mn ,th








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






















70


300.

60
- MALE 200
-. - .- FEMALE2

50- I1OO



'1' 1 2 3'4 6'7' a' 9 10'11 40- AGE (MONTHS)
I.
_1
0
- 30




20









0 T I
0-4 5-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 tO AGE GROUP (YEARS) PER 100,000 POPULATION





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






-8

the sumner mnths. Salmnella is now the rmst corrrnon etiological agent of foodborne disease in the United States.

The reported isolations of Salmonella from hiumns 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 several factors, including low irmunity, 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 increase is due to aging which causes a decrease in resistance to such organisms.

Another indication of the severity of salmnellae 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 Salmnella species exceeded those caused by all other microbiological genera. This is in contrast to the 1976 figures, which show Staphylococcus aureus responsible for rmre 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 Salmonella as a foodborne pathogen in another area of the developed world.







Eco I ogy

Salmonella is ubiquitous in the environment, being one of the, most

widely distributed overt pathogens in the vrld. The main reason for this ubiquity is the easy transmissibility of the organism. Transmission 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 comnon 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 Salmonella-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, 5. 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
















lCO0ArA(lo1INA:0RE A


AND 'CRTILIZER






I LTr ICy
FEW IN17ECTGED ON


V/,IA. rECD


F7ARM


MANY INFECTED IN IERAGEPOULTRY PROCE3SING PLANTS AND ABATTOIRS

V


BULK EGG PRODUCTSl


IFOWL AND MATI


C o r,,4 s IJrv Em D MAN AND WlS


4nrf
LOCAL
IUNPASTEUPJZr-DI 1 POCU~rJ


DOMESTIC ANIMALS


FI dTE 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 sumnary of the microbes that have been isolated from oysters and blue crabs. These organisms have not been reported in mullet or clams.









TABLE I
MICROFLCRA OF THE SEARFS STUDIED CRGAN I -W SEAFOD 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


REFEREE
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

Relations-hip of Salrnonellae to Indicator Groups

The use of specific groups of microorganisms to indicate the possible presence of pathogenic organisms had its beginning near the turn of the twentieth century (30). The purpose of these indicator organisms 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 analyses. In 1946,'the first quantitative guideline for coliform counts in water from harvesting areas was established, whiich placed a limit of seventy organisms per 100 rrL water. This was rrxodified slightly in 1965, and in 1974 the fecal coliform limit of fourteen organisms per 100 rri 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 characteristics 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 organisms 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 mde (2, 3, 55) to-determine the best indicator group to use in the seafood industry; these studies have yielded contrasting 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 interpretation 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 pathogen. Also, the increased incidence of enteroviruses in oysters has caused Ellender et al. in 1980 (21) to suggest the use of a viral analysis 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 rrL 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 mmals 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 shellfishgrowing areas.

Survival of Salmonellae During Storage

It is of interest to the seafood industry to consider the survivability 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 neat (62), and in a study involving "soul foods", S. typhimuriurn survived for five days at 10 C in all foods studied, which included collard greens, field peas, sweet potatoes, and semiprocessed 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 conrrmercial 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. typhirnurium 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 ND NDETHODS

Materials

All microbiologcial culture redia 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 anirml 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 Camission 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

Samp I es

Thirty samples of each seafood (oysters, clams, nizilet, and crabs) mere 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 nnuth 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 rrade to cool the samples (with the exception of the mullet) because of the short time.- span involved between collection and analysis.

Salmonella Analysis

The m-ethod for isolation of Salmounella generally followed the Bacteriological Analytical Manual (BAM) (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 we-re blended with lactose broth and brought to a final volurr of 900 gram s total volure. Mullet samples were not blended, but placed whole into individual plastic bags containing lactose broth in a 1:10 proportion. The bags we-re heat-sealed, the sam-ples shaken, and the bags incubated. This





20

rrethod 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-rL 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 green agar (BG), and bismuth sulfite agar (BS). These plates were incubated for 24 + 2 hours at 35 C. T%o 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, Vi). 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 (Polynesoda caroliniana) from the west coast harvest location was performed using five 10-rnL samples of the same dilution incubated in 90 rrL lactose broth (51). The analyses were performed in

























TABLE 2
BIOCHEMvICAL TESTS PERFRVED F(R ISOLATION CF SALMMELLA


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 acetylmethylcarbinol
Presence of lysine decarboxylase


NED I WL LOYED Urea broth
Phenol red dulcitol broth Brom cresol purple lactose broth Brom cresol purple sucrose broth Malonate broth

Sirmnon's citte agar slant

Tryptone broth with Kovac's reagent

KCN broth

NVRVP medium with methyl red /VRVP with alpha-naphthol and INOZ(-


Lysine decarboxylase broth






22



















TABLE 3
BASIS ICR DICARDING ISLXATES I. Urease present

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

3. Sucrose fermentation, unless acid slant on TSI

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






23

duplicate and the mst probable number technique was used to quantitate the results. Caposite samples of 100 grams for oysters and 200 grams for clams were prepared to increase the number of individual rmllusks 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 IVPN 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 comercially 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 comnonly found in the vicinity.

Aerobic Plate Count

The pour plate method using standard plate count agar was utilized to quantitate the mesophilic aerobic bacteria (AFC). This analysis was performed in duplicate using Butterfield's phosphate buffer (66) as the diluent. Ccrnposited 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 comrposited. 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 mst probable number (MPN) technique. One rrL of each dilution was transferred into either three or five 10-mL 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 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 tPN table for gassing B(B 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 thepercentage 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 DISCUSS ICN

Salmoxnella Analysis Results

Isolation of Salmonellae From Various Seafoods

Previous studies have shown that the number of seafood samples

found to contain salmoxnellae varies with respect to sampling method and the seafood studied. A me~an of 11 .1% of the oyster homogenate samples and 2.3% of the clamn homo~genate samples mere 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 Salmoxnella 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 my account for this difference, amoung which

























SALMDNELLAE

SEAFtCM LOCATION OF
HARVEST

Oysters West Coast
East Coast
Canbined

Clams West Coast
East Coast
Ca bined

Mullet West Coast
East Coast
Canbined

Crabs West Coast
East Coast
caught
purchased
Ca-nbined


TABLE 4
RECOVERED FRCM fOR SEARFlS


DATE


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 rre-thodology, and/or the fact that mullet are free-swirrming 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 (rmximmn 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 preenricihnent procedure was used, which is reconmnded 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 preenriclinent rreditim, which allowed the organisms to overcome any physiological stress.

A difference was seen in the number of salmonellae-positive

samples isolated from each coast. Salmnellae 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 humn 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 ma~ny 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 rmCre 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-swinming 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 salmo~nellae in raw-chicken carcasses. D'Aoust et al. (19) found the whole carcass rinse n-ethod to be superior to the thaw water analys-is 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 preenrichrnent broth. Carrnercially, 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 animalIs. This difference in sampling rrethoddology 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 preenriclinent and selective enrichmnent segments of the analysis for salmonellae in the east coast crabs, the incubation temperature fluctuated between 37 and 4~5 C. Because salmonellae were recovered, these results are presented.

WvN Studies

Quantitating the number of salmovnellae per 100 grams of west coast oysters using the nnst probable number (M'N) 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 salmo~nellae consumed at one nreal of raw oysters vould, therefore, be minimal. With the infective dose being dependent on the consumer, food handling and preparation,


























TABLE 5
NOST FABLE NUMBER OF SAN.MNELLAE PRESENT IN OYSTERS

DAYS CF SA'VLE IMPN PER
S100 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 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-ater 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 (CDlC) 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
NOST HWBABLE NLMER OF SL.MDNELLAE F1,ESENT


DAYS CF


0 5


10


SALE


A
B

A
B

A


IN FRESH-WATER CLAMS NPN PER 100 g

5.1
9.2 5.1
2.2 9.2
16.0











/
/

TABL.E 7
SERIOLOICAL IDENTIFICATION OF iSCLATES RA RLR SEARFDS AND SEDIMENT

NIM3ER SAMPLES CONTAINING SEYIYPE , SPECIES CLAMS OYSTERS CRABS FOLYNE SEDIMENT
\;EST EAST '%STa EAST EEEST EAST ST MST

S. agona .5 1 3
S. allandale I I
S. anatun I i
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. thonpson I

Edwardsiella tarda 2

b West coast oysters are those from the MPN study as vll as the the individual animal study. Polymesoda isolates are those from the fresh-water clam WPN 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 nonhumn or human sources in 1979.

Edwardsiella tarda, another member of the family Enterobacteriaceae, 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 RERCRTED SAVCtN'ELLA SER TYPES HRCPA HUWN AND NZ)NHMNN S)U S*, 1979


HLMN
Serotype
typhinurium** enteritidis heidelberg newport infantis 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


NDNHLM
Serotype
typhimur itf*l agona
derby infantis panama heidelberg oranienberg rmntevideo cholerae-suis v. kunzendorf weltevreden vorthington


* reported to QJC
** includes var. copenhagen
Source: Salrmonella Surveillance Annual Swmnary (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
SALMDNELLA SEIPYTYPES PREVIOUSLY REFRTED FICM SEAFDS
SERDYYPE CLAMS OxSTERS CYSTERS S-RIME
S. anatu X
S. blockley X
S. braenderup X
S. cerro X
S. derby X X
S. heidelberg X
S. hilversun X
S. infantis 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. typhimuriun X X
S. virchow X

Reference 2 3 61 18







Isolation MethodoloyAlthough 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 mused, and the laboratory personnel performing the analysis. For the purpose of this study, the procedure recornended in the BPM (66) was the mst practical in that a standard was needed. Higher recovery may have been possible by the use of an alternative rnethod, such as elevated temperatures or the use of rmre selective redia.

The problem with the isolation method was first identified during an analysis of east coast crabs. In March 1982, due to incubator failure, the preeenriclment 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% mre samples than when using the culture method employing elevated temperatures. Because of the lack of a method which is optinal, 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
2
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 GXNT, lOTAL (LIFCWv, FECAL


TABLE 10
GL I RC1M, AND SA."ELLAE RECOVERED 1=U2v FOLR SEAFOODS


TOTAL
CDLIIFCRS* M'N/g


FECAL
cLIFCM*
MPN/g


SALADELLAE
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


DATE


AFC*
CFU/g


1100 Est 800 Est

260
1100 Est

4.4 X 105 5.1 X 106

7.5 X 105 1.3 X 108 7.9 X 10 2.6 X 10 8


78 306

4700
28 0
56


3/30 2/30

13/30
4/30

0/30 0/30


11/30
9/30 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








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 coliformN'PN of the waters exceeded 200 organisms per 100 mrL that salmonellae were recovered from the clams. The range of total coliform NFN of water where salmnellae were recovered was from 490 to 11,000 organisms per 100 rrL with the fecal coliform range from 33 to 2300 organisms per 100 rrL. When studying 539 oysters, however, Andrews and cowvrkers (3) found one salmonellaepositive oyster which was harvested from water having a total coliform NPN of 11 organisms per 100 mrL water and a fecal coliform level of less than 1.8 organisms per 100 niL water. Of their oyster sarrples found positive for salmonellae, 7.5% had total coliform counts within the approved range of a maximum of 70 organisms per 100 rrL 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 rmximum of 14 organisms per 100 rrL 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 rmL seawater or in the seafood meat may not insure the absence of pathogenic microorganisms in rmllusks harvested from these waters. Therefore, the absence of pathogenic organisms from approved waters based on current analysis rrethods cannot be assured.






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 ent9r ic pathogens 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 Miorrison

(22) wre able to isolate salmonellae from a stream of high quality where the total coliform count was 30 organisms per 100 riL water. Throughout the entirety of the stream, there were no known additions of

humran 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" rmuntain 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 environrrent.

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 nay be another indication of the possible autochthonous nature of Salmonella.

The United States Department of Agriculture has had to deal with

whether salmoxnellae in foods are "added" substances or whether they are

a natural part of domestic animals and fish. For exarrple, a current court decision ban importation of Indian shrim-p, 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 mere 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 "Iray 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 shrim-p 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 vas 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 Svwden, have atterrpted 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 vay eliminated humn salmonellosis. Reported cases per 100,000 population are still high, with 10 cases per 100,000 people in Denmark, 44 in Finland, 43 in Svwden, 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 wvuld be costly to the population on the whole, and therefore would be met with opposition.






45

Significance of the Findings

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, nvre 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 rmoisture content. In most instances, foods that are to be heat processed can be made ccxrpletely safe with respect to salmonellae contamination without impairing the quality of the food (7). The specific times and temperatures required to kill salmo~nellae depend upon the number of bacteria present, the pH of the food, the species of Salmnella, 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, rmore 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 rnmst often consumed as appetizers, the pH of the stomach would inhibit the mjority of the salmonel lae.






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 wvuld 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 comon-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 vuld account for the low numbers of reported cases of salmnellosis 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 htran and nonhumn sources (Table 7). These rarer serotypes may be seldom isolated from






47

hurans because the virulence mray differ between serotypes, with the rore virulent serotypes being isolated more often because of their. capacity to survive the host defenses and produce rrore severe symptoms. There is also evidence that virulent species of Salmonella multiply intracellularly whereas avirulent species do not (16). This wuld also increase the incidence of particular species over others. Additionally, 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 mrey 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 mere 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.














SL~vMRY AND Ga ULLSIONS

Analysis for the presence of Salmonella in four seafoods harvested in the state of Florida was performed. Samples from both the east and %est coasts of Florida %ere used as being representative of the seafoods 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
2
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:

I. The incidence and distribution of Salmonella in three of the four seafoods studied mould 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 terrmperatures.

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 corrmonly 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 among 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 huran salmnellosis under normal conditions of harvesting, processing, storage and consurrption.














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56

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BIOXMAPHICAL S(ElTli

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 Collegein 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 humn 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.


a

iihn A. Koburfe, Chaian IPjfessor of Tod Science
and Humn 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 s /. 0b1 ger Prof essor ofvFood Scienqe and Humn 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 Microbiology 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


Dfean, r lege of Agricultof




Dean for Graduate Studies and Research




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