THE USE OF BACTERIOPHAGE IN

DETECTING FOODBORNE BACTERIAL PATHOGENS

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University o f Guelph

by

STACY JANE FAVRIN

In partial fdfihent of requirements

for the degree

Master of Science

May, 1998

OStacy Jane Favrin, 1998

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THE USE OF BACTERIOPHAGE IN DETECTING FOODBORNE BACTERIAL PATHOGENS

S t acy Jane Favrin University of Guelph, 1998

Advisor: Dr. M.W. GrifEths

Salmonella is the second leading cause of foodbome illness in most developed counties

causing diarrhea, crampg vomiting and often fever. Poultry, eggs and rnilk are fiequently

implicated as vehicles of infection. Simple, rapid, and specific methods are required for the

detection of Salmonella in foods. A bacteriophage assay combining the use of

irnmunornagnetic separation WS) with fluorescence or absorbance measurements, was

developed for the detection of Salmonella Enteritidis. The sensitivity of the assay in pure

suspensions, skimmed milk powder, ground beef and liquid whole egg was determined. The

MS-bactenophage assay was able to detect 3 CFU/g or ml present initidy in each of these

food samples. The assay was generdy speciflc for serogroup D Sulmonel(a; with Salmonellu

Typhimurium also testing positive. The IMS-bacteriophage assay was adapted to the

detection of E- col2 0157:H7. The assay was able to detect 2.5 CFU/g E. coli 01 57:H7

present initially in ground beef.

Thanks to God, for the perspective that lmowing Him provides, and for His strength and faithfidness which helped me to persevere. This work has been completed for His glory.

1 would Like to express my sincere thanks to my advisory cornmittee. Special thanks to my advisor, Dr. Mansel Gritnths, for his support, guidance and great suggestions. Thanks to Dr. Heidi Schraft for her support and the opporhinity to be a teaching assistant in Food Microbiology 42-323. It was a great learning experience, and a highlight of my program. The basic concepts of this study were introduced to me by Dr. Sabah Jassim and for that, and his help and guidance early in rny program, 1 am grateful.

Thanks to Dr. Joseph Odurneru and Dr. Massimo Marcone for being a part of my examination committee.

Thanks to Dr. Ted Heying and GEM Biomedical for fùnding this project, and for the opportunity to be involved in such exciting and relevant research.

Special thanks to my husband, Steve, whose patience and support, especially at the end, wiil always be appreciated. Thanks also to my parents for their interest and support throughout my Master's studies.

1 am also grateful to Jinni, Larry, Doug and Thomas, for the good laughs and the help they each have provided me in reaching this goal.

TABLE OF CONTENTS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Salmonella Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Background: 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. Classification: 1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Salmonellosis 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Clinical signs: 2

1.2.2. Infective dose: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .2.3. Incidence and costs: 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.4. Trends in Salmonella infection: 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.5. Sources of infection: 7

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .3 Ecology and control: 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Detection of Salmonella 16

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 .4.1. Conventional methods: 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .4.2. Rapid methods: 17

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .4.3. Immunomagnetic separation: 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.Bacteriophage 24

. . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 . Lytic and temperate bacteriophage: 24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 S.2. Phage-typing: 24

. . . . . . . . . . . . . . . . . . . . 1 .5.3. Bacteriophage based detection methods: 26 . . . . . . . . . . . . . . . . . . 1.5.3.1. Luminescence and reporter genes: 26 . . . . . . . . . . . . . . . . . . 1 5 3 .2 . Fluorescent bacteriop hage assay: 28

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.3. Metabolic inhibition: 28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.4. BIND assay: 29

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Fluorescence 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6.1. Principles: 29

1.6.2. Fluorescence as a means of determining ce11 viability: . . . . . . . . . 30 . . . . . . . . . . . . . . . . . . . 1.6.3. Direct Epifluorescence Filter Technique: 32

1.7. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2 . Development and characterization of a bacteriophage assay for the detection of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella Enteritidis 35

2- 1 . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Introduction 35

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Materials and methods 38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Bacterial strains: 38

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.Bacteriophageandhost.. 41

LIST OF FIGURES

Figure 1.1.

Figure 1.2.

Figure 1 -3.

Figure 1.4.

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 2.6.

Figure 2.7.

Figure 2.8.

Figure 2.9.

Figure 2.10.

Figure 2.1 1.

Number of human cases of illness fiom total Salmonella, S. Typhimurium, and S. Enteritidis reported to the National Laboratory for Bacteriology and Enteric Pathogens fiom 1985 to 1995. . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5

Infection cycle of lytic bacteriophage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Biochemistry of the bioluminescence reaction . . . . . . . . . . . . . . . . . . . . . 26

Energy level schematic diagram illustrating energy changes involved in absorption and fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

Overview of the IMS-bacteriophage protoco t for the detection of Salmonella Enteritidis in enriched samples. . . . . . . . . . . . . . . . . . . . . . . . 47

Details of the IMS-bacteriophage protocol for the detection of Salmonella Enteritidis, . . . . . . . . . . . . . , , , , , . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . - 48

Transmission electron micrograph of bacteriophage SJ2. . . . . . . . . . . . . 58

Scanning electron micrograph of bacteriophage SJ2 attached to S. Enteritidis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Resulu of one-step gowth experiment for phage SJ2. . . . . . . . . . . . . . . . 60

Average clump counts following rnicroscopy protocol for four initial populations of S. Enteritidis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Relationship between plate count and DEFT count for different dilutions (1 o-'~ IO5, 1 O', 10J) of S. Ententidis in buffer. . . . . . . . . . . . . . . . . . . . . 63

Epifluorescent micrograph of S. Enteritidis stained with Molecular Probes LIVE/DEAD@ BacLight" bacterial viability stain. . . . . . . . . . . . . . . . . . . 64

Percent of negative control value for five populations of S. Enteritidis in broth following IMS-bactenophage assay . . . . . i . . . . . . . . . . . . . . . . . . . 66

Sensitivity of the Pharmacia spectrophotometer as indicated by mean optical density values for various populations of S. Enteritidis. . . . . . . . 68

Sensitivity of the MGM Fluorometer as indicated by mean fluorescence values for variou populations of S. Ententidis. . . . . . . . . . . . . . . . . . . . . 68

Figure 2.12.

Figure 2.13.

Figure 2.14.

Figure 2.15

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 3.4.

Figure 4.1

Figure 4.2

Relationship between plate count, and plate count following IMS for different dilutions of S. ~nteritidis in buffer. . . . . . . . . . . . . . . . . . . . . . . 69

Scanning electron rnicrographs of S. Enteritidis attached to magnetic beads. . . . . . . . . . . . .- . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Cornparison of L-broth, lambda buffer and three dextrose concentrations (0.2%, 0.5% and 1 .O%) for the reduction of S. Enteritidis by phage SJ2 at 37OC . . . . . . . . . . . , . . . . . . . . . . . . . . - ~ ~ . . . . . - . . * . ~ . . . . . . . . 73

Cornparison of L-broth, lambda buffer and three dextrose concentrations (0.2%, 0.5% and 2.0%) as media for growth of S. Enteritidis at 37°C. . . 73

Percent of negative control value for skimmed milk powder samples inoculated with S. Enteritidis at four levels (0, loO, 1 ol,andl O' CFUig). . 93

Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, 10°, 1 ol,andl 0' CFU/g). . . . . . . . . . . 93

Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, loO, 10',and 10' CFU/g). . . . . . . . . . . 97

Percent of negative control value for liquid whole egg samples inoculated with S. Enteritidis at four levels (0, 1 0°, 10',and 10' CFU/ml). . . . . . . . . . 97

Percent of negative control value for four populations of E. coli 0 157:H7 following IMS protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13

Percent of negative control value for ground beef samples inoculated with E. coli0157:H7atthreelevels(O, loO, 10'CFU/g). . . . . . . . . . . . . . . . 113

LIST OF T B L E S

Table 1.1.

Table 1.2.

TabIe 1.3.

Table 2.1.

Table 2.2.

Table 2.3.

Tabfe 2.4.

Table 2.5.

TabIe 2.6.

Table 2.7.

Table 2.8.

TabIe 3.1.

Prevalence of Salmonella in foods of animal orïgin. produce and h i ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Cornparison of several existing tests for Salmonella spp . . . . . . . . . . . . . 20

Cornparison of five methods for recovery of Sa/monella fiom processed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . raw broiler carcasses 22

Non- Enteritidis SaZrnonella strains used for the specificity study . . . . . . 39

Salmonella Enteritidis strains used for the specificity study . . . . . . . . . . . 40

Non-Salmonella strains used for the specificity study ................ 40

Schedule for one-step growth experirnent- . . . . . . . . . . . . . . . . . . . . . . . . 44

Schedule for plating growth tubes (GT- 1 and GT.2) . . . . . . . . . . . . . . . . . 44

Bacteriophage assay and plaque assay specificity results for non- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella strains 76

Bacteriophage assay and plaque assay specificity results for Salmonella Enteritidis strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Bacteriophage assay and plaque assay spec i f ic i~ results for non- Salmonellastrains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Tally of Mse positive and false negative results obtained fiom skimmed rnilk powder. ground chicken and liquid whole egg samples when various

. . . . . . . . . numerical cut-offvalues are used to detemine a positive test 92

1. Introduction

1.1. Salmonella

1.1.1 Background:

Salmoneliae are small, Gram negative, non-spo~g rods that are widely distributed in

nature, with humans and animals being their p r i m q reservoirs. Salmonella was first

identined as a human pathogen in 1888 following a German outbreak caused by an

organism subsequently known as SaZrnonella Typhimurium (Tauxe, 199 1). Food

po isoning from Salmonella results when significant numbers of the appropriate strains are

ingested.

1.1.2, Classification:

The genus Salmonelh consists of around 2400 serotypes (Andrews, 1996). Antigenic

classification of Sahonellae is generally based on the Kaufham-White scheme and uses

somatic (O), capsular (Vi), and flagellar 0 antigens (Bergey and Holt, 1994). When

classification is made by this scheme, species and serovars are placed in serogroups

designated A, B, C, and D etc. according to similarities in content of one or more O

antigens. Narning of a Salmonella serovar is based on the place where it was first

isolated, for example S. London (Jay 1992).

For epidemiological purposes, Salmonella cm be divided into three groups (WHO, 1988):

1) Those that infect hurnans only, which include S. Typhi, Paratyphi 4 and S. Paratyphi

2) Host adapted serovars, some of which are human pathogens and may be conrracted

fi-om foods.

3) Unadapted serovars (no host preference). These cm be pathogenic for both humans

and animais, and include most foodborne serovars.

1 -2. Salmoneilosis

1 -2.1. Clinical signs:

With the exception of typhoid fever caused by S. Typhi, which is not normally foodbome,

there exists four main clinical manifestations of SaZmuneLIa infection (Andrews, 1996).

The most cornmon is gastroenteritis, characterized by diarrhea, cramps, vomiting and

often fever. Recovety generally occurs in two to three days. Second, the organisms

invade the bloodstrearn and settle in the liver, kidneys, gallbladder, heart and joints where

abscesses or other complications may occur. Third is a typhoid-like fever that is milder

and shorter in duration than the two to three week recovery from typhoid fever caused by

S. Typhi. Lastly there exists a carrier condition, whereby certain individuals harbour the

organism asymptomaticaily, and can spread the organism to others.

Large plasrnids have been demonstrated to be a prerequisite for the virulence and

invasiveness of various Salmonella serotypes, including S. Enteritidis (Helmuth et al.,

1993). Mouse studies indicate that virulence plasrnids enable strains to colonize and

multiply withîn animal organs (Gulig, 1990), and the fiequency of vinilence plasrnids is

almost 100% when strains are isolated from animal organs or human blood (Helmuth et

al., 1993). Although exceptions have been observed, virulence plasmids seem to play a

signïficant roIe in systemic infections of livestock and humans (Hehuth et al. 1993). It is

also thought that enterotoxins, cytotoxins, and lipopoiysaccharides are involved in the

pathogenesis of Salmonella. contributing to the local damage of the intestinal mucosa that

results in enteric syrnptoms (Suruki, 1994).

1.2.2. Infective dose:

Whether infection follows exposure to Salmonella depends on the numbers of organisms

ingested and the ability of those organisms to overcome local defences. Factors influencing

infective dose include differences in virulence among organisrns, variation in susceptibility

among hosts and conditions that might alter the number of organisms reaching the

intestine (Blaser and Newman, 1982). Early human feeding studies suggested that

salmonellosis occurred only after ingestion of large numbers of organisms but these

studies were found to be flawed for one or more reasons. Blaser and Newman (1982)

reviewed 11 outbreaks for which approximations of the infective dose were calcuiated. In

six of the 1 1 outbreaks the doses ingested were calculated to be <1 O3 organisms.

The low infective dose was confirmed more recently during a 1994 US outbreak of

Salmonella Enteritidis involkg contaminated ice cream. Using a three dilution Most

Probable Number procedure, the infective dose appeared to be no more than 28 cells

(Vought and Tatini, 1998).

1.2.3. Incidence and costs:

In 1995, a total of 6389 human cases of salmonellosis were reported in Canada through

provincial laboratones to the National Laboratory for Bacteriology and Enteric Pathogens

(NLBEP) (Health Canada, 1998). Typhirnunum and S. Ententidis were the most

frequent serortypes reported, with 1366 and 964 cases respectively. There were a

reported 244 hospital inpatient visits, 38 outpatient visits and ten deaths associated with

Salmoneh cases. Fifty two outbreaks were reported for 1995, most of which were

associated with comrnunity events, restaurants and nursing homes.

Over the past decade, the total number of Salmonella cases reported £tom the NLBEP, as

well as reported S. Typhimurium cases, has decreased (Figure 1.1). The number of annual

cases associated with S. Enteritidis has slightly increased over the same time penod. A

moderate seasonal trend was observed for all serotypes, with increased rates of

salmonellosis in the surnmertime mealth Canada, 1998). In cornpanson, there were

13,680 human cases of C m p y Z o b a c ~ infection and 1,493 human cases of pathogenic

1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 Year

Total

S. Ententidis

- - S. Typhimurium

Figure 1.1. Number of human cases of illness fiom total Suhonelltz, S. Typhimurium, and S. Ententidis reported in Canada to the National Laboratory for Bacteriology and Enteric Pathogens fiom 1985 to 1995 (Health Canada, 1998)

Escherichia coli infections reported for 1995 in the National Notifiable Diseases

databases (Heaith Canada, 1998).

The actual number of amual cases of salmoneilosis would greatly exceed the reported

numbers for several reasons including under-reporting and the large number of infected

individuals who do not seek medical attention. An estimated two million cases of

salmonellosis occur each year in the United States (USDA, 1998). It is estimated that the

average case of salmonellosis costs the US national economy $700 - $5000 (Mason and

Eebel, 1991). Total estimated costs of Salmonella infections in the United States are

about $ 4 billion annually (USDA, 1998).

1.2.4. Trends in Salmonella infection:

Tauxe (1 99 1) identifies four trends of Salmonella infection in the 1990s suggesting that

salmonellosis will present an ever increasing challenge to public health in the future. The

first is the increasing resistance of Salmonella to one or more antimicrobial agents.

Antimicrobial therapy may be compromised if the Salmonella is resistant to the agent of

choice. Infection can actually be promoted by such therapy as cornpeting flora, known to

have a protective effect against pathogen colonization, may be reduced. This reduction in

cornpetitive organisms may result in a smaller dose of Salmonella being infective.

The second trend is the exacerbation of salmonellosis symptoms in imrnuno-compromised

and HTV patients. S. Typhimurium, S. Dublin, and S. Enteritidis are particularly invasive

and can lead to bacteremia in such persons. Recurrent Salmonella bacteremia has been

included as an indicator disease of A l D S since 1987. Prophylactic use of antimicrobials

may fbrther increase nsk of salrnonellosis in this population.

The third trend is the association of S. Enteritidis with eggs. Investigations of S.

Enteritidis outbreaks have repeatedly shown that the most comrnon source is the grade A

sheIl egg, usuaily consumed raw or improperly cooked.

The fourth trend has been the occurrence of large and dispersed outbreaks linked to large-

scale food processing and widespread distribution. A massive outbreak in the United

States in 1985 was linked to contaminated pasteurized milk (Ryan et al., 1987). A strain

of S. Typhimurium, resistant to multiple antibiotics, was isolated from 17,000 people

during the course of the outbreak, with the estimated number of cases exceeding 180,000.

Such widespread outbreaks oAen require collaboration of public health authorities in many

jurîsdictions and even across national boundanes, illustrating the need for a global

approach to food safety.

1 -2.5. Sources of infection:

Sahonellae are ubiquitous and primarily reside in the intestinal tract of birds, reptiles,

farm animals and humans. Since salmonellae are intestinal organisms they can be

introduced into the environment through feces, poliuting water and foods. Transmission

to man is usudy food-borne firom eating undercooked rneat, milk or eggs, or by cross-

contamination to other foods which are eaten without further cooking. Salrnonellae grow

rapidly in foods at room temperature and cm survive refigeration and freezing but are

kdled by heat over 60°C.

Table 1.1. illustrates the results of various surveys in regards to the prevaience of

Salmonella in foods. Poultry is recognized as a major source of foodborne Salmonella.

The problem starts on the farm, where contaminated feed, water and the environment can

lead to Salmonella colonization of young chicks. Processing of poultry carcasses cm lead

to a decrease in the numbers of Salmonella on carcasses, but may also lead to an increase

in the total number of contarninated carcasses because of cross-contamination during

processing. The reported prevalence of salmonellae on poultry products varies nom 2 to

100%, with the median being about 30% positive (Bryan and Doyle, 1995).

Factors that contribute to outbreaks of poultry borne disease include irnproper cooling

(48%), foods prepared a day or more in advance of serving (34%), and inadequate

cooking (27%) @ryan, 1980). Cross-contamination occurred in 27% of the 26 poultry-

associated outbreaks investigated.

Eggs were identified in the early 1960s as the source of large outbreaks of salmone1Iosis.

As a result of egg related illnesses, it is recommended that pasteurized eggs be substituted

in recipes that require raw eggs (Morse et al.. 1994). Pasteurization of buik egg products

have become cornmon practice and the numbers of outbreaks have decreased but localized

Food Country Prevalence Reference

Melons Importedb 1 1 / 1440 (0.8%) Madden, 1992 SE = S. Enteritidis

Chicken

Unpasteurized egg

Unpasteurized egg (SEa)

SEa in eggs fiom infected flocks

Bulk tank raw milk

Pork

Lamb carcasses

Beef sarnples

Smoked fish

Japan

us us UK us

Ireland us US

Japan

Spain

Japan

US

Spain

(O, 16%) 26/292 (8.9%)

69/225 (3 1 %) 3/94 (3 -2%)

a

Tokumaru et al., 199 1

Ebel et al., 1993

EbeI et al., 1993

Humphrey et al., 199 1 Henzler et al., 1994

Rea et al., 1992 Rohrbach et al., 1992

Epling et al., 1993 Tokumani et al., 199 I

Sierra et al., 1995

Tokurnam et al., 199 1

Heinitz and Johnson, 1998

Garcia-Villanova Ruiz et al., 1987

Table 1.1 - Prevalence of Salmonella in foods of animal origin, produce and fniits

I

Imported into US fiom Central Amerka, Mexico and the Carribean

outbreaks have still been traced to the use of ungraded eggs. Eggs have been found to be

responsible for the majority of S. Enteritidis outbreaks for which a food vehicle has been

identified (Hogue et al., 1997; Gast and Beard, 1993). Eggs become contarninated

internally with S. Ententidis as a result of infection of the hens ovarïes and oviducts

(Stephenson et al., 1991). A survey by Bamhart et al. (199 1) found an overall mean of

26.6% of pooled ovary sarnples within infected flocks to be positive for Salmonella

Enteritidis, with a range of 0400%. Humphrey (1994) reported a prevalence of 0.6% of

S. Enteritidis in eggs originating nom naturally infected poultry laying flocks. Subsequent

growth of these bactena is govemed by temperature and length of storage and it appears

that high levels of contamination occur only when the yolk is invaded (Humphrey, 1994).

A cornparison of 1991 and 1995 survey results in the United States suggests there has

been no decline in S. Ententidis occurrence in the commercial egg industry between these

years .

Although Salmonella is most frequently associated with eggs and poultry, outbreaks of

Salmonella are not tirnited to these food sources, An outbreak of Salmonella Infantis

involving 500 confirmed cases in Denmark was traced to a single pig slaughterhouse

(Wegener and Baggesen, 1996). A ready to eat savoury snack produced in Israel was

responsible for an outbreak in that country as weil as numerous cases in England, Wales,

Canada and the United States (Killalea et al., 1996; Shohat et al., 1996). The municipal

water supply in a rural Missouri township was responsible for a 1993 S. Typhimurium

outbreak involving more than 650 persons with seven deaths (hgu lo et al., 1997).

Salmonella outbreaks have also been linked to goats' mik cheese (Desenclos et al., 1996),

pasteurized milk (Sun, 198 5), cured ham (Gonzilez-Hevia, 1 W6), and cheddar cheese

(D'Aoust et al., 1985; Bezanson et al., 1985). Most recently, a March 1998 outbreak in

Canada implicated cheddar cheese fiom V ~ ~ O U S prepared lunch snacks as a source of

Salmonella Ententidis infection. At the tirne of this report, over 500 children had become

il1 (Food Safety Net, 1998). In addition to foods of animal origin, outbreaks have been

linked to unpasteurized orange juice (Ekish, 1998), tomatoes (Hedberg et al., 1994), and

watermelon (Larson et al., 1979). arnong others. Fruits and vegetables may become

contaminated while growing in orchards and fields, during harvesting and post-harvest

handling, processing and distribution (Beauchat, 1996). According to D' Aoust (1994) the

prevalence of Salmonella in fiesh h i t s and vegetables remains quite low (O to 8%).

Between 1983 and 1987, the percentage of outbreaks of gastrointestinal illness caused by

fruits and vegetables in the US averaged 5.5% Wadden, 1992). This value includes al1

reported foodborne illness, not just outbreaks associated with Salmonella.

In some instances, outbreaks are the result of cross-contamination. A 1994 US outbreak

of Salmonella Enteritidis in ice cream was linked to tanker trucks which carried

unpasteurized liquid egg pnor to transporting ice cream premix (Hemessy et al., 1996)-

Ninety percent of party-goers at a catered party in Toronto, Canada became ill,

presumably following consumption of curry chicken and fish cutlets (PHERO, 1994). An

inspection of the catering premises revealed several serious violations, and cross-

contamination of ready to eat food with dirty utensils and food contact surfaces was

thought to be a contributing factor.

1 -3. Ecology and control:

It is evident that the control of foodborne illness due to infection with Salmonella is of

great national and world-wide importance. The current increase in Salmonella infection

associated with poultry suggests that reducing infection in, or contamination of poultry

could significantly decrease human illness (Sockett, 1995). The control of salmonellosis

requires action at d levels of the food chah which may be addressed by the application of

the Hazard Analysis Critical Control Point system (HACCP). Reliable testhg and

sensitive analytical methods are still an important part of the process.

Feed ingredients are recognized as being fiequent sources of Salmonelln, but the Iink

between S. Enteritidis infections in laying flocks and the consumption of contaminated

feed is not well established (Gast and Beard, 1993). Mice are highly susceptible to S.

Enteritidis infection and may serve as amplifiers by shedding large numbers of the

organism. Mice have been observed to persist through the cleaning and dissection

process in poultry houses and rnay be the main vehicle in the spread of S. Enteritidis fiom

house to house, and between flocks (Gast and Beard, 1993; Mason, 1994). Surface

water, insects, crops, pet animals, humans and other livestock contribute to the prevalence

of Salmonella in poultry (Noordhuizen and Frankena, 1994). The most important source

of contamination appears to be the resident Salmonella of the flock (Lahellec et al., 1986).

A Canada-wide survey of 295 randornly selected layer flocks found that environmental

(faecal and eggbelt) samples fiom 52.9% of the flocks were contaminated with

salmonellae (Poppe, 199 1).

Experimental infections of young chickens 16th SaZrnonelh Enteritidis leads to extensive

intestinal colonization which is sometimes followed by invasion of the interna1 organs

(Gast, 1994). Young chicks reared in disinfected production units were found to be

particularly susceptible to Salmonella colonization, as the development of protective

intestinal microflora was severely delayed. It was found, however, that adult-level

resistance could be conferred in young broilers when a bacterial preparation, given orally,

established an adult Iike intestinal ffora in these young birds (Hirn et al., 1992). This

concept later became known as the Numi concept or cornpetitive exclusion (CE).

Bacterial cornpetition in the gut for nutrients and surface receptor sites, as well as

chemical inhibition of pathogens (for exarnple by production of volatile fatty acids and

bacteriocins) are thought to be involved in the protective eEect of these cultures (Nurmi et

al., 1992; Schneitz et al., 1992).

Until recently, there has been only one cornmercially available CE product. ~roilact"

(Orion Corportaion, Farmos, Finland) is an undefined culture, usually administered in the

first drinking water. A recently developed product, preernptm (MS Biosciences Lnc.

Dundee, Illinois), is applied as a spray on newly hatched chicks.

Competitive exclusion has been used in Sweden since 1981 as part of their national

control program for SaZmoneZZa. A 1990 nationwide study in Sweden demonstrated that

less than 1% of broiler chickens were contaminated with Sahonelia after slaughter

(Wierup et al., 1992). Competitive exclusion is not seen as a panacea for the control of

Salmonella, but in conjunction with good hygiene and rearing practice it has been

demonstrated to have a profound effect on the incidence of Salmo~ella infection in treated

flocks.

Once the broilers leave the farm, adequate hygiene must be rnaintained to prevent cross-

contamination and proliferation dunng transport and processing. A study by Jones et al.

(1991), concluded that catching, loading and live haul procedures appear to be major

contributors to the contamination rates seen in ready-to-cook broilers. A significant factor

affecting the persistence of Salmonella throughout processing is that of attachment and

entrapment. Bacteria that become attached to the skin or muscle cannot be easily

removed by subsequent processing steps, and enteric bactena are sometirnes M y

attached to the skin before the birds arrive at the plant (Lillard, 1989; Bryan and Doyle,

1995).

Hygiene problems associated with poultry processing are similar to that of other meat

animals. There are certain features, however, that make microbial control with poultry

more dificult. First is the rapid rate of processing; up to 6000 birds per hour. Secondly,

the carcass remains whole during processing therefore it is difficult to remove the

intestines without breakage through a reiatively small hole in the abdomen (Mead, 1989).

The main problem in poultry processing is limiting the spread of surface and faecal

bacteria. In positive birds, Salmonella can be found fiom the crop to caeca, but most

frequently in the caecal content (Riemann, 1993). Cross-contamination can occur at any

point in the process. Scalding, plucking, evisceration, washing, and chilling are cntical

control points in the process (Mead, 1980). The number of Salmonel/a positive carcasses

can Vary at dflerent stages throughout processing. Percentage of positive carcasses pre-

evisceration, pre-CM, post-chi11 and post-automatic cut were 58%, 48%, 72% and 77%

respectively (James et al., 1992). The use of biocides in the washing and chilling water

have been investigated as a means of reducing the microbial Ioad on carcasses. Agents

such as chlorine, hydrogen peroxide and lactic acid have been demonstrated to reduce the

prevalence of Salmonella on carcasses, but are often associated with adverse quality

effects (Izat et al., 1989; Lillard, 1989). No processing step completely destroys

Salmonella and no definitive solution to poultry carcass contamination is available unless

the flocks are Salmonella fkee (Lahellec et al., 1986).

A more direct approach would be decontamination of processed carcasses prior to retail

distribution. Irradiation is the only suit able treatment currently available which will

eliminate Salmonella fiom carcasses and retain the desired characteristics of raw poultry

(Simonsen et al.. 1987). A cost-benefit analysis of poultry irradiation (costs of

salmonellosis iess costs of irradiation) performed in England and Wales indicates that only

under the most severe assumptions regarding irradiation costs and effectiveness was the

net benefit negative (Sockett, 1995). The mode1 presented indicated that even for a

modest 25% reduction in human salmonellosis, the minimum benefits wouId be substantiai

(£261 million - £540 million over 15 years).

Epidemiological information can also be used to aid in prevention. If the contribution of

the major components to infection occurrence could be quantified, risk factors could be

ranked and preventative measures prioritized (Noordhuizen and Frankena, 1994). A

monitoring and surveillance system (MOSS) can provide accurate and reliable information

about infection and disease occurrence over time and thus provide a means of predicting

infection and evaluating the effectiveness of control measures. The sensitivity and

specîfïcity of diagnostic tests applied would be of great importance here.

A remaining option is to increase public awareness of food poisoning hazards by providing

relevant Somation and instruction regarding the proper handling and preparation of

foods. This may ensure the steps needed to make food safe could be applied more

fiequently and reliably both commercially and at home.

1.4. Detection of Salmonella

1.4.1. Conventional methods:

Detection of Salmonella by conventional methods consists of four phases. The first is a

non-seiective pre-enrichment which permits recovery of stressed organisms, and growth of

al1 organisms present. The second is a selective e ~ c h m e n t which allows for the survival

or growth of Salmonella while reducing the numbers of non-SaImonelZa in the media.

Next is isolation using selective agar to produce presumptive isolates. The last is a

confirmation step, often employing serological and biochemical tests to codirrn that the

isolate is Salmonella and to determine its serotype (Mansfield and Forsythe 1993; van der

Zee 1994). The total time for deiection is around 72 to 96 hours. Conventional methods

can provide a theoreticai sensitivity of one Salmonella cell per 25 g of food, however the

presence of cornpetitive flora can prevent detection. van der Zee (1994) notes that fdse

negative results may be obtained when non-salmonellae outgrow salmoneilae in pre-

e~chment , seiective enrichment or plating agars.

Regarding the specinc isolation of S. Enteritidis, conventional methods are scarce. This is

mainiy attributed to the minor differences that exist between S. Enteritidis and other

serovars. Such differences are not detectable by cultural methods (van der Zee 1994).

1.4.2. Rapid methods:

The tirne required for Salmonella detection by conventional methods can cause prolonged

and expensive storage of foods prior to distribution. Ideaiiy, the goal is to detect smdl

numbers of target bacteria in the presence of large numbers of non-target cells, after they

have been injured by manufacturing processes or disinfectants, rapidly enough to prevent

the manufacture or sale of contaminated product (Wolber and Green, 1990). The genus

Salmonella is a 'zero tolerance' contaminant and detection methods should be able to

detect one viable bacterium in 25g of food. A need exists for methods that provide results

quicker and with equal or greater sensitivity than conventional methods. Such rapid

methods should be robust, reliable, cost-effective and specific, minimizing false positive

results (Blackburn, 1993). Rapid methods could also facilitate routine monitoring (Mossel

et al., 1994).

Shortening non-selective or selective ennchment times from 16- 18 hours to 6-8 hours for

recovery of Salmonella ftom foodstuffs has been considered as a means of simplifving

methodology for tirnely identification of contarninated foods (D'Aoust et al., 1992). In

some cases this strategy was found to be as reliable as standard methods, but in others it

has led to a high number of false negative resuks (Allen et al., 1991; D'Aoust et ai., 1992;

Patel and Williams, 1994). Results of a 16 to 24 hour selective enrichment were found to

consistently exceed that obtained with 6 hour selective enrichment cultures for recovery of

foodbome SalmoneZZa (D' Aoust et al., 1992). Because resuscitation of injured celis does

not occur in selective media, the benefit of direct selective enrichment is also questionable

(Andrews, 1 986).

New media and modifications of existing media have improved recovery of Salmonella.

In a three-way study selenite cystine broth, Tetrathionate broth and Rappaport-Vassiliadis

(FW) medium were evaluated for the recovery of Salmonella from highly contaminated

foods (June et al., 1996). RV medium was found to be supenor for al1 food types tested.

SaZrnonella can be also isolated by their motility in a semi-solid version of RV medium.

In regards to rapidity, Mossel et al. (1994) noted three traits of microorganisrns that

hinder attempts to speed up detection methods. First is the sublethally stressed state of

microorganisms which may be an inevitable consequence of production and distribution

chains. This forces at least a short penod of enrichment for recovery. Secondly, target

organisms are often present in very low numbers. Lastly, the organism itself, rather than a

signal indicating its presence, is often required as proof in commercial or legal disputes.

This necessitates some confinnatory procedures.

Table 1.2. outhes some existing rapid methods for the detection of SuZmonelZu in food.

Enzyme-linked immunosorbent assays (ELISA) tests typically have a sensitivity of 1 o4 to

106 CFW/ml and often have very good agreement with conventional cuitUral methods

(Patel and Wiuiams, 1994), but they of€en suffer £?om a lack of specificity (Blackburn,

1993). Manual ELISA'S often require a high level of technical ski1 but automated

systems are available. A study by Juune et al. (1992) compared two AOAC-approved

enzyme irnmunoassays, S almonella-TekTM and ReportTM, wit h the standard culture

method of the AOAC and the Food and Dmg Administrations Bacteriological Analytical

Manual @AM), for the recovery of Salmonella spp. fiom four low-moisture foods. Of

the 300 inoculated food samples, 199 were contirrned positive by Salmonella-TekN, 193

by R e p o r P , and 206 by the AOACDAM method. When preenrichments were

inoculated after incubation, the lowest concentration identified by Salmonella-TekTM was

2.0~10' ceils per ml, and 2.0 x108 for Reportm. The TECRA LNnue,capture ELISA

uses an antibody coated dipstick to recover Salmonella fiom the food pree~chment.

This is followed by a five hour e ~ c h r n e n t to increase the number of Salmonella in the

sample. Cornparison of the TECRA ELISA with the FDA culture method indicated close

agreement over 176 tests (Flint and

CFU/ml of S. Typhimunum after the

Hartley, 1993). These authors detennined that 30

16 h preenrichrnent was sufficient for a positive test.

The TECRA procedure incorportates an additional 5 h incubation followhg

preenrichrnent.

'abIe 1.2: Cornparison of several existing tests for Salmonella spp (Wolber and Green, 1990).

Test Manufacturer Assay Method Approximate Time cost

(hours) (US$/test)

Gene-Trak SalmonelIa

Salmonella-Tek

Salmonella 1-2

Tecra Salmonella

Q-Trol Salmonel f a

Oxoid Salmonella Ra~id Test

M a y

Gene-Trak

Organon Teknika

BioControl Systems

Dynatech La bs

Oxoid

Standard Microbiology 2.00

DNA probe; 8.75 Radioisotope or colorimetric detection

ELISA: Co lorimetric 5.00 detection

Immunodifision and 20.00 irnmobilization; fluorescence

ELISA; 6.25 Colorimetric detection

Enzyme immunoassay; 3 -00 Fluorescence

Standard Microbiology; 6.00 Latex agglutination

" BAM-AOAC: based on the US Food and Drug Administration Bacterial AnaZysis Man& and approved by the Association of Officia1 Analytical Chemists

Latex agglutination tests are usually used to confirm suspect colonies on solid media and

require high levels (10' to lo9 CFU) to give a positive agglutination reaction (Patel and

Williams, 1994). They are very rapid, being perfomed in about 3 minutes. Feng (1992)

notes that the sensitivity and specifkity of the antibodies used to prepare the latex reagents

needs improvement.

Nucleic acid-based tests can be highly specific, but are often labour intensive and require a

skilled technician to perform the assays. Commercial test kits such as the GENE-TRAK@

DNA hybndization test are applied after 48 hour incubation and Save approximately 18-24

hours relative to conventional methods (PateI and Williams, 1994). An evaluation of

GENE-TRAKB with 600 artificially contaminated, and 404 naturally contaminated food

samples showed excellent agreement with the BAMIAOAC culture method (Wilson et al,

1990).

Polymerase chah reaction (PCR) is a technique whereby a small amount of target DNA is

amplified, giving a theoretical detection lirnit of one molecule of target DNA

(Swaminathan and Feng, 1994). PCR is highly sensitive but generally requires a pure

culture and considerable laboratory tirne and expertise (Yu and Stopa, 1996), thus

offsetting PCR's high sensitivity advantage. PCR also has a disadvantage in that it cannot

dserentiate viable and non-viable bacteria (Mossel et al., 1994).

Rapid biochemical profile systems, such as Analytical Profile Index (AH) strips

(Biomerieu, St. Laurent, Quebec) are available but require isolated colonies on solid

media. These tests generdy provide information to the genus level for Salmonella.

TabIe 1.3 outlines the results of a study by Bailey et al. (1991) comparing confirmed

culture with enzyme immunoassay (Salmonella-Tekm), DNA Hybndkation (GENE-

TRAK"), antibody immobilization (1-2 Testm) and the Food Safety Inspection Service

(FSIS) culture rnethod for the recovery of Salmonella fiom naturally contaminated

processed broiler carcasses.

Table 1.3. Cornparison of five rnethods for recovery of salmonellae fiorn processed raw

Test Positive (%) False + (Yo) False - (%) - - -- - - - - -- - -

Confirrned culture 71

FSIS Culture 65.1

Salmonella-Tekm 75.6

GENE-TRAP 71.3

1-2 Test- 66- 1

broiler carcasses @ d e y et al., 199 1) r -

-

Table 1.3. illustrates the difficulty in comparing methods. Feng (1992) noted that the

complexity of food matrices make cornparisons between test kits difficult. Test sensitivity

and the incidence of false positive and negative results are infiuenced by the food types

analysed. Feng reviewed various comparative studies of commercial assay kits for

Salmonella detection and found that some rapid rnethods performed better than other

methods in some instances but not in others. This highlights the importance of choosing a

rnethod appropriate to the application, and validating that method. He noted that despite

22

the advantages and limitations of each method, most assays exarnined were found to be

rapid, simple and at least as sensitive as conventional methods.

1.4.3. Imrnunomagnetic separation:

Irnrnunomagnetic separation (IMS) has been used as an alternative to selective enrichment

broths for a variety of bacteria including Salmonella. Paramagnetic beads are coated with

polycional antibodies which can target and separate Salmonella fiorn a rnixed suspension

without loss of viability, producing a normal isolate for further confirmation. IMS can

eliminate the need for selective enrichment and reduce the time required for conventional

methods by one day. Mansfield and Forsythe (1993) compared IMS as an enrichment

method with approved selective enrichment protocols for Sulmorrella using selenite

cystine, Rappaport-Vassiliadis and Muller-Kaufham tetrathionate broths. The results of

the 120 food samples tested indicated that Dynabeadsa gave comparable results after a 10

minute capture compared with 24 and 48 hour exuichrnent periods with the approved

media. The authors concluded that MS could be a reliable alternative to standard

methods for Salmonella screening. A collaborative ring-trial compared the use of IMS

with conventional enrichment broths for the recovery of stressed Salmonella fiom herbs

and spices (Mansfield and Forsythe, 1996). IMS was found to be a suitable alternative to

conventional enrichment and reduced the time required for detection by 24 hours.

IMS has also been used in conjunction with other rapid detection methods. Dziadkowiec

et al. (1995) found that Salmonella was successfùlly isolated and enumerated in skimmed

milk powder by IMS and indirect conductance despite the 1000-fold greater number of

non-target cells. IMS has been successfuiiy used in conjunction with ELISA'S,

conductance rnicrobiology, electrochemilumlnescence, and PCR (Cudjoe et al., 1995; Holt

et al., 1995; Parmar et al., 1992; Yu and Stopa, 1996; FIuit et ai, 1993).

1 S. Bacteriophage

1 S. 1. Lytic and temperate bactenophage:

Bacteriophage can generally be divided into two categories, temperate and Iytic. Infection

by lytic phage, such as Felix-O1 of Salmonella spp., always results in the imrnediate

production of progeny phage. The infection cycle of lytic phages is illustrated in Figure

1.2. The total infection cycle can take as little as 25 minutes with the number of progeny

phage being highly variable. Temperate phages, such as the P22 phage of Salmonella

Typhimurium have two possible outcornes (Poteete, 1994). It can grow lytically, as

above, or form a lysogen. In the latter case, instead of unrestrained DNA replication and

phage assembly, a stable relationship is established with the host ceii which is maintained

over many generations. In this temperate state, the viral DNA replicates at the same rate

as host ce11 DNA and is distributed to daughter celis at each ce11 division (Birge, 198 1).

Phage DNA Phage head Mature phage proteins Tailandfibre particie

proteins

l nfection Phage DNA Repl. Setf assembly

Eariy mRNA Late rnRNA

!

v Early proteins l Late proteins Lysis

Time (minutes)

Figure 1.2 Infection cycle of lytic bactenophage. During infection, the viral DNA is injected into the host cell. The early proteins produced are enzymes necessary for the replication of the viral nucleic acid. Late proteins include proteins required for the virus coat.

Bactenophages have been used in the identification and classification of bacteria since

1925. The specificity of bacteriophage for their hosts have made them ideal agents for

what has been cailed 'phage-typing'. Phage-typing schemes have significance in medicine,

epidemiological studies, as well as industrial fermentations and cheese formation (DuBow,

1994).

1.5.3. Bacteriophage based detection rnethods:

1 -5 -3.1. Luminescence and reporter genes:

Many marine organisms, including Vibrio and Photobacteritrm, are biolurninescent, that is

they are capable of emitting light. The light reaction requires oxygen, a source of energy,

a luciferase enzyme and a long chain fatty aldehyde. The reaction is illustrated in Figure

1.3.

E;MN-H2 + O, + RCOH -+ luFife- + FMN + RCOOH + H,O + Light

Figure 1.3 Biochemistry of the bioluminescence reaction.

Bacterial luciferase is encoded by the lm A and lzrx B genes. In bacterial cells, detection

based on bioluminescence has the advantage of being non-invasive, non-destnictive and

can offer high sensitivity in real-time analysis (Stewart and Williams, 1992).

Bacterial luciferase has been used in reporter gene expression, particularly for Gram

negative organisms whose light output is typicafly one hundred-fold that of Gram positive

organisms. It has also been used as a reporter of cellular viability. Since light output

would be dependent on a functional intracellular biochemistry, it was reasoned that

compromised cells with impaired biochemical activity would elicit a reduction in light

emission (Stewart and Williams, 1992). This may have application in the field of

predictive Mcrobiology, where light output in geneticdy engineered bioluminescent

bacteria can be monitored as cells are exposed to diffierent stresses (Baker et al., 1992).

Chen and Griffiths (1996a) successfùliy used luminescent SaZmoneZZu as real tirne

reporters of growth and recovery from sublethal injury in foods.

In addition to these applications, bacterial luciferase has been used as a reporter for

bacterial detection and enurneration. In this case, lux genes are introduced into the

genome of bactenophage. Bactenophage lack the intracellular mechanisms necessary for

light production and thus remain dark. Upon host infection the phage genes, including the

additional lux genes, are expressed and within an hour of infection the host bacteria are

bioluminescent (Stewart, 1990). This method exploits bacteno phage-host specificity.

Recombinant lux+ bactenophage can detect target bacteria in a food matrix without

enrichment provided they are present at levels greater than 103 CF'U/ml (Stewart, 1990).

Kodikara et al. (1991) has used ZZJ& recombinant phage for near on-line detection of

enteric indicator organisms. If the target is present at levels greater than IO" C N per g or

cmZ, it can be detected without enrichment in less than an hour. A 4 h o u enrichment was

found to be suscient for detection when counts were 10 CFU per g or cm2. Chen and

Griffiths (1996b) used lm+ recombinant bacteriophage for the detection of Salmorrelia in

eggs. Recombinant transducing phage were introduced into artificially inoculated eggs.

Infection of target Salmonella by the phage resulted in luminescence, which could be

detected through the egg shell using a BIQ Bioview Image Quantifier. A 6 hour

preenrichrnent was sufficient for the detection of as few as 10 Salmonella cells per ml

present in the original sample

An advantage of these luminescent applications is the sensitivity of instrumentation

availabte to detect and quant* light output. A detection limit of less than 10

biolurninescent bacteria per millilitre of sample has been reported (Stewart and Williams,

1992), with inexpensive luminorneters detecting as few as 102 luminescent bacteria per ml

(Stewart, 1990). A limitation of these methods is the genetic manipulation required to

prepare the bacteriophage for the test.

1 -5.3 -2. Fluorescent bacteriophage assay:

In addition to using genes as reporter molecules in phage, Goodndge (1997) developed a

fluorescent bactenophage assay based on a method described by Hennes et al. (1995),

whei-e the genetic material of phage LGl of Escherichia coli O1 57:H7 was labelled with a

fluorescent probe. Attachent of the labelled phage to the surface of the target celk

could be visualized by epinuorescent rnicroscopy or altematively quantified by flow

cytornetry. The assay was successfûliy applied to artificially inoculated ground beef and

raw milk samples.

1.5 -3 -3. Metabolic inhibition:

Bacteriophage has been used in conjunction with turbidity and colonmetric rnethods as

well as conductance rnicrobiology (McIntyre, unpublished data). Listeria, Sahonella,

and E. coli were prepared for detection in semi-selective media according to their

respective protocols; with and without bacteriophage added. If the target organism was

present, the time for detection of sarnples containhg phage increased, if it occurred at d,

relative to the sarnples without phage. In this case, the lytic activity of bactenophage was

exploited as a means of confhming the target organism of interest was indeed responsible

for the positive test.

1.5.3.4. B W assay:

The only commercially available bacteriophage assay for Salmonella is the Bactenal Ice

Nucleation Diagnostic (BIND) test. The Salmonella-specific bacteriophage P22 has been

engineered with the bacterial gene responsible for an ice-nucleating protein. When

infected, the bactena produce ice crystals in supercooled water (Worthy, 1990).

Detection of fkeezing is aided by addition of a dye that fluoresces in supercooled water,

but is quenched and changes colour when the water freezes. The test can be completed in

2-6 hours and has a sensitivity of 10 CFU/rnl. No false positives were observed with non- . salmoneiiae and food material did not interfere with the assay (Wolber and Green, 1990).

1.6. Fluorescence

1.6.1. Principles:

Fluorescence has been used for decades in the field of microbiology, and more recently in

food microbiology. The term fluorescence refers to the light (luminescence) emitted when

a substance retums f?om an excited or higher energy state to its normal or lower energy

state. A fluorescent molecule is raised to a higher energy state by the absorption of

radiant energy such as W rays. Some of the absorbed energy is dissipated in collisions

with other molecules. When the molecule drops back to it's normal ground state, much of

the absorbed energy is emitted at a Iower energy (longer wavelength) than the absorbed

energy (White and Argauer, 1970). The change in energy states results in fluorescence

and is illustrated in Figure 1.4. Instruments for fluorescence analysis consist of two

essential units; a source of exciting energy, and a means of observing or measuring the

intensity of the fluorescence emission.

1.6.2. Fluorescence as a means of determining cell viability:

Fluorescence has been used as a means of determining ce11 viability. To this end, several

fluorescent stains have been investigated. Rhodamine 123, which stains viable cells based

on transmembrane potential, was a moderate predictor of ce11 viability (Matsuyama,

1984). Dead ceUs or cells with a dissipated transmembrane potential showed markedly

diminished fluorescence. Gram positive bacteria stained well with rhodamiie 123, but half

of the 14 Gram negative strains tested stained sparsely (Matsuyama, 1984). Kasprelants

and Kell (1992) concluded that flow cytometry using rhodamine 123 was an effective

method for the rapid assessrnent of bacterial viability. In another study rhodamine 123 and

carbocyanine dyes were found to exhibit only small changes in fluorescence between

GROUND

Figure 1.4. Energy level schematic diagram illusmting energy changes involved in absorption and fluorescence. A photon of energy hv,, is supplied by an extemal source such as an incandescent lamp or a laser and is absorbed by the fluorophore, creathg an excited singlet state (S,, S2, S3). The excited state exists for only a fraction of a second, during which time some of the energy is dissipated. The dotted lines indicate energy dissipation without producing fluorescence. A photon of light, hv,, is emitted, rehiming the fluorophore to its ground state, S,. Due to energy dissipation, the fluorescence ernission is at a longer (less energy) wavelength than the absorbed energy (Adapted fiom White and Argauer, 1970; Johnson, 1996).

viable and non-viable populations of bacteria using flow cytometry (Mason et al., 1995).

In the same study, calcafluor white and oxonol dye (bis 1,3-dibutylbarbituric acid

trimethine oxonol) were much more usefùl (Mason et al., 1995). Ethidium bromide, a

nucleic acid stain, was investigated to assess the viability of Pseudomonas after fkeeze

thawing (Puchkov and Melkozemov, 1995). Ethidium fluorescence increased with

decreased viability, as disruption of the ce11 led to interaction of the fiuorochrome with

intracellular nucleic acids. Ethidium bromide was determined to be a simple, low cost and

rapid means of comparative bacterial viability assessment. A novel viability stain has been

developed by Molecular Probes that involves two fluorescent cornponents, a live stain,

SYTO 9, and a dead stain, propidium iodide (Molecular Probes Product Information).

The live and dead staining is based on the permeability of the ce11 membrane. #en used

together, live cells fluoresce green and dead cells fluoresce red when excited by blue light.

1 -6.3. Direct Epifluorescence Filter Technique:

One of the earliest applications of fluorescence in food microbiology was the direct

epinuorescence filter technique (DEFT). It was onginally developed for counting bactena

in milk and was suitable for milk containing between 5x10~ to 5x10~ bacteria per mi

(Pettipher et al., 1980). DEFT has subsequently been used for a vanety of other

applications, including estimating bacterial counts on meat, poultry and food contact

surfaces (Shaw and Farr, 1989; Holah et al., 1988). Microorganisms are recovered kom

the sample by the use of membrane filtration, and stained with the fluorescent nucleic acid

stain, acridine orange. Cells are then counted by means of a fluorescent microscope.

Acridine orange has been used as a vital stain, based on the colour of fluorescence

reflecting the RNA to DNA ratio in the cell, but this has not always been proven reliable

(Pettipher, 1983).

In this present study, the advantages of IMS as an alternative to selective enrichment, the

speciscity of bacteriophage and the sirnplicity of the novel Molecular Probes fluorescent

viability stain were exploited in the development of a rapid assay for the detection of

Salmonella Enteritidis in food. The assay exploits the normal infection cycle of a lytic

bacteriophage, thus requiring no genetic manipulation. The endpoint of the assay is

evaluated by monitoring phage infection. When bacterial cells are lysed by phage, they

are no longer viable, and the amount of green fluorescence in the sample decreases. This

decrease in fluorescence can be quantified, either by microscopie count by a modified

DEFT count, or by fluorescence measurements of cells in suspension.

1.7. Objectives

The objectives of this study were to:

Develop a bacteriophage-based assay to detect Salmonella Enteritidis in a timely,

inexpensive and technically simple manner without the need for manipulation,

genetic or othenuise, of the biological species involved.

Determine the endpoint of the assay by simple, standard, and inexpensive

laboratory instrumentation.

Evaluate the specificity of the assay for SaZrnonelia Enteritidis, other Salmonella

serotypes as well as other bacteria.

Investigate the use of the assay for the detection of Salmonella Enteritidis in

skimmed milk powder, gound chicken, and liquid whole egg.

Investigate the use of the assay with a different phagehost systern, namely phage

LGl and host Escherichia coli 0 157:H7.

The assay would capitalize on the inherent specificity of bacteriophage for its target

bacterium, and be based on the normal infection cycle of bacteriophage in its host. As

such the assay should be easily applicable to other bacteriophage and host combinations.

In addition, the assay would detect only viable bacteria, leaving those target bactena

killed by disinfectants or other bacteriocidal activities undetected.

2. Development and characteruation of a bacteriophage assay for the detection of Salmonella Enteritidis.

2.1. Abstract

Salmonella is the second most common source of foodborne illness in Canada and the

United States, perhaps responsible for millions of cases of gastroenteritis a year. Children,

the elderly and the immunocompromised are particularly susceptible. Many rapid methods

are available for the detection of SnIrnoneIla in foods but are often insensitive, expensive

or require a high degree of technical ability to perform. This study describes the

development and characterization of a novel assay that utilizes the normal infection cycle

of bacteriophage SJ2 for the detection of Salmonella Enteritidis in broth. Initial

experiments with a modified direct epifluorescent filter technique (DEFT) demonstrated

that the bioiogy of the bacteriophage could be exploited for the detection of S. Enteritidis.

To make the assay more applicable to food samples, subsequent experiments were

performed using irnrnunomagnetic beads as a separation method, and fluorescence and

optical density as the endpoint. The lower detection limit in broth was calculated to about

104 CFU/ml. The results of this study demonstrate that the bacteriophage assay is a rapid,

simple and sensitive technique for the detection of Salmottella Enteritidis in broth culture.

2.2. Introduction

Salmonella was first identified as a human pathogen in 1888 following a German outbreak

caused by an organism subsequently known as Salmonella Tyhimurhm (Tauxe, 199 1).

The most cornmon clinical manifestation of salmonellosis is gastroententis, characterized

by diarrhea, crarnps, vomiting and often fever. Salmonellae are ubiquitous in the

environment and primarily reside in the intestinal tract of birds, reptiles, f m anirnals and

humans. Transmission to man is usually foodbome from eating undercooked meat, miik

or eggs, or by cross-contamination to other foods which are eaten without cooking.

Four trends in Salmonella infection have been identified in the 1990s suggesting

Salmonella will continue to be a challenge to public health (Tauxe, 1991). These trends

include increased antirnicrobial resistance of SuZmoneIZa to one or more rnicrobial agents,

the exacerbation of salmonellosis in irnmunocompromised individuals, the association of S.

Enteritidis with grade A shell eggs, and lastly the occurrence of large and dispersed

outbreaks linked to large-scale production and distribution of foods. A multiple-antibiotic

resistant strain of S. Typhimurium found in pasteurized milk was responsible for a large

outbreak in the U.S. involving an estimated 180,000 cases (Ryan et al. 1987).

SalmonelIu is most fiequently associated with eggs and poultry, but outbreaks have been

linked to other sources such as cheese, pork products, water, and citrus juices (D'Aoust et

al. 1985, Wegener and Baggersen, 1996, Angulo et al,, 1997, Parish, 1998). In many

cases, improper handling and preparation of food in homes and institutions are responsible

for salmonellosis outbreaks (Bryan, 1980).

Conventional cultural methods for the enurneration of SalmonelZu require 72 to 96 hours.

This cm cause prolonged and expensive storage of foods prior to distribution. Advances

have been made in shortening the time required for Salmonella detection in food.

Antibody-based tests, such as ELISA tests, have decreased detection times. Non-specific

binding of competing organisms can lead to a lack of specificity, however, and long

enrichment steps may be required to reach detection limits (Patel and Williams, 1994).

Nucleic acid-based tests, although they can be highly specific, are often labour intensive

and require a high degree of technical skill. As such they may not be suitable for routine

analysis of food samples.

Irnmunomagnetic separation uses paramagnetic beads coated with covalently bound

afitibodies against specific surface markers on the target rnicroorganism. With the aid of a

magnetic particle separator, target cells can be pulled out of a food pree~chment,

eliminating the need for selective enrichment and shortening assay time by as much as 24

hours (Mansfield and Forsythe, 1993).

In addition to these immunological and genetic applications, bacteriophage assays have

been developed, where reporter genes, such as the lux genes responsible for bacterial

luminesence, are introduced into the phage DNA, producing light when these genes are

expressed in the target cells. Stewart (1990) reports that target bactena present at levels

greater than IO3 per mi, can be detected by recombinant lm+ bacteriophage in a food

matrix without enrichment.

The BIND assay is the only comrnercially available bacteriophage assay for the detection

of Salmonella in food, and is based on the production of ice nuclei upon infection by

phage. (Worthy, 1990). A sensitivity of 10 CFU/ml have been reported, and food

material was found not to interfere with the assay (Wolber and Green, IWO). The major

limitation of these bacteriophage assays is the genetic manipulation required to produce

the detecting agent.

This study describes the development and characterization of a bacteriophage assay for the

rapid detection of Salmoizella Enteritidis in broth cultures. IMS was employed to separate

and concentrate target Salmonella. Additional assay specificity was inherent with the use

of bacteriophage. The end point of the assay was detected either by optical density at 600

nm or by fluorescence using Molecular Probes LIVE/DEAD' BacLight" bactenal viability

stain.

2.3. Materials and methods

2.3.1. Bacterial strains:

Forty-one strains were used for enurneration, recovery and specificity studies. Tables 2.1.

2.2 and 2.3 outline the 30 strains of non-Enteritidis Salmonella, 5 strains of Salmonella

Enteritidis and 6 strains of non-Salmonella bacteria used in this study. Cultures were

obtained from Health Canada, the Center for Disease Control and the University of

Guelph, Department of Food Science culture collection.

Stock cultures were maintained fiozen at -20°C in 15% glycerol. Fresh bacterial cultures

for use in experiments were produced by inoculation of frozen stock cultures ont0 L-agar

able 2.1. Non-Enteritidis SaZrnonella strains used for the specincity shidy.

Serogroup Bacterial Species Strain Ongin

Group B

Group C

S. Typhimurium

S. Typhimurium

S. Heidelberg

S. Saintpaul

S. Bredeney

S. Schwartzengmnd

S. Schwartzengnuid

S. Agona

S. Indiana

S. Brandenburg

S. Reading

S. Infântis

S. Thompson

S. Mbandaka

S. Braenderup

S. Montevideo

S. Ohio

S. Oranienburg

S. Tenenssee

S. Johannesburg

S. Urbana

S. Rubislaw

S. Hadar

S. Kentucb

Hedth Canada

LCDCa

Heaith Canada

Heaith Canada

Heaith Canada

LCDCa

Heal th Canada

Health Canada

Health Canada

Health Canada

Health Canada

Heaith Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada

Health Canada " Laboratory, Center for Disease Control.

39

Table 2.1. continued. Non-Enteriticlis SuZmonelh strains used for the specificity study.

Serogroup Bacterial Species Strain Ongin

Group C S. Haardt SA 9 14074 HeaIth Canada

I S. Choleraesuis S-870 LCDCa

1 Group D S. Berta SA 941 123 HeaIth Canada

I S. Panama SA 93 1592 Health Canada

S. Sendai S-5 13 LCDCa " Laboratory, Center for Disease Control.

Table 2.2 Bacteriophage assay and plaque assay specificity results for Salmonella Enteritidis strains.

Bacterial species S train Ongin

S. Enteritidis SA 932451 Health Canada

1 S. Enteritidis Laboratory strain Egg

1 S. Enteritidis ATCC 13076 ATCCa

1 S. Enteritidis SA 94245 1 Health Canada

S. Enteritidis EN 2588 LCDC~ I " Amencan Type Culture Collection

Laboratory, Center for Disease Control

Table 2.3. Bacteriophage assay and plaque assay specificity results for non-Salmonella strains.

Bacterial species S train Origin

Serratia marsescens ATCC 8 100 ATCCa

Klebsiella pneumoniae ATCC 1388 ATCC

Citrobacter fieundii Laboratory S train University of ~ u e l p h ~

Shigella flemeri Laboratory Stmh University of ~ u e l p h ~

Escherichia coli ATCC 25922 ATCC

Escherichia coli 015 7:H7 EC 920333 Health Canada " Amencan Type Culture Collection

Department of Food Science

plates (1% tryptone (Difco Laboratories, Detroit, MI), 0.5% sodium chloride @$CO),

0.5% yeast extract (Difco) and 1% agar (Difco)). Plates were incubated overnight at

37T. Broth cultures were prepared by inoculating L-broth with cells from an L-agar

plate and incubating them ovenllght, with shaking, at 37OC.

2.3 -2. Bacteriophage and host:

The bacteriophage and host used in this study, SJ2 and S. Enteritidis respectively, were

obtained £tom Dr. Sabah Jassim, Department of Food Science, University of Guelph.

Both the bacteriophage and the host had been isolated from egg. Phage SJ2 was amplified

in its host by the plate method. Ten-fold serial dilutions of undiluted phage stock (10''

PFU/ml) were prepared in lambda buffer (0.25% MgSOJHfl, 0.0005% gelatin, and 0.6%

1 M TRIS pH 7.2 in distilled water, adjusted to pH 7.2). From each dilution tube, 0.1 ml

of phage suspension was added in duplicate to 0.1 ml of an overnight culture of host.

Tubes containing phage and host were incubated for 10 minutes at 37°C to allow

attachment. Samples were then added to 2.5 ml of melted top layer agar (1% tryptone,

0.5% yeast extract, 0.5% NaCI, and 0.4% agar) cooled to 45°C. The mixture of agar,

phage and cells was poured quickly onto a Petri plate containing dried L-Agar and the

plate was swirled so that the mixture covered the entire plate. When the top layer had

hardened, plates were incubated overnight at 3 P C . Phage was recovered fiom the plates

by adding 8-10 ml of lambda buffer to a plate containing the highest dilution of phage.

Phage was harvested with a glass hockey stick and the resulting lysate (bufer, phage, host

cells, and media residue) was transferred to the next highest dilution. Plates were washed

sequentially, transferring lysate to sterile tubes and using ffesh buffer as required.

Following phage recovery, chloroform was added to the lysate tube at a 1:3

chloroform:lysate ratio. Lysate was put on ice for 10 minutes to facilitate recovery of

phage from unlysed cells. The lysate suspension was centrifuged at 5000x g for 15

minutes. The supernatant was withdrawn and filtered through 0.2 prn pore size syringe

filters (Nalgene, Rochester, New York).

Phage was enumerated by the plaque assay method. Ten-fold serial dilutions of phage

were prepared in lambda buffer. From appropriate dilution tubes, 0.1 ml of phage

suspension was added in duplicate to 0.1 ml of an ovemight culture of host S. Enteritidis.

Tubes containing phage and host were incubated for IO minutes at 37°C to ailow

attachment. SampIes were then added to 2.5 ml of melted top layer agar cooled to 4S°C.

The mixture of agar, phage and cells was poured quickly ont0 a Petri plate containing

dned L-Agar and the plate was swirled so that the mixture covered the entire p!ate. When

the top layer had hardened, plates were incubated overnight at 37OC. Plaques were

counted, and the phage titre in the original tube was calculated.

2.3.2.1- One-step growth experiment:

In order to charactenze phage SJ2, a one-step growth experiment was performed to

determine a) the 'burst tirne' (the period between adsorption and lysis) and b) the 'burst

size' (the average number of phage particles released per infected host cell). The method

used was adapted from Maramorosh and Koprowski (1967). Forty microlitres of an

overnight culture of S. Ententidis was inoculated into 40 ml of L-broth. The culture was

incubated at 37OC, shaking, for 2.5 hours. The tube was centrifuged at 5000x g for ten

minutes in a Sorvall RT6000 refngerated centrifuge (DuPont Inc., Mississauga, Ontario).

The pellet was resuspended in 2 ml of L-broth. The phage population used was 5x10'

PEZl/ml. Three tubes containing 10 ml of L-broth were labeled D L , GT-1, and GT-2

(dilution tube, first growth tube, and second growth tube).

The schedule of the growth experiment is outlined in Tables 2.4 and 2.5. Plating occurred

by adding 0.1 ml of sarnple from GT-1 or GT-2 to 2.5 ml of top layer agar containing 0.1

ml of host culture. Samples were immediately poured ont0 solidified L-agar plates and

incubated ovemight at 37OC. Following counting of plaques, the relative titre of each

plate was divided by the titre of plate 1 and ptotted linearly as a function of time. The

percentage of adsorption during the first four minutes was calculated by comparing the

count on plate 13 with the count on plate 1.

2.3.3. Procedure for analysis - Microscopy protocol:

Ten microlitres of bacteriophage SJ2 (2x10~ PFUI10 pl) was added to a microfuge tube

containing 100 pl of stationary phase culture of S. Enteritidis. Tubes were incubated for

15 minutes at 37OC to allow for phage at tachent and were then centrifùged at 14,000

rpm for 10 minutes to remove unattached phage. The pellet was resuspended in 100 pl

stationary phase host (approxùnately 2x10~ CFU/lOOpl) to act as a 'helper'. Samples

were incubated for 1 hour at 37°C. Dunng this incubation penod, progeny phage fiom

Time (minutes)

Table 2.4. Schedule for one-step growth experiment (adapted from Maramorosch and Koprowski, 1967).

O 0.1 ml phage was added to 0.9rnl of cells in Eppendorf tube, incubated at 3 7OC, shaking

4 0.1 ml fiom Eppendorf to DIL (1 : 100 dilution)

4.5 0.1 ml fiom DIL to GT-1 (1 : 10,000 dilution)

" DL, dilution tube; GT-1, first growth tube; GT-2, second growth tube.

5 2 ml sample fiom GT-1 was cenûifbged for 8 minutes. Supernatant was titrated for phage

7 0.1 ml fiom GT- 1 to GT-2 (1 : 1,000,000). Sarnples (O. 1 ml) are taken in sequence fiom GT-1 and GT-2 and plated for plaque forrning units according to the schedule in Table 2.5.

Tabte 2.5. Schedute for plating growth tubes (GT-I and GT-2) (adapted fiom Maramorosch and Koprowski, 1967).

Tirne" (minutes) Frorn GT-1 From GT-2

Plate I

-

Plate 3

-

Plate 5

-

Plate 7

-

Plate 9

-

Plate 1 1

-

-

PIate 2

-

Plate 4

-

Plate 6

-

Plate 8

-

Plate 10

-

Plate 12

a Note: Between minutes 12 and 20, the centrifuged supernatant sample was ready, and 0.1 ml was assayed on Plate 13.

the original cell population would be released and the helper population would be

influenced based on the number of target cells present initially. Following incubation, 900

pl of lambda buffer and 10 pl of Molecular Probes LIVE/DEAD@ Baclight- bacterial

viability stain solution were added. Each 10 pl of dye solution contained 1.5 pl dye A

(live stain) and 1.5 pl dye B (dead stain) in lambda buEer, which was consistent with

Molecular Probes recornmendation of 3 pl of dye AB per millilitre of sample.

2.3.3.1. Epifiuorescent microscopy:

One millilitre samples were filtered using a lOcc syringe (Becton Dickinson & Co.

Franklin Lakes, NJ) ont0 13 mm diameter, 0.2 pl black polycarbonate membrane filters

(Millipore Corporation, Bedford, MA) housed in Swimex filtration devices (Millipore).

Foliowing filtration, the membrane was removed fiom the filtration device and placed on a

glas slide, a drop of Baclight Mounting oil (Molecular Probes, Eugene, OR) was added,

and the filter was covered with a cover slip. The samples were viewed with a Nikon

Labophot Microscope with epduorescence equipment (Nikon Corporation, Mississauga,

ON) with an excitation range of 450-490 nm. Clumps were counted according to a

modified DEFT procedure.

2.3 -3.2. Bacterial counts:

The micoscopy protocol relied on the ability to accurately enurnerate fluorescently stained

bacterial cells. The epifluorescent technique (DEFT) described by Pettipher (1983), was

modified to enumerate Live and dead cells using the Molecular Probes LIVE/DEAD@

BaclightTM bacterial viability stain. The resulting DEFT counts were CO rrelated to

standard plate counts. Ten-fold dilutions of an overnight culture of S. Enteritidis were

prepared in lambda buffer. CeUs were stained according to the procedure outlined in 2.3.3

and lei? in the dark for 15 min. Ten randomly selected fields of view were counted and

averaged. The DEFT count per ml was calculated by multiplying the average clump count

by a microscope factor. The microscope factor was calculated as folows:

Microscope factor = Area of membrane throueh which sample is filtered !mm2) Microscopy field area (mm2) x Sarnple volume (ml)

The filter area was calculated from the interna1 radius of the filter membrane (n x radius2)).

The area of the microscope field of view was calculated from the radius of the field of

view determined with a hemocytometer.

2.3.4. Procedure for analysis - IMS protocol:

2.3 -4.1. IMS, phage attachent and amplification:

Flowcharts for the IMS protocol are illustrated in Figures 2.1 and 2.2. Twenty microlitre

volumes of Anti-Salmonella ~ ~ n a b e a d s @ @ynal Inc., Lake Success, NY) were added to

rnicrofbge tubes containhg 1 ml of stationary-phase culture of S. Enteritidis. Samples

were rotated at 30rpm for 30 min at room temperature on an Orbitron Rotator II (Fisher

Scientific, Mssissauga, ON). Sampies were placed in a magnetic rack @pal Inc., Lake

Success, NY) in order to separate the magnetic beads from the broth. The beads were

washed two times in lambda buffer, and resuspended in 500 pl L-broth. One hundred

microlitres of phage were added and samples were incubated for 15 min at 37OC to allow

for attachent. Magnetic beads were separated and washed twice in lambda buffer to

remove unbound phage. Beads were resuspended in 100 pl of lambda buffer and

incubated for 30 min at 37OC to allow for release of progeny phage. Magnetic beads were

captured and the progeny phage recovered in the supernatant was added to a 1 ml volume

of helper S. Enteritidis ceUs in L-broth, adjusted to an optical density of 0.100

(approximately 1x10~ CN/rnl). Helper population and progeny phage were incubated at

37°C for one hour.

2.3 -4.2. Optical density:

Following incubation of progeny phage with the helper cells, the samples were transferred

to 1.5 ml disposable plastic cuvettes (Fisher Scientific, Mississauga, ON). Absorbance

was read at 600nrn in a Pharmacia Novaspec II Spectrohotometer (Arnersham Pharmacia

Biotech Inc, Uppsala, Sweden).

2.3 -4.3. Fluorescence staining:

Alternatively, samples were centrifùged at 14,000 rpm for 10 minutes in an Eppendorf

54 15 C bench top centrifuge (Brinkmann Instruments Inc., Westbury, NY). Supernatant

was discarded and the pellet was resuspended in 900 pl lambda buffer. Samples were

stained wit h 1 00 pl Molecular Probes LIVE/DEAD@ ~ a c ~ i g h t " bacterial viability stain

solution. Dye was prepared such that a 100 pl portion of dye solution contained 1.5 pl

each of the [ive stain (component A) and the dead stain (component B) in lambda buffer.

Sarnples were left at room temperature in the dark for 15 minutes.

2.3 -4.4. FLSOO fluorometer:

Four 200pl portions of each lm1 sample were distributed into Coming clear 96 well

microtitre plates (Fisher scientific, Mississauga, ON). Samples were read in the FL500

fluorescence plate reader (Bio-Tek Instruments, Winooski, VT). Excitation was 485 nm

and emission was 530 nm, with band-widths of 20 and 25 nm respectively.

2.3.4.5, MGM fluorometer:

Samples were distributed into 6 x 50 mm glass tubes (Fisher ScientSc, Mississauga,

Ontario) and read immediately in the MGM fluorometer (MGM Instruments, Hamden,

Connecticut). Excitation wavelength was 460 nm and emission wavelength was 5 10 nm.

2.3 -4.6. Efficiency of immunomagnetic separation:

An ovemight culture of S. Ententidis was serially diluted IO-fold in L-Broth to a dilution

of 10". The cell concentrations in the six dilutions were estimated to be 1 08, IO', 106, los,

1 04, and lo3 CFU/ml. Irnrnediately after dilution, the efficiency of magnetic capture was

tested on each dilution in duplicate. To determine the ce11 populations in the initial

dilutions, duplicate spread plate counts were performed. Following magnetic capture, the

beads were resuspended in 1 ml of lambda buEer and duplicate counts of each sample

were performed. Dilutions were made as required to enumerate sarnples containing high

numbers of ceus. Ail plates were incubated ovemight at 37°C. colonies were counted and

the resulting counts were compared to the number ofCFU/rnl in the original dilutions.

2.3.5 Assay parameters:

2.3 -5.1. Temperature:

The IMS assay was tested at three temperatures (30°C, 37OC, and 42°C) to determine the

optimum temperature for the assay. Each incubation period. attachent, amplification,

and helper incubation were performed at one of the three experimental temperatures.

Positive and negative samples were tested in duplicate at each experimental temperature.

2.3.5 -2. Phage population:

The IMS assay was performed using three phage populations (Io6, 10'. and 108 PFU/100

pl) to determine the optimum phage concentration for the assay. Positive and negative

samples were tested in duplicate at each phage population. The experirnent was

conducted twice.

2.3 -5.3. Media:

The L-broth used for the finai step in the assay interfered with the fluorescent dye,

necessitating a centrifugation step to wash cells and remove media residue pnor to

fluorescent staining. An alternative to L-broth, whkh would not interfere with fluorescent

staining, was sought in order to eliminate the washing step. The media would have to

support both the growth of helper cells in the absence of phage, and the reduction of

helper by phage. Prelirninary studies suggested that lambda buffer was not a suitable

alternative- It was thought that the addition of sugars to the buffer rnay increase the

performance of the buffer. Two sugars were tested (dextrose and maltose), at three

concentrations (02.%, OS%, and 1.0%) in duplicate. These samples were compared with

L-broth and lambda buffer, also tested in duplicate. Optical density readings at 600 nm

were taken of al1 sarnples at time O, 1 h, 1.5 h and 2 h.

2.3 -5 -4 Freeze-dried helper:

Because of the potential problems with maintainhg and manipulatirtg iive SaImortella

stock cultures in a food lab, the use of a fieeze dried helper population was investigated as

an alternative to an overnight culture. An ovemight culture of S. Enteritidis was diluted 1

in 10 in L-broth and 1 ml samples were distributed into stenle Eppendorf tubes. Samples

were fiozen ovemight at -20°C on a slant, and then transferred to a -80°C freezer for 30

min. Small holes were pierced in the top of the tubes and they were placed in a Lyph-

~ o c k @ flask (Labconco Corp., Kansas City, MO) and freeze-dried for 21 h at 5 . 7 ~ 1 0 ~ ~

mBar vacuum pressure in a Labconco ~ ~ ~ h - ~ o c k @ Freeze Dry System (Labconco Corp.).

The titre of the cell population in two samples was determined before and aîter freeze-

drying by duplicate plate counts on L-agar. Samples were reconstituted in 1 ml of lambda

buffer immediately prior to use.

The feasibility of freeze-dried helper cultures for use with the bacteriophage assay was

determined. At the start of the protocol, four freeze-dried samples were reconstituted in 1

ml of lambda buffer and incubated at 37°C until ready for use. The IMS assay was

conducted with four negative sarnples (broth only) and four positive samples (1 in 10

dilution of an overnight S. Enteritidis culture) as outlined in section 2.3.4.1. Following the

amplification step, the average optical density (600 nrn) of the fieeze-drïed helper ce11

samples was determined. An ovemight culture of S. Enteritidis was adjusted to the same

optical density. The progeny phage recovered h m the assay samples were divided such

that there were duplicate positive and negative sarnples for each helper type. The

overnight helper population and the fieeze-dned helper population were compared for

their ability to support the growth of helper bactena in negative samples, and the reduction

in helper ceil numbers in Salmonella positive sarnples.

2.3 -6. Scanning electron rnicroscopy :

Four samples containing 1 ml of ovemight S. Ententidis culture and 20 pl

immunomagnetic beads were incubated at room temperature on an Orbitron Rotator II at

30 rpm for 30 minutes. The samples were washed twice in lambda buffer and resuspended

in 0.5 ml of lambda buffer. To two of the samples, 100 pl of bacteriophage SJ2 were

added, and sarnples were incubated at 30 rpm on an Orbitron Rotator II for 10 minutes at

room temperature. Samples were washed once and beads were resuspended in 0.5 ml

lambda buffer. The samples were filtered with the use ofa Buchner vacuum filtration unit

ont0 a 0.2 pm polycarbonate membrane filter (Poretics Corp. Livermore, California)

situated in a 13 mm syringe apparatus (MiIlipore, Bedford, Massecheusses). The filter

was transferred to 2% glutaraldehyde in Sorensen's phosphate buffer (SPB, 1: 1 ratio of

0.07 M Na2B0, 7H20 and 0.07 M KHJ'O, pH 6 . 9 for one hour a t room temperature

to fix proteins. Filters were rinsed three times in SPB, flooded with 2% osmium

tetraoxide and left in the dark for one hour at room temperature for lipid fixation.

Samples were rinsed three times in SPB and gradually dehydrated in a graded senes (50%.

70%, 80%, 90%, 95%, 100% (three times)) of ethanol for 10 minutes at each level. The

sarnples were cntical point dried with CO, in a LADD cntical point dryer (LADD

Research Industries, Burlmgton, Vermont). The samples were rnounted on a specimen

stubb, sputter coated with goldlpalladium in a Polaron SC500 sputter coater ( Soquelec,

Montreal, Quebec), and scamed in a Hitachi S-4500 Scanning electron microscope

@tachi, Tokyo, Japan) at a 7 and 25 keV accelerating voltage.

2.3.7. Transmission electron microscopy:

Bacteriophage SJ2 was prepared for transmission electron microscopy by negative

staining. A drop of the phage suspension was placed on parafilm and a formvar/carbon

coated copper grid (200 mesh) (Marivac Ltd., Halifax, Nova Scotia) was floated on top of

the sample for 2 minutes. The copper grid was blotted dry, and was floated, sample side

down, on a drop of 1% wt/vol aqueous uranyl acetate (Fisher Scientific, Nepean, ON) for

2 minutes and blotted dry. The sample was viewed in a Phillips EM300 transmission

electron microscope (Phillips Electrical Corp. New York, NY) operating at 60 keV with a

liquid nitrogen cold trap.

2.3 -8. Bacteriophage specificity:

Bacteriophage SJ2 was tested against 37 bacterial stains (Table 2.1-2.3) to determine its

specificity. Plaque assays were performed, based on a modified method for the

quantitative assay of phage outlined by Maramomsch and Koprowski (1967). Phage SJ2

suspension (0.1 ml) and 0.1 ml of an ovemight culture to be tested were added to top

layer agar cooled to 45OC. The mixture of phage, cells and agar was quickly poured onto

Petri plates containing hardened L-agar and swirled to cover the plate. When the top

layer agar had hardened, plates were incubated overnight at 37OC. Plates were observed

for the presence of plaques. The IMS bacteriophage protocol was performed on overnight

cultures of al1 41 strains, diluted 1 in1 O in L-broth.

2.3.9. Statistical analysis:

Linear regression comparing plate counts with DEFT counts and IMS plate counts was

performed using Quattro-Pro (Corel Corporation, Ottawa, Ontario). Unless otherwise

indicated, all statistical analysis involving treatment cornparisons was performed using a

one way analysis of variance (ANOVA) at a signincance level of a = 0.05 in Quattro-Pro

(Corel). Where statisticdly significant ciifferences existed, post hoc analysis was

performed using Scheffe' s contrast test (Scheffe, 1959).

2.4. Results

2.4.1. Bacteriophage and host:

A transmission electron rnicrograph of bactenophage SJ2 showed a head and a long tail

(Figure 2.3). The base plate was visible but tail fibres, if they exist, were not. Scanning

electron rnicrographs of bactenophage SJ2 attached to S. Enteritidis are shown in Figure

2.4.

2.4.2. One-step growth experiment:

The propagation curve of phage SJ2 (Figure 2.5) shows a burst time of approximately 30

minutes with a burst size of approximately 100. Companng the plaque count of plate 13

with plate 1 indicates that approximately 97% of the phage had adsorbed to the host celis

during the first four minutes.

2.4.3. Microscopy protocol:

Ten-fold dilutions of S. Enteritidis were evaluated by the centrifugation and epifluorescent

rnicroscopy protocol. The average counts obtained fiom Salmonella positive samples

were compared with samples containing no Sa/moneZZa initially. Live (green) celis were

counted, as prelirninary studies (not shown) indicated dead (red) ce11 counts were a poor

representation of what was occurring in the assay. DEFT counting methodology requires

Figure 2.3. Transmission electron micrograph of bactenophage SJ2. Bar =IO0 nrn. Ma@cation, x 207,850.

Figure 2.4. Scanning electron microgaphs of bacteriophage SJ2 attached to S. Enteritidis. A) Bar = 900 nm. Magnification, x 20,000. B) Bar = 360 nrn. Magnification, x 50,000.

20 25 30 35 Time (minutes)

Figure 2.5. Resdts of one-step growth experirnent for phage SJ2. Lysis occurs d e r approximately 3 0 minutes, with a burst size of about 1 00, observed within 42 minutes.

that any cell, or group of cells, separated by a distance less than twice the diameter of the

cells nearest each other, be counted as one clump. Theoreticaiiy, one clump would be

responsible for one colony forming unit on solid agar. This convention was adopted in

the assay. The results of one of the trials is illustrated in Figure 2.6. At initial ce11

populations of 1o3, Io4, and 10' CFU/ml, the expected decrease in clump counts was

observed relative to the Salmonella negative sample. A one way analysis of variance

(ANOVA) showed a statistically significant difference between samples. The calcuiated

F-value was 154, giving a p value <0.001 (F,, = 2.9). Post hoc analysis using Scheffe's

contrast test demonstrates there was a significant difference between the negative samples

and the positive samples at the 95% confidence level. Likewise, the 103 CFU sample was

significantly different from the IO4 and 10' CFU levels. The latter two were not

signifïcantly dEerent from each other. These results were confirmed in two additional

trials.

2.4.3-1. Bacterial counts:

Figure 2.7 illustrates the relationship between standard plate counts and DEFT counts. In

al1 instances, live (green) cells were counted, as dead (red) ce11 counts were not a good

indicator of phage activity. DEFT counts generally correlated well with plate counts (i =

0.89). Figure 2.8 is an epinuorescent rnicrograph of a stationary phase S. Enteritidis

culture stained with the BacLightm bacterial viability stain and filtered on a 13mrn black

polycarbonate filter.

O 7x1 O= 7x1 o4 7x1 o5 Initial Population (CFUI100pf)

Figure 2.6. Average clump counts following microscopy protocol for four initial populations of S. Enteritidis. Bar represents one standard deviation.

4 5 6 Log,, , plate countlrnl

Figure 2.7. Relationship between plate count and DEFT count for different dilutions (1 O-', 1 05, 1 O-4, 1 O") of S. Enteritidis in buffer, tested in triplicate. Line represents fitted regression line (y = 0 . 8 3 ~ + 0.52, ? = 0.95)

Figure 2.8. Epifluorescent micrograph of S. Ententidis stained with Molecular Probes LISEDEAD@ BacLight" bacterial viability srain. Live ceiis fluoresce green, dead ceUs fluoresce red.

2.4.4. IMS protocol:

2.4.4.1. Optical density:

Ten-fold serial dilutions of S. Enteritidis were prepared in L-broth. Dilutions representing

plate counts of 102, 10" 104, los, and 106 CFUIml, were tested in duplicate. Three trials

were performed. For each sample, the absorbance value was calculated as a percentage of

the mean of the negative control values for that trial. The results over al1 three trials were

pooled and are illustrated in Figure 2.9. The sensitivity of the assay with pure cultures in

broth is approximately 104 CFU/rnl. If a value of 70% of the mean of the negative control

value is used as the cut-off for a positive sample (determined in section 3.4.1), then none

of the samples tested in the 1o3 CFU/ml range were positive. Two of the 10' CFU/ml

samples were determined to be 1.5xl0'CFU/ml by plate count. These samples were both

exactly 70% of the negative control after two hours incubation. The other four samples in

the 104 range were determined by plate count to be above 5x10' CN/ml and had values

55% or lower. Samples above IO4 CFülrnl were al1 clearly positive as weli.

2.4.4.2. Fluorescence:

The assay was performed as indicated in section 2.4.3.1. Following optical density

readings, cells were stained and read in the FL-500 fluororneter. As expected, the

resulting distributions were very similar to those obtained by the absorbance readings.

The sensitivity of the assay with pure cultures in broth is comparable to the optical density

6x10' 6x10' 6 x 1 0 ~ 6x1 0' 6 x 1 0 ~

Mean Initial Population (CF Ufrnl)

Figure 2.9. Percent of negative control value for five populations of S. Enteritidis in broth following TMS-bacteriophage assay. Values s h o w are averages of three trials. Endpoints were determined by optical density. Bar represents one standard deviation.

results of approximately104 CFU/ml. The lowest population for which a positive test was

observed was 8xid CFU/ml. When the MGM tluorometer was used for one of the trials,

it was noted that the detection Limit was closer to 1x10' CFU/ml, even though absorbance

readings taken before fluorescent staining indicated a lower detection S i t by more than

two log cycles. A second trial showed a difFerence in detection limit by more than one log

cycle in favor of the spectrophotometer.

The sensitivity of the MGM Fluorometer and the spectrophotometer were compared to

determine their ability to detect low numbers of cells. An ovemight culture of S.

Enteritidis was washed in lambda buffer and was serially diluted. Each 10-fold dilution

was then further diluted 2:3, and 1:3 in buffer to represent populations between full log

cycles. Tnplicate sarnples of each dilution were analysed in each machine according to

sections 2.3.4.2. and 2.3.4.4. The results are illustrated in Figure 2.10. and Figure 2.1 1.

The spectrophotometer was able to detect a bacterial population of approximately 6.35

log,, CN/ml . The MGM Fluorometer was more sensitive by approximately one log

cycle. At the lowest dilution tested (5.35 log,, CFU/ml), the MGM fluororneter signal

was more than one standard deviation over the blank.

2.4.4.3. Efficiency of irnrnunomagnetic separation:

A cornparison of plate counts following IMS was compared to spread plate counts for six

dilutions of S. Enteritidis. The results are illustrated in Figure 2.12. There was a good

correlation between plate counts and IMS plate counts (? = 0.99). The efficiency of IMS

Figure 2.10. Sensitivity of the Pharmacia spectrophotometer as indicated by mean optical density values for various populations of S. Enteritidis. Bar represents one standard deviation.

Figure 2.1 1. Sensitivity of the MGM Fluorometer as indicated by mean fluorescence values for various populations of S. Enteritidis. Bar represents one standard deviation.

I L I 1 1

3 4 5 6 7 8 9 Log,, plate countlml

Figure 2.12. Relationship between plate count, and plate count following MS for different dilutions of S. Enteritidis in buffer. Two replicates from each 10- fold dilution (1 03, 1 04, 1 os, 1 06, 10' and 1 o8 CFU/ml) were counted. Line represents fitted regression line (y = 0 . 8 9 ~ + 0.39, $ = 0.99)

was reduced at higher celi populations, presumably because of the larger ce11 to bead ratio.

Figure 2.13 shows scanning electron micrographs of S. Ententidis attached to

irnrnonumagnetic beads.

2.4.5. Assay parameters:

2.4.5.1- Temperature:

The bactenophage assay was tested at 30°C, 37OC and 42T. There was no significant

difference in helper reduction between 37°C and 42T, but these temperatures were

significantly better than 30°C (px0.05). Growth of helper appeared to be reduced at 30°C

relative to the other temperatures, but this was not significant at the 95% confidence level

(p = 0.15). As a result, 37°C was used for the assay, as this is a common incubation

temperature and there was not a 42OC incubator routinely available in the laboratory.

2.4.5.2. Phage population:

Three phage populations (Io6, IO7 and 108 PFU/100 pl) were tested in duplicate to

determine the optimum phage population for the assay. There was no si,-cant

difference in helper reduction when 10' and 10' PFU were used, but these phage

populations performed signincantly better than 106 PFU @< 0.05). This was confirmed in

a second trial. Efficient removal of unbound phage after the attachrnent step is very

important to the success of the protocol. As a result 10' PFU was considered optimal for

Figure 2.13. Scanning electron rnicrographs of S. Ententidis attached to immmunomagnetic beads A) Bar = 2.0 Pm. Magnification, x 9,000. B) Bar = 4.5 Pm- Madcat ion, x 4,000.

the assay, as removal of phage would presumably be more complete if a lower initial

population was added.

2.4.5.3- Media:

The L-broth used for the helper incubation step in the assay interfered with the fluorescent

dye, necessitating a centrifugation step to wash cells and remove media residue prior to

fluorescent staining. An alternative to L-broth, which would not interfere with fluorescent

staining, was sought in order to eliminate the washing step. In addition to cornparhg L-

broth and lambda buEer, two sugars were tested (dextrose and maltose), at three

concentrations (0.2%, OS%, and 1.0%). Results for dextrose are shown in Figure 2.14

and 2.15. Regarding reduction of helper by phage, an ANOVA perfomed on results at

1.5 hours resulted in an F value of 424 with a calculated p value of < 0.00 1 (F,, = 5.19).

Post hoc analysis using Scheffe's test indicated that at 1.5 hours. L-broth perfomed better

than al1 the other media, lambda buffer was the least acceptable, and there was no

significant difference between the rhree dextrose concentrations (p = 0.05). Results

obtained with the maltose samples were very similar to dextrose.

Regarding growth of helper, L-broth was superior to the other treatments at ail time

intervals, with the dextrose solutions perforrning only marginally better than the buffer

afone (Figure 2.15). Again, distributions for the maltose samples were similar to those

for dextrose.

- L-Broth --F

g 0.04 ' Buffer Cu -- -5- e g 0.03 - 6,

-'\ 0.2% Dextrose

2 - - - - -----,< - 0.02 - 0.5% Dextrose

- -t

0.01 O 0.5 1 1-5 2 1,0% Dextrose

Time (Hours)

Figure 2.14. Cornparison of L-broth, lambda buffer, and three dextrose concentrations (0.2%, 0.5% and 1.0%) as media for the reduction of S. Ententidis by phage S J2 at 3 7OC.

- O ' ,

- 0 0.5 1 1.5 2

Time (HOUE)

-e

L-Broth

3-

Buffer - 0.2% Dextrose

-E-

0.5% Dextrose

-t

1 .O% Dextrose

Figure 2.15. Comparison of L-broth, lambda buffer, and three dernose concenirations (0.2%, 0.5% and 1 -0%) as media for the growth of S. Enteriticlis at 37OC.

In another study, ammonium chlonde was added as a nitrogen source to lambda buffer

containhg 0.2% dextrose. When compared to 0.2% dextrose in buffer, there was no

significant dflerence for both growth of helper population and reduction of helper by

phage (p <O.OS).

None of the media treatments tested compared well with the L-broth, particularly for

facilitating growth of the helper population. A washing step will continue to be necessary

when fluorescence is used as an endpoint.

2.4.5.4. Freeze-dried helper:

One millilitre samples of helper bacteria were fieeze-dried and tested with the IMS assay,

in order to possibly eliminate the need for overnight growth of Salmonella cultures for the

completion of the protocol. A cornparison of the titre of samples before and after fieeze-

drying indicated a one log reduction in ce11 numbers following freeze-drying.

The recovery penod of fieeze-dried samples was investigated. Freeze-dried samples were

reconstituted in 1 ml of lambda buffer and incubated at 37T. When phage was added

following reconstitution, a decrease in optical density (600 nrn) was observed within one

hour. Growth, as measured by opticaI density, was observed within two hours.

Freeze-dried helper was compared with an overnight helper culture in the IMS assay.

Freeze-dned samples were reconstituted at the start of the protocol and incubated at 37OC

until required (approximately a 2.5 hour recovery period). The desired trend was

observed with the fieeze-dned sarnples, but the ovemight helper population perfomed

significantly better for both growth of the helper, and reduction of the helper by phage (p

< 0.001)

2.4.6. Specificity of bacteriophage SJ2:

Bacteriophage SJ2 was tested against 41 strains of bacteria to detennine its host range.

The results are presented in Tables 2.6-2.8. Al1 six non-Salmonella strains tested were

negative by both plaque assay and phage assay. Phage SJ2 infected al1 five of the S.

Enteritidis strains tested as detemined by plaque assay, and resulted in a positive test

when these strains were tested in the IMS phage assay. Phage SJ2 is not specific to S.

Ententidis however. Strong positive results for both tests were observed for S.

Typhimurium SA 942256 and S. Sendai. Interestingly, S. Typhimunum 94-51 was not

positive by either method. Other serotypes belonging to group D,, namely S. Berta and S.

Panama, showed somewhat inconsistent results for both the plaque assay and the phage

assay. Weak lysis was observed by the plaque method on two of four trials for both of

these organisms. In addition, these strains were positive by the phage assay the fïrst time

they were tested, but this was not confirmed in a second trial. It folIows then, that these

two strains would not be reliably detected by the IMS assay.

Table 2.6. Bactenophage assay and plaque assay specificity results for non-Enteritidis

Serogroup Bacterial Species Strain Plaque assay / Phage assay

Group B

Group C

S. Typhimurium

S. Typhimurium

S. Heideiberg

S. Saintpaul

S. Bredeney

S. Schwartzengnuid

S. Schwartzengnrnd

S. Agona

S. Indiana

S. Bmdenburg

S. Reading

S. Infantis

S. Thornpsoa

S. Mbandaka

S. Braenderup

S. Montevideo

S. Ohio

S. Oranienburg

S. Tenenssee

S. JO hannes burg

S. Ubana

S. Rubislaw

S. Hadar

S. Kentucky

S. Newport

Table 2.6. continued. Bacteriophage assay and plaque assay specificity results for non- Ententidis Salmonella stiains. I

Serogroup Bacterial Species S train Plaque assay I Phage assay

Group C S. Haardt SA 914074

S. Choleraesuis S-870

Group D S. Berta SA 941 123

S. Panama SA 93 1592

S. Sendai S-5 13 -- -- -

a Weak lysishot consistently positive by bacteriophage assay.

Table 2.7. Bactenophage assay and plaque assay specificity resdts for SolmoneZZa Enteritidis strains.

Bacterial species S train Plaque assay/Phage assay

S. Enteritidis SA 932451 +/+ S. Enteritidis Laboratory Strain +/+

S. Enteritidis ATCC 13076 +/+ S. Enteritidis SA 94245 1 +/+

S. Enteritidis EN 2588 +/+

Table 2.8. Bacteriophage assay and plaque assay specificity results for non-Salmonella strains.

Bacterial species Sîrain Plaque AssayPhage assay

Serratia marsescens ATCC 8 1 O0 -1-

Cino bacter fieundii -/-

Sh ige lla flexne ri -/-

Escherichia coli ATCC 25922 4-

Escherichia coli 0 1 5 7:H7 EC 920333 -1-

2.5. Discussion

2.5.1. Bacteriophage specificity:

It became clear early on that phage SJ2 was not specific for only Salmonella Ententidis

nor was it's host range sufficient for a generic SalmonefZa test. SpecScity testing was

nonetheless camied out to further characterize the phage. The phage was not infectious

for any of the non-Salmonella strains tested. AI1 five of the S. Enteritidis strains tested

showed strong positive reactions by both methods. Strong positive results were also

observed for S. Typhirnurium SA 942256 and S. Sendai. S. Typhimunum 94-5 1 was not

positive by either method. This particula. strain of S. Typhimunum is highly resistant to

multiple antibiotics. Perhaps the mutations responsible for antibiotic resistance also

imparts some resistance to infection by phage. Other serotypes belonging to group D,,

namely S. Berta and S. Panama, showed somewhat inconsistent results for both the plaque

assay and the phage assay. There are no diagnostically important somatic or flagellar

antigens among cornrnon among the strains testing positive that were not also present in

strains that were not sensitive to phage SJ2. Somatic (O) antigen 09 was cornmon to the

serogroup D Salmonella tested (which includes S. Enteritidis), but this antigen is not

present on S. Typhirnurium. O-antigen 01, and 12 are common to al1 the strains testing

positive, but are also found on other group C strains tested (Bergey et al., 1984). Since

phage SJ2 was isolated fiom the environment, and has not been characterized, it is

unknown what constitutes a receptor. A more suitable choice for a genenc Salmonella

test may be bactenophage Felix-01, which has been shown to uifect 98-99.5% of the

greater than 5000 SaIrnonella strains tested (Bergey et al., 1984)

2.5 -2. One-step growth experiment:

The one step growth experiment was important in that it dictated some of the incubation

tirnes required for the assay. Since lysis began to occur around 30 minutes, the attachent

and washing steps could not exceed this time, ~îherwïse progeny phage rnay be lost. As a

result, 15 minutes was permitted for attachment of phage, followed by washing. The

results of the growth experiment suggest the 30 minute incubation penod for amplification

would be sufficient for one complete round of infection.

It is important to note that results of this procedure are estimates only. Samples taken

more frequently would give greater precision for determining the latent period. Also, if

adsorption was not swifi and very high, burst-size calculation may not be very accurate

(Mararnorosch and Koprowski, 1967). The 97% adsorption rate caiculated in this

experirnent indicated that adsorption occurred very rapidly. The plates representing times

greater than 42 minutes were too numerous too count, so it is difficult to estimate how

much higher the relative plaque titre would be. If this increase is due to late-bursting cells,

the burst size may be underestimated.

2.5.3. Microscopy protocol:

This protocol demonstrated the feasibility of the basic principles of the assay. That is,

progeny phage from Salmonella present initially in the sample could be indirectly detected

by its effect on a helper population. When this effect was compared to samples containing

no Salmonella initially, it became clear which sarnples were positive. Furthemore, the

greater the initiai Salmonella population, the greater the effect on the helper population.

There are several reasons the centrifugation protocol would not be suitable for testing

food samples. First, there is no means of dealing with the background microflora that

would be in a food sarnple. At the very least, a selective ennchrnent step would be

necessary. Second, centrifugation is not desirable as it would require additional

instrumentation to perform the assay. Lastly, direct microscopie counts are labour

intensive, time-consuming and not suitable for routine analysis, not to mention the capital

costs of an epifluorescent microscope. If the bactenal population of the sample exceeded

the upper lirnit for the filter used, accurate counts could not be obtained and there would

be no opportunity to diiute the samples for additional counting. Each sample would also

require some mathematical manipulation to calculate averages which would become

cumbersorne with large numbers of samples.

2.5.3.1. Bacterial counts:

Molecular Probes LIVE/DEAD' BacLightTM bacterial viability stain for microscopy and

quantitative anaiysis was employed in the assay. The stain i s a two-colour fluorescence

assay consisting of SYTO 9 green fluorescent nucleic acid stain, and the red fluorescent

nucleic acid stain propidiurn iodide (Molecular Probes, Product Information). The stains

ditfer in their spectral characteristics and their ability to penetrate healthy bacterial cells.

SYTO 9 is able to penetrate healthy bacterial cells and thus will label al1 cells in a sample,

those with and without intact membranes. Propidium iodide is unable to penetrate healthy

cells and cornpetes for binding sites with SYTO 9 only in cells with damaged membranes.

As a result, the appropriate mixture of the two stains results in Iive cells staining

fluorescent green and the dead cells staining fluorescent red. Both stains excite in the blue

range (470nrn). The live stain emits between 510 and 540 nrn, and the dead stain emits

between 620 and 650 nrn.

There was good correlation between DEFT counts and plate counts (3 = 0.95) in this

present study. Pettipher et al. (1980) reported a correlation of r = 0.83, when comparing

plate counts with acridine orange clump counts of bacteria in raw milk. It is reasonable to

assume that food samples would have a lower correlation coefficient given the greater

dieculty in recovering bacteria from food samples relative to cells in broth. For example,

Shaw and Farr (1989) used DEFT for the analysis of meat and poultry. They reported

that orangelred debris on the DEFT filter overestimated the actual count by 5 to 1000-

fold.. The use of 1% vlv Tween 80 has been found to improve the correlation to more

acceptable values however. This indicates another potential drawback of the present

microscopy/centrifugation protocol in that pretreatment of food samples would be

necessary to rernove fat and debris.

2.5.4. IMS protocol:

The IMS protocol was developed to make the bacteriophage assay suitable for the analysis

of food sarnples. The use of IMS eliminates the need for selective e~chment , thereby

reducing the total assay tirne by as much as 24 hours. Many authors have reported the

utility of IMS in increasing the sensitivity and specincity of Solmonella detection, as well

as significantly reducing total assay time (Mansfield and Forsythe, 1993; Holt et al., 1995;

Parmar et al., 1992). The application of IMS as a separation technique in this assay also

eliminates the need for centrifugation. Magnetic particle separatioii is important, not only

for initial recovery of target cells, but for permitting subsequent washing steps (phage

removal), and for imrnobolizing the beads and cells for recoveiy of progeny phage in the

supernatant.

Regarding endpoint detection, a Buororneter is considerably easier to use than obtaining

microsco pic counts and requires less sample manipulation. In addition, results obtained

require only minimal mathematical manipulation.

Optical density was evaluated as an endpoint when it was observed that, in many cases,

positive and negative samples could be visually differentiated by differences in the turbidity

of the samples. Using optical density has sorne further advantages. First. a

spectrophotometer is a relatively inexpensive and common piece of laboratory equipment.

Second, sarnple manipulation and total costs are reduced as there is no need for

fluorescent staining. There is no need for a washing step to remove media, as samples can

be read directly fiom the incubator. The assay also becomes more flexible, since samples

can be re-incubated after initial readings, if necessary, to further distinguish between

positive and negative samples. The average incubation tirne was about 1.5 h.

Optimal parameters for the assay were determined to be an assay temperature of 37"C, a

phage population of 10' P m , with L-broth being the most suitable media for helper

incubation. Freeze-dried helper cells show some promise as an alternative to overnight

cultures, but a longer recovery period (>2.5 hours) may be required in order to approach

the performance of the ovemight helper population. Andrews (1986) reviewed the

resuscitation of injured Salmonella spp. and coliforms from foods. He reported that slow

(drop-wise) rehydration of dried cultures result in higher viable counts when compared to

rehydration by rapid addition of water. This is Iikely attributed to ce11 wall damage

occuning dunng rapid rehydration. Temperature was also found to be an important

factor. Spray-dried culture recovered best when rehydrated at 50°C, while freeze-dried

cultures were maximally recovered at a rehydration temperature of 20 to 25°C. These

factors should be taken into consideration when contemplating the use of a fkeeze-dned

helper population in the present assay.

2.5.5 Efficiency of immunomagnetic separation:

The eficiency of IMS was evaluated. There was a good correlation between JMS and

plate counts over the range of ce11 populations tested (r = 0.99). At the 104 dilution

(1 .4x103 CFU/ml), 86% of the bacteria in the sarnple were recovered. This dropped to

15.7% for the 10-' dilution (1 .4x108 CFU/ml), presumably because of the increased ce11 to

bead ratio in the sample. There are approximately 1.3 x10' beads added per 1 ml sample

(Dynal Inc., personal communication). At the highest dilution tested, tbis meant a cell to

bead ratio of about 10 to 1. Vermunt et al. (1992) reported a recovery of 5 1% * 7.8%

following IMS. The etticiency of IMS in this experiment was better than the 10%

recovery reported by Hanai et al. (1997).

One of the problems cornmon to antibody based tests is non-specific binding. Parmar et

al. (1992) evaluated an IMS-conductance method for the detection of Salmonella in milk

powders. In evaluating the specificity of IMS, they noted that exposure to Cipobacfer

freundii resulted in a Salmonella-type conductance curve and concluded the magnetic

beads were only partially specific for the Salmonella strains tested. Non-specific binding

of Citrobacter has been reported by other authors (Cudjoe et al., 1995). . ~ i t h the use of

bactenophage providing additional specificity, non-specific binding should not interfere

with the assay provided an appropriate number of Salmonella can be recovered on the

beads. The Citrobacferfrerndii tested as part of the phage specificity test in this study

did not result in a positive test. Factors affecting irnmunologicai and non-specific binding

of bactena to magnetic particles include the culture medium, the bacterial strain and the

particlekell incubation time and temperature (Blackburn, 1993).

2.5.6. Assay sensitivity:

Optical density was chosen as the means of determining assay sensitivity, since incubation

time was flexible. The assay sensitivity is approximately 104 CFU/rnl, as

indicated in Figure 2.9.

Another Salmonella detection assay based on the principle of assaying progeny phage was

reported by Hirsch and Martin (1983a; 1983b). They describe a method for the detection

of SuZmoneIZu using Felix-O 1 bacteriop hage and high performance liquid chromatograp hy

(HPLC). The interaction of bacteriophage Felix-O1 with S. Typhimurium resulted in

increased numbers of bactenophage which were subsequently detected by HPLC. It was

determined that 106 Salmonella per ml need to be present to be detected. The detection

limit of the present assay is about two log cycles lower. The lower detection lirnit of

ELISA'S have been reported to range frorn 104 CFU/ml up to 10' CFU/d (Patel and

Williams, 1994; Cudjoe et al. 1995; June et al., 1992). This present assay has a detection

limit comparable to or better than other available detection methodg excluding gene

amplification methods..

2.5 -6 . Conclusion:

The bacteriophage assay described here is based on the cornbined specificity of

immunomagnetic beads and bacteriophage SJ2 for SalmoneIIa spp. The study

demonstrates that the normal biology of bacteriophage SJ2 in its host bacterium can be

exploited, with progeny phage subsequently assayed indirecty by its eEect on a helper

population of healthy S. Ententidis cells. The assay endpoint can be determined either by

optical density or fluorescence measurements. The assay is not specific to S. Ententidis,

nor is it suitable for a genenc Salmonella test, but provides a good working mode1 for the

future development with other host/phage systems. The assay is simple to perform, can br:

completed in under 4 hours, and has a sensitivity in the range of 1 O' CFU/rnl.

3. The use of the IMS-bacteriophage assay for the detection of Salmonella Enteritidis in skimmed mük powder, ground chicken and liquid whole egg.

3.1 Abstract

Salmonella infection is the second most prevalent cause of foodborne illness in most

developed countries. Poultry products, eggs and milk are ftequently implicated in

outbreaks. The objective of this study was to appty an Unmunomagnetic separation @MS)

bacteriophage assay to the detection of S. Enteritidis in artificially inoculated skimmed

milk powder, ground chicken, and liquid whole egg. In al1 food types tested, the IMS

assay was able to detect S. Enteritidis at the lowest initial inoculation level tested, 10"

CFU/g, following a preenrichment ranging fiom 10 to 18 hours. These results indicate

that the IMS assay is a rapid and sensitive means of detecting S. Ententidis in these foods.

3.2 Introduction

Salmonella is widely recognized as an important cause of foodborne illness. Poultry

products, eggs and milk are frequently implicated as vehicles of infection in cases and

outbreaks of Sa~monelZu infection (Bryan and Doyle, 1995; Henzler et al., 1994; Lin et al.,

1988; Ryan et al., 1987). Detection methods can play a role in stopping the transmission

of disease by preventing the sale of contaminated product. There are many available

methods for the detection of Salmonella in foods. Conventional cultural methods are very

slow, and require expensive storage of foods before results are available. The largest

group of rapid methods for Salinonellu detection are antibody-based tests such as enzyme

linked immunosorbent assays (ELISAs), latex agglutination, imrnunodifision and

methods incorporating M S . Drawbacks of these methods include non-specific binding

leading to false positive results, the high degree of technical skill required, and in some

cases, high detection Limits. Nucleic acid-based tests such as PCR can be very sensitive

and specific but can be expensive and labor intensive-

The specificity of bacteriophage has been exploited in the detection or typing of several

bacterial species (Stewart, 1990; Wobler and Green, 1990; Hirsch and Martin, 1983a;

Chen and Grifliths, 1996; Dubow, 1994).

This study describes the use of a novel IMS-bactenophage assay for the detection of S.

Ententidis in skimmed milk powder, ground chicken, and liquid whole egg.

3.3 Materiais and Methods

3 3.1 Bacteriophage and host:

The bacteriophage and host used in this study, SJ2 and S. enterifidis respectively, were

obtained fkom Dr. Sabah Jassim, Department of Food Science, University of Guelph.

Both the bacteriophage and the host were isolated fiorn egg. The method of isolation is

unknown. Bacteriophage SJ2 and host S. Ententidis were maintained as previously

descnbed in sections 2.3.1 and 2-3 -2.

3 -3 -2. Food enrichment preparation:

Ail food samples were purchased from a local supermarket. Overnight cultures of S.

Enteritidis were serially diluted IO-fold in lambda buffer. The population of S. Enteritidis

in the dilution tubes was determined by triplicate plate counts. Tluee replicates of

skimmed milk powder, six replicates of ground chicken, and three replicates of Liquid

whole egg were inoculated to give initial ce11 concentrations of 102, 10' and 10' C m / g or

ml. In each case, 1 millilitre of the appropriate dilutions of S. Enteritidis were added to 25

g or ml samples to give the required final ce11 concentrations. A negative control for ali

experirnents included uninoculated food. For each experiment, the results of food samples

were compared to a negative control consisting of 1 ml of L-broth, tested in triplicate.

For skimmed milk powder, spiked samples and unspiked skirnrned milk powder samples

were added to 225 ml of L-broth in a 500 ml Erlenmeyer flask and incubated, with

shaking, for 10 hours at 37OC. On the day of testing, each inoculation level was tested in

duplicate.

For ground chicken, controls, spiked samples and unspiked samples were added to a 500

ml Erlenrneyer flask containing 225 ml of L-broth. Samples were incubated for 16 to 18

hours at 37OC. For trial one and two, each inoculation level was tested in triplicate. In

trial three and four, samples were tested uninoculated and inoculated at the 10' CFU/g

level only. Each inoculation level was tested in tnplicate. A second package of ground

chicken was used for trials five and six. Sarnples were incubated for 10 hours in a

stomacher bag with a filter at 37OC and each of the four inoculation levels were tested in

duplicate. Negative controls @roth ody) were tested in triplicate.

For liquid whole egg samples, eggs were dipped in 95% ethanol and left to dry in a

laminar flow hood under W light for 10 minutes to disinfect the shells. Eggs were

asepticaiiy cracked and the contents of four eggs were pooled. Twenty five millilitre

portions were spiked as indicated above, added to 225 ml of L-broth in a 500 ml

Erlenmeyer fiask and incubated, with shaking, for 16 hours at 37°C. On the day of

testing, each inoculation level was tested in duplicate.

3 -3 -3 . Testing of food samples with the IMS bacteriophage assay:

Enrichment cultures from the skimmed milk powder, ground chicken and liquid whole egg

were tested according to the IMS protocol as previously described in section 2.3.4. The

results of some experiments were determined using optical density as the endpoint, others

employed both optical density and fluorescence as the endpoint.

3.3 -4. Determination of the assay endpoint:

A quantitative means of distinguishing positive from negative samples was determined.

The results h m the testing of d three food types were pooled, and a numerical cut-off

was determined based on the value that gave the minimal number of false positive and

false negative results. The results of al1 food samples tested were compared to the mean

of the negative control samples in each experiment. Cut-offs based on 80%, 75%, 70%,

65%, and 60% of the mean of the negative control samples for each experiment were

calculated the number of false negative and false positive results at each cut-off were

ascertained.

3.4. Resdts

3 -4.1. Determination of the assay endpoint:

Table 3.1 shows the results when five cut-off values were used to determine the endpoint

of the assay. False positive results occur when an unspiked sample has a value less than or

equal to the calculated cut-off. False negatives occur when a spiked sample has a value

above the calculated cut-off. A cut-off of 70% was chosen as the cut-off for a test to be

considered positive. At this level, one of 18 uninoculated samples (5.5%) gave a false

positive result. Two of 69 of the inoculated food sarnples gave a false negative result. AU

three of these samples were fiom liquid whole egg.

3 -4.2. Skimrned rnilk powder sarnples:

Samples of skirnrned rnilk powder inonilated with four levels of S. Entedidis (0, loO, 1 01,

and IO* CFU/g) were evaluated by optical density and in trial two by fluorescence, in

duplicate over three experirnental trials. Figure 3.1 shows the pooled results from the three

trials. Al1 four inoculation levels are reported as a percentage of the mean negative

Table 3.1. Tally of false positive and false negative results obtained fiom skimmed milk powder, ground chicken and liquid whole egg sarnples when various numerical cut-off values are used to determine a positive test.

Cut-OP 60%

positive, it must be less than or equal to the cut-off value,

65% 70% 75% 80%

False positive (+) and False negative (-) results are reported as the number of false positive or false negatiie samples out of

Skimmed mitk powder False +b False -b 015(0%) 1115(7%)

the number of true negative and true positive samples tested. Corresponding percentages are indicated in brackets.

a Cut-off was calculated from the mean of the negative control values in each experiment. In order for an experimental result to be considered

O15 (0%) 1/15 (7%) 0/5(0°h) 0/15(0°h) 115 (20%) 011 5 (0%) 215 (40%) 01 15 (0%)

In trials 2 ,3 and 4, uninoculated ground chicken sampler gave a strong positive result. Chicken was confirnied to be naturally contaminated with Salmonella. Results from these uninoculated samples are not included in the table.

Ground chickenC False t False - 0/3(0%) 0124(0%)

Second set of hiais with ground chicken; new package.

O13 (0%) 0124 (0%) 0/3(0%) 0124(0°/o) 013 (0%) 0124 (0%) 013 (0%) 0124 (0%)

Ground chickend False -I- False - 014(0%) 3/12(25%) 014 (0%) 2112 (16.6%) OI4(0%) 0/12(0%) 014 (0%) O112 (0%) 014 (0%) O11 2 (0%)

Liquid w h o u False + False -

116(16.7%) 3118(16,7%)

Tota b False + False - 1/18(5.5%) 7/69(10%)

116 (16.7%) 311 8 (16.7%) 116(16.7%) 2118(ll.1°/o) 116 (16.7%) 111 8 (5,5%) 116 (1 6.7%) 011 8 (0%)

111 8 (5.5%) 6169 (8.7%) 1/18(5.5%)2169(2,9%: 211 8 (1 1,1%) 1169 (1.4%) 311 8 (1 6,6%) 0169 (0%)

O 3 35 350

Mean Initial lnoculum (CFUIg)

Figure 3.1. Percent of negative control value for skimmed milk powder samples inoculated with S. Enteritidis at four levels (O, 10'. 1 o', and 10' CFU/g). Values shown are averages of three trial S. Endpoints were determined using optical density. Bar represents one standard deviation.

O 1 6 60

Mean Initial InocuIurn (CFUIg)

Figure 3 -2. Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, IO', IO', and 10' CFU/g). Values shown are averages of four trials. Endpoints were determined using the FL500 fluororneter. Bar represents one standard deviation.

93

control value. The assay was consistently positive at the lowest inoculation level tested

(10' CFU/g) and there were no fdse positive or false negative results in any of the three

trials as determined by optical density measurements. For trial two, fluorescence was

measured using the MGM fluorometer. Only one of the six inoculated samples (102

CFU/g) was positive at the 70% cut-off. At the 75% and 80% cut-off, the number of

positive sarnples increased to three and four respectively. Duplicate fluorescence samples

from each inocuiation level were pooled and transferred into cuvettes. Optical density

measurements were taken, resulting in all three inoculation levels being positive at the

70% cut-off level. The MGM fluorometer may be very sensitive to small amounts of

media residue that are not completely removed dunng the washing step, which may

account for the readings observed. No false positives were observed for optical density or

fluorescence.

3 -4.3. Ground chicken samples:

Samples of ground chicken inoculated with four levels of S. Enteritidis (0, loO, IO', and

102 CFU/g) were evaluated by fluorescence with the F'L500 fiuorometer in tnplicate. A

subsequent attempt to confirm these results showed a positive test at al1 inoculation levels,

including the uninoculated samples. Two subsequent trials were conducted with an

uninoculated sample tested in triplicate, and a sample inoculated at 10' CFUfg (confirmed

by plate count), also tested in tnplicate. Al1 six uninoculated samples, as well as the

spiked samples showed strong positive results, confhming the results of the second trial. It

was suspected that the ground chicken sample was naturally contarninated with

Salmonella, which may be responsible for the false positives observed. To test this

hypothesis, the IMS samples fiom the unspiked chicken were retained and analyzed

according to the Health Protection Branch method for the isolation and identification of

Salmonella fiom foods (Anon., 1989). Two hundred microlitres of the magnetic beads

were transferred in duplicate into selenite cystine broth (DIFCO, Detroit, Michigan) and

tetrathionate brilliant green broth @ECO) and incubated ovemight at 37°C. Samples

were streaked ont0 brilliant green sulfa agar @ECO) and bismuth sulfite agar

@IFCO)and incubated overnight at 37OC. Suspect colonies were streaked ont0

MacConkey agar @ECO) and incubated ovemight at 37OC. Suspect colonies were

streaked ont0 Tryptic Soy Agar (DIFCO) and incubated ovemight at 37°C. Two suspect

colonies were biochernically characterized by analytical profile index (API) 20E

(Biomerieux, St. Laurent, Quebec) and serology. The first API profile indicated an

excellent identification of Sulmonelh spp. Serology testing indicated positive

agglutination with Anti-Salmonella Factor 8 (DIFCO, Detroit, Michigan) confirming the

presence of a group C Salmonella. The result of the second API was Proleus mirdilis.

The results of this testing indicated the ground chicken was naturally contarninated with

Salmonella. The pooled results of these four trials are presented in Figure 3.2. The

uninoculated samples which were responsible for the positive test were not included in

Figure 3 -2, or Table 2.1.

A second package of ground chicken was purchased in an attempt to demonstrate the

utility of the bacteriophage assay on a ground chicken sarnple which was not naturally

contaminated. This would also elirninate the possibility that matrix eEects, perhaps via

the trapping of phage in unspiked sarnples, were responsible for the false positives

observed. Ground chicken was tested as indicated above with one exception. Samples

were incubated in stomacher bags with a filter in an attempt to reduce the fat and

particulate material introduced into the assay tubes. In addition, each inoculation level

was tested in duplicate, and compared to negative control tubes (broth only), tested in

triplicate. Results are shown in Figure 3.3. There were no fdse positives or fdse

negatives observed at the 70% cut-off level. These results suggest that the 'false-

positive' results obtained in the previous trials with unspiked ground chicken may be the

result of natural Salmonella contamination, and not matrix effects of the food sampie.

Regarding the instrumentation used for determinhg the assay endpoint, optical density

measurements were consistent and reliable. In addition, fluorescence results for ground

chicken trials one through four were obtained using the FL-500 fluorometer. These

results were also very predictable, and distributions corresponded well to those obtained

by the spectrophotometer. In trial five and six, results were obtained using optical density

and the MGM fluororneter. The optical density results were as expected. The

fluorescence results did not correspond to the optical density readings, with inoculated

chicken samples showing much higher readings than the negative control and uninoculated

chicken samples

3 -4.4. Liquid whole egg:

O 4 29 290

Mean Initial lnoculurn (CFU/g)

Figure 3 -3. Percent of negative control value for ground chicken samples inoculated with S. Ententidis at four levels (0, loO, IO', and 102CFU/g). Values shown are averages of two trials. Endpoints were determined using optical density. Bar represents one standard deviation.

Mean Initial lnoculum (CFUIml)

Figure 3.4. Percent of negative control value for liquid whole egg samples inoculated with S. Ententidis at four levels (0, 1 oO, 1 o', and 102 C F U / ~ ~ ) . Values shown are averages of three trials. Endpoints were detennined using optical density. Bar represents one standard deviation.

The pooled results of three trials with liquid whole egg are illustrated in Figure 3.4. Al1

three triais were evaluated by optical density. Three of the negative control samples had

values rnuch lower than expected. This is presumably because of insufficient removal of

phage after the attachrnent step, resulting in an inappropriate reduction in helper ce11

population. As a result, the uninoculated samples averaged over 120% of the negative

control values. There was one false positive sample in trial two (Table 3.1). This value

was quite low, again likely due to inefficient phage removai. This sample contributed

greatly to the large standard deviation shown in Figure 3.4 for the unspiked samples. Two

false negative samples were obsenred in trial three at the 70% cut-off level (Table 3.1).

Interestingly, these false negative sarnples were observed at the rniddle inoculation level

(10' CFU/ml). Had one of the negative control samples been closer to the expected value,

these samples would Wcely have been positive. This highlights the importance of carefùl

and efficient removal of unbound phage fiom the tubes following the attachent step.

3 -5. Discussion

3 -5.1. General comrnents:

The purpose of this study was to test artificially inoculated food samples using the IMS-

bacteriophage assay. A cut-off of 70% of the mean negative value for each trial was

arbitrarily chosen to indicate a positive sample. This value was determined based on the

cut-off that gave the most acceptable number of false positive and false negative results,

when al1 of the food sample results were pooled.

False positive results Likely occur when unattached phage are not efficiently removed

during the washing steps. The phage may then be recovered in the supernatant and added

to the helper population, resulting in an undesirable reduction in ce11 numbers. This was

observed with the egg samples, where unexpectedly low values for three of the negative

control samples were observed. This highlights the great importance of thÏs washing step,

and the care that must be taken to maximize the removal of unattached phage.

The food matrix may be responsible for inhibiting the efficient removal of phage.

Unspiked samples sometirnes have reduced endpoint values compared to the broth only

controls (Figure 3.2). This dflerence is seldom responsible for a false positive result, but

suggests that the food matrix may somehow inhibit removal of phage during the washing

steps. This phage may be recovered later and effect a small change in helper relative to

the negative control.

Experimental trials are not meant to be directly compared between days, but for the

purpose of this report, results were pooled. Variations in incubation times and dserences

in inoculation levels between experimental trials may be responsible for some of the larger

standard deviations depicted in Figures 3.1 through 3.4. Values of replicate samples

within trials are generally very consistent.

In most cases, a final incubation time of 1.5 h was sufficient to distinguish positive from

negative samples. An evaluation of the actual absorbance values following the IMS assay

indicates the progeny phage will not imrnediately reduce the helper population below it's

original reading of 0.100. In most cases, growth of helper will occur in both positive and

negative samples, but the rate of growth will be slower in positive samples. After a 70 to

80 minute incubation, two rounds of infection have occurred and the values for positive

samples will generally decline, while the helper fiom negative samples will continue to

grow. Incubation cannot occur indefinitely, however, as srnaIl numbers of phage nom

negative samples may eventually 'catch-up' to the growth and effect a negative change in

these samples.

Results of the IMS-bacteriophage assay are presumptive, and plating of the beads would

provide a viable isolate for fûrther confirmation.

3.5.2. Skirnmed d k powder:

Many outbreaks have been associated with rnilk products (El-Ganar and Martb 19921,

including milk powders (Rowe et al., 1987). Skimmed milk powder was chosen as an

opportunity to test the assay in a processed food. The IMS-bacteriophage assay worked

consistently with the skimmed milk powder samples. The assay was positive over al1 three

trials at the lowest inoculation level tested (10' CFU/g).

Parmar et al. (1992) investigated an IMS-conductance test for the rapid detection of S.

Enteritidis and S. Typhimuriurn in skirnrned milk powder. Low numbers of Salmonella

(<20 CFU/ml) were detected in a total time of 13.5 hours, consisting of a 6 hour

preenrichment followed by IMS and conductance rneasurements. For lower ce11 numbers

(one cell in 25g) the authors suggest that ovemight enrichment followed by IMS and same

day conductance may be necessary.

Dziadkowiec et al. (1995) compared conventional methods with IMS-conductance for the

detection of Salmonella in skimmed milk powder emichrnents. The IMS-conductance

assay was positive following a minimum 5 hour pree~chment, when the Salmondla in

the sample had reached 50 celldml. The initial inoculum was very low (20 CFU in 250 ml

broth). These authors noted that non-Salmonella skirnrned rnilk flora increased fiom

lx1 o3 to 3x 10' CFU/rnl after 2 and 12 hours incubation respectively, while the Salmonella

count did not exceed 3x106 CFU/ml throughout the 24 hour e ~ c h r m n t period. This

suggests that after the 10 hour incubation in this current study potentially large numbers of

background flora are present, dernonstrating the utility of the IMS-bactenophage in the

presence of high numbers of background flora. Detection limits of these studies ptior to

IMS cannot be compared directly with the present study, as conductance requires an

additional incubation period after IMS .

3 -5 -3. Ground chicken:

The IMS-bactenophage assay was successful at detecting S. Enteritidis at the lowest

inoculation level tested (10' CFWg). It also appears that the assay identified SalmonelIa

in a naturally contarninated product. Al1 of the group C Salmonella strains that were used

in the specificity testing of bactenophage SJ2 (section 2.5.1) were negative by both the

plaque assay and the phage assay. Another possibility is the presence of multiple

Salmonella serovars in the ground chicken sarnple. This is quite possible, since the

chicken tested is a comminuted meat product and particularly susceptible to

contamination. Individual flocks have been reported to harbor up to five Salmonella

serovars. (J3arnhart et al., 199 1)

Coleman et al. (1995a) evaluated IMS for the detection of salmonellae in raw chicken

carcasses. They found that IMS was superior to traditional culture methods in isolating

salmonellae frorn naturally contaminated poultry. In contrast, a study by Ripabelli et al.

(1997) found that selenite cysteine was slightly better than IMS for recovery of

Salmonella nom pork and chicken samples, giving false negative rates of 16.6% and

33.3% respectively. Studies with IMS indicate that high fat levels c m lead to loss of

organisms at the separation stage (Skjerve and Olsvik, 1991; Coleman et al., 1995a).

They suggested the use of stomacher bags with an inner filter may dirninish the amount of

fat and particulate matter in the homogenate. This was observed in the present study with

the ground chicken. Ground chicken samples which had been incubated in Erlenmeyer

flasks almost invarïably had food particulate matter in the sample tubes. The food

particulate persisted through washing steps, being separated with the magnetic bead

portion of the sarnple. This seemed to interfiere with the assay. It is reasonable to assume

the presence of such particulates would hinder the attachment of the phage to the

captured target cells. Efficiency of Salmonella capture rnay also be reduced, as beads

become trapped in the particulate material. In addition to food particulates, fat in the

sample made the walls of the tubes rather 'sticky' and it was difficult to wash beads f?om

the sides of the tubes. The use of stomacher bags with a filter in the second set of trials

considerably improved the problem caused by food particulate although the effects of fat

were still apparent. Skjerve and OlNik (1991) found that IMS was not suitable for al1

food products tested. For some food products (eg. yoghurt and chicken liver), a

substantial loss of beads was observed during initial separation and washing steps. Other

foods produced a visually impure pellet (soft cheese), although most beads were still

recovered. Preliminary studies with changes to broth and washing buffers using different

proteins and detergents were found to be ineffective.

When the IMS product fiom the unspiked ground chicken sample was tested by

conventional methods, a group C Salmonella was confirmed to be present. In addition,

Proteus mirubilis persisted throughout the isolation procedures as confirmed by API

identification. Several other isolates fiom the selective media showed negative

agglutination with polyvalent Salmonella anti-sera poly IA and Vi @ECO. Detroit, MI).

Other authors have reported problems of specificity with the magnetic beads, indicating

interference of salmonellae binding by coliforms, Citrobacterfrez~ndii, pseudomonads and

Proteus spp. (Coleman et al., 1995b; Cudjoe et al., 1995). The time and temperature of

incubation, the media used and the bactenal strain can affect irnmunological and non-

specitic binding to irnrnunomagnetic particles (Blackburn, 1993).

The combined effects of fat and particulate matter as well as the non-specific binding of

non-Salmonella organisms to the beads may have an effect on the magnitude of the

positive reaction seen in the second round of ground chicken samples by reducing the

efficiency of target ce11 capture and subsequent attachent of pnage. Conversely, the

trapping of phage by fat and food particulates may contribute to the reduction in the

helper population seen in uninoculated food samples, relative to the negative control.

Non-specific binding should not produce false positive results because of the specificity of

the bacteriophage employed.

3 5 4 . Liquid whole egg:

Grade A shell eggs have emerged as major sources Salmonella infection. Seventy-seven

percent of 35 S. Enteritidis outbreaks in the US between 1985 and 1987 were linked to

eggs alone or egg-containing foods (St. Louis et al., 1988).

Isolation of Salmonella fiom eggs presents a special problem because egg albumin

contains one or more factors that can interfere with detection (Andrews, 1996). The

growth of bacteria in egg albumin is limited by the presence of lysozyme, enqme

inhibitors such as ovomucoid, avidin, conalbumin and a high pH (Banwart, 1979). Such

stresses may necessitate a long preenrichment to allow recovery of injured cells.

Stephenson et al. (1991) investigated the recovery of S. Enteritidis fkom shell eggs and

noted that egg albumin seemed to inhibit survival, as numbers of Salmonella inoculated

irito egg albumen decreased during refngerated storage. Salmonella introduced into the

egg yolk, however, increased under the same storage conditions, indicating possible

growth of Salmonella in this rnatrix. While Stephenson and others (Hammack et al.,

1993) suggest testing yolk, others report the albumen to be more frequently contaminated

(Humphrey et al., 199 1).

Holt et al. (1995), investigated a magnetic bead-ELISA systern for the detection of

Salmonella Ententidis in pooled liquid egg samples. The IMS-ELISA was compared to

an IMS-direct protocol, where the lMS product was plated on brilliant green agar

supplemented with novobiocin. They found that 100% of pooled egg initially

contaminated with 10 cells per ml were positive following a 24 h enrichment at 37OC. In

pooled egg initially contaminated with 1 ce11 per ml, 61% and 72% were positive by IMS-

ELISA and IMS-direct respectively. A PCR procedure for detection of Salmonella in

whole shell egg by Burkhalter et al. (1995), indicated a detection Iirnit of 1-10 ceils per

egg when a 16-24 h preenrichment and selective enrichment were used. This present

assay compares favourably with these results.

3.6. Conclusion:

One false positive and two false negatives were observed out of the 18 negative, and 69

positive samples tested, al1 from liquid whole egg. The IMS-bacteriophage assay was

positive at an initial inoculation Ievel of 10' CFU/ml for al1 three food types tested.

Efficient removal of unbound phage was proven to be criticai to the success of the assay,

since the results of food samples were compared to negative controls tested with each

trial. Optical density is the preferred endpoint, given the flexibility in extending the

incubation period and the reduced sample manipulation required. The FL-500 fluorometer

provided consistent results that were veiy sirnilar to that obtained with a

spectrophotometer. The MGM fluorometer, while being the most sensitive o f the three

instruments in terms of lower detection lirnit, did not provide reliable results when applied

to the food samples. The IMS-bactenophage assay was demonstrated to be a rapid and

sensitive test for the deteetion of Salmonella Ententidis in skimmed milk powder, ground

chicken, and iiquid whole egg.

4. Application of the IMS-bactenophage assay to the detection of Escherichia coli 0157:H7 in ground beef.

Escherichia coli 0 157:H7 gained widespread attention as a human pathogen following a

large US outbreak in 1992 involving contaminated hamburgers. Ulness caused by E. coli

O I57:H7 can range Eom watery diarrhea, to life-threatening conditions such as hernolytic

uremic syndrome or thrombotic thrombocytopenic purpura. Ground beef is frequently

identified as the vehicle of transmission in E. coli 0157:H7 outbreaks. The purpose of

this study was to investigate the application of a recently developed immunomagnetic

separation ( M S ) bactenophage assay, originally developed for the detection of

Salmonel[a Enteritidis, to Escherichia coli 0 157:H7. After demonstrating the assay

could be successfully applied to pure cultures of E. coli 0157:H7 in broth, ground beef

was tested at two inoculation levels. The ground beef assay was positive for the lowest

inoculation level tested (2.5 CFU/g). The results of this study dernonstrate that the IMS-

bacteriophage assay can easily be applied to the detection of other pathogens in foods.

4.2. Introduction

Escherichia coli is considered part of the normal intestinal flora of humans and other

warm-blooded animals, generally residing as harrnless commensals. Some strains are

pathogenic and cause distinct diarrhed syndromes. Foodbome diarrheagenic strains are

divided into four groups baseà on virulence properties, clinical syndromes, and distinct

O:H serogroups (Padhye and Doyle, 1992). The first main category is enteropathogenic

E. coli (EPEC), which is mainly associated with neonatal and infantile diarrhea. Adult

carriers are usually asyrnptomatic. The second group are enteroinvasive E. coZi (EEC)

which invade epithelial cells resulting in bloody diarrhea. This group of organisms are

similar to shigellae. The third group of pathogenic E. coli is enterotoxigenic E. coli

ETEC), which is responsible for watery diarrhea caused by adherance to the intestinal

epithelial and production of one or more toxins. The fourth category is

enterohemorrhagic E. coli (EHEC), which includes E. coZi 0157:H7. A principal

manifestation of E. coli 0 157:H7 is hemorrhagic colitis, characterized by sudden onset of

abdominal cramps, and watery diarrhea which later becomes grossly bloody. Other

manifestations include hemolytic uremic syndrome (HUS), and thrombocytopenic purpura

(TTP). HUS is a combination of hemolytic anemia, thrombocytopenia, and renal failure

that can occur acutely on otherwise healthy individuals. TTP is similar to HUS except

that the central nervous system is involved (Padhye and Doyle, 1992). Although human

illness associated with E. coli 0 157:H7 is infiequent compared to other pathogens such as

Salmonella, the severity of clinical symptoms and the potential for serious complications

and death, makes it a noteworthy food safety issue (USDA, 1994).

Various types of foods have been implicated in 0157 associated illness. The rnajority

(71%) have been linked to bovine products including ground beet raw milk and roast beef

(USDA, 1994). Cross-contamination of other foods such as apple cider, vegetables and

mayonnaise have been confirmed or suspected in other outbreaks &JSDA, 1994).

Improvements to culture media have been successful in irnproving the recovery of E. cdi

0157:H7 from foods. Weagant et al. (1995) describe a revised e~chmen t and agar-

plating system involving a 6 h enrichrnent in EHEC enrichrnent broth (modified tryptic soy

broth supplemented with vancomycin, cefsulodin, and cefixime) foilowed by spread plating

on sorbitol-MacConkey agar supplemented with tellurite and cefixime (TCSMAC).

The use of immunomagnetic separation combined with electrocherninuminescence (ECL)

was investigated for the detection of E. cok 0157 in food (Yu and Bruno, 1996).

Artificially inoculated rnilk, juices, senim. and supernatant fluids from ground beef, minced

chicken and fish were evaluated by IMS-ECL, giving a detection lirnit of 1000 to 2000

bacteria per ml of food. Detection limits in buEer ranged from 100 to 1000 bacteria per

ml. IMS was found to be more sensitive than direct culture for isolation of E. coli O 157

from inoculated meat samples and artificially mixed cultures (Wright et al. 1994;

Fratamico et al. 1993).

Nucleic acid based tests are available for the detection of E coli 01 57:H7. Polymerase

chain reaction (PCR) is a sensitive and highly specific method based on the amplification

of specific DNA fragments. Meng et al. (1996) describes a PCR protocol that was

successful in detecting as few as 25 CFU of E. coli 0157:H7 in three hours.

These test suffer some of the same drawbacks as described for SaIrnonelln tests including

lack of specificity, sensitivity, and tirne and technical ski11 required.

Goodridge (1997) developed a fluorescent bacteriophage assay for the detection of E. coli

O L57:H7 in foods, based on the work of Hemes et al. (1995). Bacteriophage DNA was

labeled with the fluorescent dye YOYO-1. Attachment of bacteriophage to target celis

could be visualized by epifluorescent mîcroscopy or quantified by flow cytometry. An

initial inoculum of 2.2 CFU/g in ground beef and between 10' and 1 O2 CFU/rnl in raw milk

could be detected by flow cytometry.

This present study involves the application of an IMS-bacteriophage assay, originally

developed for the detection of Salmonella Enteritidis, to the detection of E. coli O 157:H7

in ground beef

4.3. Materials and methods

4.3.1. Bacteriophage and host:

Escherichia cok 0 157:H7 strain EC920333, obtained from Health Canada, was used in

this study. Culture was maintained as described in section 2.3.1. The bacteriophage used

in this study, LG1, was obtained from Lamy Goodridge, in the Department of Food

Science, University of Guelph. The phage was amplified in its host as descnbed in section

4.3.2. Procedure for analysis:

The detection protocol was carried out as described for the Salmonella assay described in

section 2.3.4 with a few exceptions. Immunomagnetic separation was achieved using

~ y n a b e a d s ~ anti-E. coZi 0 1 5 7 (Dynal Inc., Lake Success, New York). Preliminary tests

indicated that phage LGl did not appear to be as infectious as phage SJ2. Perhaps phage

LGI has a longer burst time, or progeny phage are released more slowly. In any case, the

phage amplification step during the assay occurred in broth and the incubation time was

extended to one hour. The phage population used was 4x10' PFU/lOOpl, and the helper

population was and ovemight culture of E. coli 0157:H7 EC920333 adjusted to an

optical density of 0.100. The endpoint of the assay was determined by absorbance at 600

nm. A negative control consisting of broth only, and four initial inoculation levels (106,

105, IO', and 1 O3 CFU/ml) in broth were tested in duplicate.

4.3 -3. Food enrichment preparation:

Extra lean ground beef was purchased fiom a local supermarket. An ovemight culture of

E. coli 0 157:H7 was diluted 10-fold .in lambda buffer. Twenty five gram samples of

ground beef were inoculated with 1 ml of E. coli 0157:H7 to give inoculation levels of

102 and 10' CFU/g, as determined by tripiicate plate counts. A negative (unspiked) beef

sample was tested as well. Samples were placed in a stomacher bag with a filter and 225

ml of L-broth was added. Samples were homogenized by hand and incubated for 12 hours

at 37°C. Each sample, including negative controls consisting of broth only, was tested in

duplicate.

4.4. Results

4.4.1. Sensitivity test:

The assay was performed using four levels of E. coli 0157:H7 in broth. Triplicate plate

counts codinned the initial populations to be between 8.5~10' and 8 . 5 ~ 1 0 ~ CFU/ml. The

results are iuostrated in Figure 4.1. Based on the 70% cut-off determined in section

3 -4.1 ., the 8 . 5 ~ 1 0 ~ CFU/ml sarnple was negative, but the next lowest dilution was positive,

indicating that the detection IKnit is about 10' CFU/ml.

4.4.2. Ground beef:

The results of the ground beef experiment are illustrated in Fi y r e 4.2. Based on the 70%

cut-off for positive samples established in Chapter 3, ground beef samples were positive at

both inoculation levels, with no fdse positives observed.

4.5. Discussion

The purpose of this study was to investigate the feasibility of applying the IMS-

bacteriophage assay developed for Salmonda Enteritidis to the detection of another

foodbome pathogen. The application of the assay for the E. coli 0 1 57:H7 host and phage

was quick and simple, requiring only a few minor modifications. Since the characteristics

of different bacteriophage and subsequently the dynarnics of the host/phage relationship

8.5~1 O' 8 S x l O' 8 .5~1 os 8Sxlo6

Mean Initial Population (CFUlml)

Figure 4.1. Percent of negative control value for four populations of Ecoli O157:H7 in broth following IMS-bacteriophage assay. Endpoints were deterrnined by optical density. Bar represents one standard deviati on.

O 2.5 220

Mean Initial lnocuturn (CFUIg)

Figure 4.2. Percent of negative control value for ground beef samples inoculated with E - d i 0157:H7 at three levels (0, 1 oO, and 10' CFU/g). Endpoints were determined by optical density. Bar represents one standard devi ati on.

would v q , assay parameters would have to be optùnized for each new system

investigated.

The sensitivity of the E. coli 0 157337 assay for pure cultures in broth (104 CFU/ml) was

very similar to the detection iimit determined for the S. Ententidis assay.

Wright et al. (1994) reported that an initial inoculum of 2 CFU/g of E. coli 0 157337 in

minced beef could be detected by an IMS-direct plating protocoi following a 24 hour

pree~chment. This present assay had a comparable detection limit (2.5 CFU/g) with only

a 12 hour e~chrnent. An initial inoculurn lower than 2.5 CFU/g was not tested. Perhaps

the detection lirnit would actually be lower. The sensitivity of an ELISA developed for the

detection of E. coii 0157:H7 was found to be 0.2 to 0.9 cells per g in ground beef

(Padhye and Doyle, 1995). A similar detection limit (0.76-2.64 CFU/g) was determined

by an evaluation of the TECRA irnmunoassay for E. coli 0 157:H7 in dairy products (Flint

and Hartley, 1995). Again, this assay compared favorably.

4.6. Conclusion

The IMS bacteriophage assay, developed for the detection of S. Ententidis, was applied to

the detection of E. coli 0157:H7 in ground beef. The assay, employing the use of phage

LG1, was successful at detecting 2.5 CFU/g of E. coli 0157:H7 present initially in ground

beef. The total detection tirne was under 17 hours. The results of this study ïndicate that

the IMS-bacteriophage assay can be easily adapted to other foodbome pathogens

provided an appropriate bacteriophage is available.

5. Conclusion

Recent trends in SulmoneIIa infection suggest it will continue to be a public health

challenge (Tauxe, 1991). Poulhy, eggs and milk have been fiequently identified as

vehicles of infection. IZapid, simple and sensitive detection rnethods can play a role in

reducing the incidence of salmonellosis by preventing the sale of contarninated product. A

combination of immunomagnetic separation (IMS), bactenophage, and two assay

endpoints were investigated for the detection of Salmonella Enteritidis; first in broth, then

in food samples. Experiments with pure cultures in broth indicated that the Iower

detection limit of the assay was approximately 10' CF'U/ml. The assay detected al1 of the

S. Enteritidis strains tested, one of the S. Typhimurium strains tested, S. Sendai, and

showed weak reactions with two other group D Salmonella (S. Berta, and S. Panama).

Optical density is the preferred endpoint because of the ease of use and the flexibility it

imparts.

Artificially inoculated skimmed milk powder, ground chicken, and liquid whole egg were

evaluated by the IMS bactenophage assay. Three CFU/g or ml present initially in the food

samples were successfully detected in al1 of these foods. One false positive, and two false

negative results were obsenred for the liquid whole egg samples.

Since no special manipulation of the bacteriophage is required, it was reasoned that the

assay could be easily and rapidly applicable to other phage and host combinations. This

was demonstrated with the LGI phage and its host E. coli 0157:H7. The detection limit

in broth appeared to be very similar to the lirnit determined for the S. Entedidis assay.

The E. coli IMS successfülly detected E. coli 0157:H7 in ground beef at the lowest

inoculation level tested (2.5 C M g ) following a 12 h e ~ c h m e n t . The total time for

detection was under 17 hours.

The IMS bacteriophage assay has several advantages. Bacteriophage are very inexpensive

to pro~ahn+a ,,., ,.d an maintzii.. There is no manipulation of the phage, genetic or otherwise,

required; the assay simply exploits the normal infection cycle of the bacteriophage in it's

host. This makes the assay easily and rapidly applicable to other phagehost systems as

indicated by the E. coli 0 157:H7 assay. The assay is technically simple to perfomi, and

can be compieted in about four hours.

5.2. Limitations of the assay

A potential stumbling block in the assay is the eficiency of phage removal. This step is

absolutely critical, as false positives have been observed when unattached phage is not

sufficiently removed. Since the food sampies are compared to the mean of the negative

control samples to determine a positive or negative test, any inappropriate decrease in the

helper population in negative samples will influence the cut-off for a positive test, perhaps

leading to false negative results. Great care m u a be taken to ensure maximum removal of

unattached phage, without the loss of beads.

The current assay is also not suitable for the processing of large numbers of samples.

Twelve samples is the highest number of samples that can be comfortably processed at one

time, as the magnetic rack used in this study can accornrnodate 12 tubes. In addition,

increasing the number of samples tested will require more tirne for the washing steps. This

becomes important when considering that progeny phage are released in about 30 minutes

following infection. A 15 minute incubation for phage attachent leaves only 15 minutes

for the washing steps. It would be difficult to wash more than 12 tubes in that time

period. The tirne for attachent could be reduced to accornrnodate this, however.

It has been suggested in the literature that IMS is not suitable for al1 food types. Food

particulate matter and high background flora can inhibit the binding of target organisms,

although this was not thought to be a significant problem with the food samples tested in

this study. The specificity irnparted by the bactenophage should overcome any problems

of non-target organisms binding non-specifically to the immunomagnetic beads.

The IMS bacteriophage assay should be considered a presumptive test, and culture

confirmation should accompany a positive test. The IMS product can be plated on

selective media, and suspect colonies confirmed by serology.

5 -3. Future research

The total detection time of the assay is about 14-20 hours, depending on the

pree~chment tirne used. Manipulation of the assay steps, and shortened enrichment

times could be investigated to determine the potential of the IMS bacteriophage assay as a

same-day test. The potential use of a fieeze-dried helper population should also be fùrther

investigated to eliminate the need for maintenance of Solmonella cultures, and the

potential problems that would pose in an industriai food laboratory.

A washing step is currently required pnor to fluorescent staining to remove the broth

media which interferes with the stain. An alternative medium, which facilitates the growth

of the helper population in negative samples, and the reduction in ce11 numbers in positive

samples without interferhg with the fluorescent dye should be investigated. Such a

medium would reduce sample manipulation time, and perhaps provide the flexibility of

extending the helper incubation times, which is the case when optical density is used as

the endpoint.

Testing bacteriophage SJ2 against additional Salmonella serotypes would further

characterize the host range of the phage. It is clear however that SJ2 is not suitable for a

genenc Salmonella test, nor is it specific for S. Ententidis. A bacteriophage with a more

suitable host range, such as Felix-01, should be considered for the assay. The utility of

the assay in other food types, such as carcass nnses and raw milk, should also be

investigated.

A modified capture step, using bactenophage îmmobilized on magnetic beads should be

investigated. Some success of passive irnmobilization of phage on solid surfaces for the

capture of Salmonella has aiready been reported in the literature. This would be a

significant improvement over the current protocol. If phage could be immobilized by the

head, leaving the tail fibres free for capture and subsequent infection, the problerns

associated with residual phage in the tubes would be elirninated. The total assay tirne

would aiso be reduced, as target ce11 capture and infection would occur simultaneously.

Other formats for the assay c m be evaluated, such as a microtitre format or the use of

antibody coated tubes.

The experiments with E. coli 0157337 indicate that the IMS bacteriophage assay can

easily be applied to other foodbome pathogens. The potential for the assay to be adapted

to other pathogens, such as Lisferia and Campylobacter, should be investigated. There

are potential diagnostic applications as well, such as the detection of antibiotic resistant

organisms.

6. References

Allen, G., V.R. Bruce, P. Stephenson, F.B. Satchell, and W.H. Andrews. 1991. Recovery of Suimonella fforn high moisture foods by abbreviated selective enrichment. J. Food. Prot. 54:492- 495.

Andrews, W.H. 1986. Resuscitation of injured SaIrnonelia spp. and coliforms fiom foods. J. Food Prot- 49162-75.

Andrews, W.H. 1996. Evolution of methods for the detection of Salmonella in foods. J. AOAC Int. 79:4-12.

Angulo, F.J., S. Tippen, D.H- Sharp, B.I. Payne, C. Collier, J.E. Hill, T.J. Barret, R.M. Clark, E.E. Geldreich, H.D. Donnell, and D.L. Swerdlow. 1997. A community waterborne outbreak of salmonellosis and the effectiveness of a boil water order. Am. J. Public Hedth. 8758 1-585.

Anonymous. 1986. Methods for the isolation and identification of Salmonella fi-om foods W B - 2 0 ) . In Compendium of andytical methods. HPB methods of microbiological analysis of food. Polyscience publications Inc., Morin heights, Quebec.

Bailey, S.J., N.A. Cox, and L.C. Blankenship. 1991. A cornpanson of an enryme irnmunoassay, DNA hybridization, antibody irnmobilization, and conventional methods for recovery of naturally occurring salmonellae nom processed broiler carcasses. J. Food Prot. 54:354-356.

Baker, J.M., M.W. W t h s , and D.L. Collins-Thompson. 1992. Bacterial bioluminescence: Applications in food rnicrobiology. J. Food Prot. 55:62-70.

Banwart, G.J. 1979. Basic Food Microbiology. AVI Publishing CO., Westport CT.

Bamhart, H.M., D.W. Dreesen, R. Bastien, and OC. Pancorbo. 199 1. Prevalence of Suimortella enteritidis and other serovars in ovaries of layer hens at slaughter. J. Food Prot. 54488-49 1.

Beauchat, L.R. 1996. Pathogenic rnicroorganisms associated with fresh produce. J. Food Prot. 591204-2 16.

Bergey, D.H., J.G. Holt, N.R. Kreig, and P. Sneath. 1984. Bergey's manual of systematic bacteriology. Williams and Wilkins. Baltimore.

Bergey, D.H., and J.G. Holt. 1994. Bergey's manual of determinative bacteriology. Williams and Wilkins. Baltimore.

Bezanson, G.S., R. Khakhria, D. Duck, and H. Lior. 1985. Molecular analysis cordïrrns food source and simultaneous involvement of two distinct but related subgroups of S h o n e l l a

typhizimurÎum bacteriophage type 10 in major interprovincial Salmonella outbreak. Appl. Environ. Micro. 50: 1279- 1284.

Birge, E. A. (ed.). 198 1. Bacterial and bacterio phage genetics. Springer-Verlag Inc. New York.

Blackburn, C. de W. 1993. Rapid and alternative methods for the detection of salmonellas in foods. J. Appl. Bacteriol. 75: 199-2 14.

Blaser, M.J. and L.S. Newman. 1982. A review of saImonellosis: Infective dose. Rev. Infect. Dis. 4: 1096-1 106.

Bryan, F.L. 1980. Foodborne diseases in the United States associated with meat and poultry. J. Food Prot. 43: 240-150.

Bryan, F.L., and M.P. Doyle. 1995. Health risks and consequences of Salmonelln and CampyIobacterjejuni in raw poultry. J. Food Prot. 58:326-344.

Burkhalter, P.W., C. Müller, J. Lüthy, and U. Candrian. 1995. Detection of Salmonella in eggs: DNA analyses, culture techniques, and serology. J. AOAC. 78: 153 1 - 1537.

Chen, J., and M.W. GrEths. 1996a. Luminescent Salmonella strains as real time reporters of growth and recovery from sublethal injury in food. Int. J. Food Micro. 3 1 :27-43.

Chen, J., and M.W. Griffiths. 1996b. SalmonelZa detection in eggs using lux+ bactenophages. J. Food Prot. 59:908-9 14.

Coleman, D.J., K.E. Chick, and K.J. Nye. 1995a. An evaluation of immunomagnetic separation for the detection of saIrnonellas in raw chicken carcasses. Lett. Appl. Micro. 2 1 : 152- 154.

Coleman, D.J., K.J. Nye, K.E. Chick, and C.M. Gagg. 199Sb. A cornpanson of immunomagnetic separation plus enrichment with conventional salmonella culture in the exarnination of raw sausages. Lett. Appl. Micro. 2 1 :246-248.

Cudjoe, K. S ., T. Hagtvedt, and R Dainty. 1995. Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (1M.P)-ELISA. Int. J. Food Micro. 27: 1 1-25.

D'Aoust, J.Y., D.W. Warburton, and A.M. Sewell. 1985. Salmonella typhimuritm phage-type 10 Rom cheddar cheese implicated in a major Canadian foodbome outbreak. J. Food Prot. 48: 1062- 1066.

D'Aoust, J.Y., AM. Sewell, and E. Daley. 1992. Inadequacy of small transfer volume and short (6 h) selective enrichment for the detection of foodbome Salmonella. J. Food Prot. 55:326-328.

D'Aoust, J.Y. 1994. Salmonella and the international food trade. Int. J. Food Micro. 24: 1 1-3 1.

Desenclos, J-C., P. Bouvet, E. Benz-Lemoine, F. Grimont, H. Desqueyroux, 1. Rebière, and P. A. Grimont. 1996. Large outbreak of Salmonella enterica serotype parutphi B infection caused by goats' milk cheese, France, 1993: A case finding and epidemiological study. Brit. Med. J. 3 1291- 94.

DuBow, M.S. 1994. Bacterial identification - Use of bactenophages, p. 78-81. R.G. Webster, and A. Granoff (ed.), Encyclopedia of Virology. Vol 1. Academic Press. San Diego, California.

Dziadkowiec, D., L.P. Mansfield, and S.J. Forsythe. 1995. The detection of Salmonella in skimrned milk powder enrichments using conventional methods and imrnunomagnetic separation. Lett. Appl. Micro. 2O:36 1-364.

Ebel, E.D., J. Mason, L A Thomas, K.E. Fems, M.G. Backmen, D.R. Cummins, L. Schroeder- Tucker, W.D. Sutherlin, R.L. Glasshoff, and N.M. Smithhisler. 1993. Occurrence of Salmonella enteriridis in unpasteurized liquid egg in the United States. Avian Dis. 3 7: 13 5- 142.

El-Gazzar, F.E., and E.H. Marth. 1992. Salmonellae, salmonellosis, and dairy foods: A review. J. Dairy Sci. 75 :2327-2343.

Ephg, LX., J.A. Carpenter, and L.C. Blankenship. 1993. Prevdence of Campylobacter spp. and Mnzonella spp. on pork carcasses and the reduction effected by spraying with lactic acid. J. Food Prot. 56536-537.

Feng, P. 1992. Commercial assay systems for detecting foodborne Salmonella: A review. J . Food Prot. 59927-934.

Flint, S.H.,and N.J. Hartley. 1993. Evaluation of the TECRA imrnunocapture ELISA for the detection of Salmonella tphimurium in foods. Lett. Appl. Micro. 17:4-6.

Flint, S.H., and N.J. Hartley. 1995. Evaluation of the TECRA Escherichia coli 0 157 visual immunoasssay for tests on dairy products. Lett. Appl. Micro. 2 1 :75-78.

Fluit, A.C., M.N. Widjojoatmodjo, A.T.A. Box, R. Torensma, and J. Verhoef. 1993. Rapid detection of salrnonellae in poultry with the magnetic immuno-polymerase chah reaction assay. Appl. Env. Micro. 59: 1342-1346.

Fratarnico, P.M., F.J. Schultz, and R.L. Buchanan. 1992. Rapid isolation of Escherichia coli 0 157:H7 fiom e ~ c h m e n t cultures of foods using an imrnunomagnetic separation method. Food Micro. 9: 105-1 13.

Garcia-Villanova Ruiz, R., R.G. Vargas, and R. Garcia-Villanova. 1987. Contamination on fiesh vegetables dunng cultivation and marketing. Int. J. Food Micro. 4:285-291.

Gast, R.K. 1994. Understanding Salmonella enteritids in laying chickens: the contributions of experimental infections. Int. J. Food. Micro. 2 1 : 107- 1 16.

Gast, R.K., and C.W. Beard. 2993. Research to understand and control Salmonella enteritidis in chickens and eggs. Poultry Sci. 72: 1 157- 1 163.

Gonzalez-Hevia, M.A., M.F. Gutherrez, and MC. Mendoza. 1996. Diagnosis by a combination of typing methods of a Salmonella ~himunnzirrm outbreak associated with cured harn. J. Food Prot. 591426428.

Goodridge, L.D. 1997. A fluorescent bacteriophage assay for detection of Escherichia coli 0157:H7 in ground beef and raw milk. Thesis. University of Guelph. Guelph, Ontario.

Gulig, P .A., 1990. Virulence plasmids of SuZrnonelln tphimurim and other salmonellae. Microb. Pathogen. 8:3 -1 1.

Gunnarsson, A., B. Hurrell, and E. Thal. 1977. Recent experiences with the salmonella-O 1 phage in routine diagnostic work. Zentralbl. Baltenol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A. 23 7:222-227.

Harnrnack, T.S., P.S. Sherrod, V.R. Bruce, G.A. June, F.B. Satchell, and W.H. Andrews. 1993. Research Note: Growth of Salmonella enteritidis in Grade A eggs during prolonged storage. Poultry Sci. 72:373-377.

Hanai, K., M. Satake, H. Nakanishi, and K. Venkateswaran. 1997. Cornparison of cornmercially avaiiable kits with standard methods for detection of Salmonella strains in foods. Appl. Env. Micro. 63 :775-778.

Health Canada. 1998. Canadian integrated Salmonella, CampyIlobacter and pathogenic E. coli surveillance report - Preliminary report.

Hedberg, C.W., K.L. MacDonald, and MT. Osterholm. 1994. Changing epiderniology of food- borne disease: a Minnesota perspective. Clin. Infect. Dis. 18:671-682.

Heinitz, M.L., and J.M. Johnson. 1998. Incidence of Listeria spp., Salmonella spp., and Clostrzdium botzilinzrm in smoked fisb and sheltfish. J. Food Prot. 61 :3 1 8-323.

Helmuth, R., G. Morelli, and M.A. Montenegro. 1993. Properties and biological role of Salmonella virulence plasmids, p.35-40. F. Cabello, C. Hormaeche, P. Mastroeni, and L. Bonina (ed.), Biology of Salmonella. Plenum Press, New York.

Hennes, K.P., C.A. Suttle, and A.M. Chan. 1995. Fluorescently labelled virus probes show that natural virus populations can control the stmcture of manne rnicrobial communities. Appl. Env. Micro. 61:3623-3627.

Hennessy, T.W., C.W. Hedberg, L. Slutsker, K.E. White, J.M. Besser-Wiek, M.E. Moen, J. Feldrnan, W.W. Coleman, L.M. Edmonson, K.L. MacDonald, M.T. Osterholm, and the Investigation Team. 1996. A nationd outbreak of Salmonella enteritidis infections fiom ice cream. New Eng. J. Med. 334: 128 1-1386.

Henzler, D.J., E. EbeI, J. Sanders, D. Kradel, and J. Mason. 1994. Salmonella enteritidis in eggs nom commercial chicken layer flocks implicated in human outbreaks. Avian. Dis. 38:37-43.

Him, J., E. Numû, T. Johansson, and L. Nuotio, 1992. Long-term experience with cornpetitive exclusion and salmonellas in Finland. Int. J. Food Micro. 15:28 1-285.

Hirsch, D.W., and L.D. Martin. 1983a. Rapid deteciion of Salmonella spp. by using Feiix-O1 bacteriophage and high-performance liquid chromatography. Appl. Environ. Micro. 45260-264-

Hirsch, D.W., and L.D. Martin. 1983b. Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-performance liquid chromatography. Appl. Environ. Micro. 46:1243- 1245-

Hogue, A.T., E.D. Ebel, L.A. Thomas, W. Schlosser, N. Bufano, and K. Fems. 1997. Surveys of Salmonella enteritidis in unpasteurized liquid egg and spent hens at slaughter. J. Food Prot. 10:1194-1200.

Holah, J.T., R.P. Bets, and R.H. Thorpe. 1988. The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent mter technique (DEFT) to assess microbial populations on food contact surfaces. J. Appl. Bact. 65215-221.

Holt, P. S ., R.K. Gast, and C.R. Greene. 1 995. Rapid detection of Salmonelh enteritidis in pooled liquid egg samples using a magnetic bead-ELISA system. J. Food Prot. 58:967-972.

Humphrey, T.J., A. Whitehead, A.H. Gawler, A. Heniey, and B. Rowe. 1991. Numbers of Salmonella enteriiidis in the contents of naturally contaminated hens eggs. Epiderniol. Infect. 106:489-496.

Hurnphrey, T.J. 1994. Contamination of egg shell and contents with Salmonella enteritidis: a review. Lnt. J. Food. Micro. 2 1 : 3 1-40

Izat, A.L., M. Colberg, M.H. Adams, M.A. Reiber, and P.W. Waldroup. 1989. Production and processing studies to reduce the incidence of saImonellae on commercial broiIers. J. Food Prot. 521670-673.

James, W.O., W.O. Williams, J.C. Pnicha, R. Johnston, and W. Christensen. 1992. Profile of selected bacterial counts and Salmonella prevalence on raw poultry in a poultry slaughter establishment. J. Am. Vet. Med. Assoc. 20057-59.

Jay, J.M. 1992. Modem Food Microbiology. Chapman and Hali, New York.

Johnson, I.D. 1996. Introduction to fluorescent techniques, p. 1-4. R-P. Haughland (ed.), Handbook of fluorescent probes and research chemicals. Molecular Probes, Eugene, OR.

Jones, F.T., R.C. Axtell, F.R. Tarver, D.V. Rives, D.E. Scheideler, and M.J. Wineland. 1991. Environmental factors contributing to Salmonella colonization of chickens. III L.C. Blankenship (ed.), Colonization control of human bacterial enteropathogens in poultry. Academic Press, San Diego, California. p. 3-20.

June, G.A., P.A. Sherrod, and W.H. Andrews. 1992. Cornparison of two enzyme immunoassays for recovery of Salmonella spp. from four low-moisture foods. J. Food Prot. 55:60 1-604.

June, G.A., P.A. Sherrod, T.S. Hammack, KM. Amaguana, and W.H. Andrews. 1996. Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for recovery of Salmonella spp. fkom raw flesh, highly contaminated foods and poultry feed: Collaborative study. J. AOAC Int. 79: 1307- 1323.

Kaprelyants, AS., and D.B. Kell. 1992. Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. J. Appl. Bactenol. 72410-422.

Killalea, D., L.R. Ward, D. Roberts, J. de Louvois, F. Sufi, J.M. Stuart, P.G. Wall, M. Susman, M. Schwieger, P.J. Sanderson, I.S.T. Fisher, P.S. Mead, O.N. Gill, C.L.R. Bartlett, and B. Rowe. 1996. Intemational epidemiological and microbiological study of outbreak of Salmonella agoiza infection from a ready to eat savoury snack -1: England and Wales and the United States. Brit. Med. J. 3 13: 1 105-1 107.

Kodikara, C.P., H.H. Crew, and G.S.A.B. Stewart. 1991. Near on-line detection of enteric bacteria using lux recombinant bacteriophage. FEMS Micro. Lett. 83126 1-266.

Lahellec, C., P. Colin, G. Bennejean, J. Paquin, A. Guillerm, and J.C. Debois. 1986. Muence of resident Salmonella on contamination of broiler flocks. Poultry Sci. 65:2034-2039.

Larson, A., J. Wallis, C. Lewandowski, D. Jenson, S. Potsic, M.K. Nickels, M. Lesko, C. Langkop, R.J. Martin, T. Endo, H.B. Ehrhard, and B.J. Francis. 1979. Salmonella Oranienburg gastroententidis associated with consumption of precut watermelons - Illinois. Morb. Mortal. Wkly. Rep. 28522-523

Lillard, H.S. 1989. Factors afEecting the persistence of Salmonella during the processing of poultry. J. Food Prot. 52:829-832.

Lin, F.C., J.G. Morris Jr., D. Tmmp, D. Tilghman, P.K. Wood, N. Jackman, E. Israel, and J.P. Libonati. 1988. Investigation of an outbreak of Salmonella enteriridis associated with consumption of eggs in a restaurant chain in Maryland. Am. J. Epid. 128:839-844.

Madden, J.M. 1992. Microbial pathogens in fresh produce - the regdatory perspective. J. Food Prot. 55:821-823.

Mansfield, L.P., and S.J. Forsythe. 1993. Irnmunomagnetic separation as an alternative to enrichment broths for SalmonelZiz detection. Lett. Appl. Micro. 16: 122-125.

Mansfield, L., and S. Forsythe. 1996. Collaborative ring-trial of Ilynabeadsa anti-Salmonella for irnrnunomagnetic separation of stressed SuZmonelIu cells from herbs and spices. Int. J. Food Micro. 29A1-47.

Mararnorosch, K., and H. Koprowski. 1967. Methods in virology. Academic Press. New York, New York.

Mason, J., and E. Ebel. 199 1. The APHIS Salmonella enteritidis control program. An. Health Insight. Winter. p 1-9.

Mason, J. 1994. Salmonella enteritidis control programs in the United States. Int. J. Food Micro. 21~155-169.

Mason, D.J., R Lopez-Arnoros, R. Allman, J.M. Stark, and D. Lloyd. 1995. The ability of membrane potential dyes and calcafiuor white to distinguish between viable and non-viable bactena. J. Appl. Bacteriol. 78:309-3 15.

Matsuyama, T. 1984. Staining of living bacteria with rhodarnine 123. FEMS Micro. Lett. 2 1 : 153- 157.

Mead, G.C. 1980. Microbiological control in the processing of chickens and turkeys, p. 9 1-104. h Meat quality in poultry and game birds. Proceedings of the joint 15" poultry science symposium on poultry meat quality. British Poultry Science Limited.

Mead, G.C. 1989. Hygiene problems and control of process contamination, p. 183-220. h G.C. Mead (ed.), Processing of poultry. Elsevier Applied Science. London.

Meng, J., S. Zhao, M.P. Doule, S.E. Mitchell, and S. Kresovich. 1996. Polymerase chain reaction for detecting Escherichia coli 0 157:H7. Int. J. Food Micro. 32: 103-1 13.

Molecular Probes Product Monnation. LIVEDEAD" ~ a c ~ i ~ h t " Bacterial Viability Kits. Molecular Probes Inc. Eugene, OR.

Morse, D.L., G.S. Birkhead, J. Guardino, S.F. Kondracki, and J.J. Guzewich. 1994. Outbreak and sporadic egg-associated cases of Salmonella enterifidis: New York's experience. Am. J. Pubic Health. 84359-860.

Mossel, D.A.k, C.M.L. Marengo, and C.B. Struijk. 1994. History of and prospects for rapid and instrumental methodology for the microbiological examination of foods, p. 1-28. P.D. Patel (ed.), Rapid analysis techniques in food microbiology. Chapman & Hall, Glasgow.

Noordhuizen, J.P., and K. Frankena. 1994. Salmonella enferitidis: Clinical epiderniological approaches for prevention and control of S. enteritidis in poultry production. Int. J. Food Micro. 2l:l3 1-143.

Nurmi, E., L Nuotio and C. Schneitz. 1992. The competitivie exclusion concept: Development and fiiture. ht. J. Food. Micro. 15 :237-240.

Padhye, N.V., and M.P. Doyle. 1992. Escherichia coli 0 157:H7: Epidemiology, pathogenesis, and methods for detection in food. J. Food Prot, 55555-565-

Padhe, N.V., and M.P. Doyle. 1995. Rapid procedure for detecting enterohemorrhagic Esherichia coli 0 157:H7 in food. Appl. Env. Micro. S7:2693-2698.

Parish, M.E. 1998. Coliforms, Escherichia coli and Salmonella serovars associated with a citms- processing facility implicated in a salmonellosis outbreak. J. Food Prot. 6 1 :280-28 1.

Parmar, N., MC. Easter, and J.J. Forsythe. 1992. The detection of SalmonelZa enteritidis and Salmonella typhimurilrrn using immonomagnetic separation and conductance microbiology. Lett. Appl. Micro. 15: 175-178.

Patel, P.D., and D.W. Williams. 1994. Evaluation of commercial kits and instruments for the detection of foodborne bacterial pathogens and toxins, p. 60-103. In P.D. Patel (ed.), Rapid analysis techniques in food microbiology. Chapman & Hall, Glasgow.

Pettipher, G.L., R. Mansel, C.H. McKinnon, and C.M. Cousins. 1980. Rapid membrane filtration- epifluorescent microscope technique for direct enumeration of bacteria in raw mik. Appl. Env. Micro. 3 9:423 -429.

Pettipher, G.L. 1983. The direct epinuorescent filter technique for the rapid enumeration of micro-organisms. Research Studies Press Ltd. London.

PHERO. 1994. Birthday party blues. An outbreak of Salmonella typhimzrrizrm. 5: 139-140.

Poppe C., R.J. M n , C.M. Forsberg, R.C. Clarke, and J. Oggel. 1991. The prevalence of SuZrnonella enferitidis and other Salmonella ssp. among Canadian registered commercial layer flocks. Epiderniol. Infect. 106:259-270.

Poteete, A.R. 1994. P22 bactenophage, p. 1009-1013. R.G. Webster, and A. Granoff (ed.), Encyclopedia of Virology. Vol 2. Acadernic Press. San Diego, California.

Puchkov, E.O., and AN. Melkozernov. 1995. F1uorimetx-i~ assessrnent of Pseudomonas fluorescens viability f ier freeze-thawing using ethidium bromide. Lett. Appl. Micro. 2 1:368-372.

Rea, M C , TM. Cogan, and S. Tobin. 1992. Incidence of pathogenic bacteria in raw milk in Ireland. J. Appl. Bact. 73 :33 2-336.

Riemann, H. 1993. Control of Salmonella in poultry. Department of Epidemiology and Preventative Medicine. University of California, Davis.

Ripabelii, M.L. Summarco, A. Ruberto, G. Iannitto, and G.M. Grasso. 1997. Imrnunomagnetic separation and conventional culture procedure for detection of naturally occuning Salmonella in raw pork sausages and chicken meat. Lett. Appl. Micro. 24:493-497.

Rohrbach, B.W., F.A. Draughon, P.M. Davidson, and S.P. Oliver. 1992. Prevalence of Listeria monocytogenes, Cmpylobacterjejuni, Yersinia enterocolitica, and Salmorda in bulk tank rnilk: Risk factors and nsk of human exposure. 3. Food Prot. 55:93-97.

Rowe, B., N.T. Begg, D.N. Hutchinson, H.C. Dawkins, R.J. Gilbert, M. Jacob, B.H. Hales, F.A. Rae, and M. Jepson. 1987. Salmonella ealing infections associated with consumption of infant dried rnilk. Lancet 2:900-903.

Ryan, C.A., M.K. Nkels, N.T. Hagrett-Bean, M.B. Potter, T. Endo, L. Mayer, C.W. Langkop, C. Gibson, R.C. McDonald, R.T. Kemey, N.D. Puhr, P.J. McDomeil, R.J. Martin, M.L. Cohen, and P.A. Blake. 1987. Massive outbreak of anti-microbial resistant salmonellosis traced to pasteurized milk. J. Am. Med. Assoc. 258:3269-3274.

Scheffe, H. 1959. Analysis of Variance. John Wiley and Sons hc . New York.

Schneitz, C., L. Nuotio, G. Mead, and E. Nurmi. 1992. Cornpetitive exclusion in the young bird: Challenge models, administration and reciprocal protection. Int. J. Food Micro. l5:24 1-244.

Shaw, B.G., and L.J. Farr. 1989. The rapid estimation of bacterial counts on meat and poultry by the direct epifluorescent filter technique, p. 47-57. C.J. Stannard, S.B. Petitt, and S.B. Skinner (ed.), Rapid microbiological methods for foods, beverages and pharmaceuticals. Blackweil Scientific Publications. Odord.

Shohat, T., M.S. Green, D. Merom, O.N. Gill, A. Reisfeld, A. Matas, D. Blau, N. Gd, and P.E. Slater. 1996. International epiderniological and microbiological study of outbreak of Salmorzella agona infection fiom a ready to eat savoury snack -II: Israel. Brit. Med. J. 3 13 : 1 10% 1 109.

Sierra, M., E. Gonzalez-Fandos, M. Garcia-Lopez, M.C.G. Femandez, and M. Prieto. 1995. Prevalence of Salmonella, Yersinia, Aeromonm, CmpyZo bacter and cold-growing Escherichia coli on eeshly dressed lamb carcasses. J. Food Prot. 58: 1 183- 1 1 85.

Sirnonsen, B., F.L. Bryan, J.H.B. Christian, T . A Roberts, R.B. Tompkin, and J.H. Süliker. 1987. Prevention and control of food-borne salmonellosis through application of Hazard Analysis Critical Control Point (EWCCP). Int. J. Food Micro. 4:227-247.

S kjerve, E., and 0. Olsvik. 199 1. Immunomagnetic separation of SaZrnonella from foods. Int. J. Food Micro. 14: 1 1- 18

Sockett, P.N. 1995. The epidemiology and costs of diseases of public health significance, in relation to meat and meat products. J. Food Safety. 1 2 9 1 - 1 12.

Stephenson, P., F.B. Stacheli, G. Allen, and W.H. Andrews. 1991. Recovery of SuZrnonella fiom shell eggs. J. AOAC. 74:82 1-826.

Stewart, G.S.A.B. 1990. In vivo bioluminescence: New potentials for microbiology. Lett. Appl. Micro. 10: 1-8.

Stewart, G.S.A.B., and P. Williams. 1992. l m genes and the applications of bacterial bioluminescence, J. Gen. Micro. 13 8: 128% 1300,

St. Louis, M.E., D.L. Morse, M.E. Potter, T.M. DeMefi, J.J., Guzewich, R-V. Tauxe, P.A. Blake. 1988. The emergence of grade A eggs as a major source of Salmonella enteritidis infections: New implications for the control of salmonellosis. J. Am. Med. Assoc. 259:2103-

Sun, M. 1985. Desperately seeking SaIrnonella in Illinois. Science. 228:829-830.

Suzuki, S. 1994. Pathogenicity of Salmonella in poultry. Int. J. Food Micro. 21 :89-105.

Swarninathan, B., and P. Feng. 1994. Rapid detection of food-borne pathogenic bacteria. Am. Rev. Micro. 48 :40 1-426.

Tauxe, R.T. 199 1. Salmonella: A postmodem pathogen. J. Food Prot. 54563-568.

van der Zee, H. 1994, Conventional methods for the detection and isolation of Salmonella enterifidis. Int. J . Food Micro. 2 1 (1/2):4 1-46.

Tokumaru, M., H. Konuma, M. Umesako, S. Konno, and K. Shinagawa. 199 1. Rates of detection of Salmonella and Campylobacter in meats in response to the sample size and the infection level of each species. Int. J. Food Micro. 13 :41-46.

USDA:APHIS:VS. 1994. Escherichia coli 0 157:H7. Issues and ramifications. Centers for Epidemiology and Animal Health. Fort Collins, CO.

USDA. March 19, 1998. ARS News SeMce.

Vermunt, A.E.M., AAJ.M. Franken, and R.R Beumer. 1992. Isolation of salmone~as by immunomagnetic separation. J. Appl. Bad. 72: 1 12-1 18.

Vought, K. J., and S .R. Tatini. 1998. Salmonella enteriitidis contamination of ice cream associated with a 1994 multistate outbreak. J. Food Prot. 61 5-10.

Weagant, S.D., J.L. Bryant, and KG. J i e m a n . 1995. An irnproved rapid technique for isolation of Escherichia coli 0 1 57:H7 fiom foods. J. Food Prot. 58:7-12,

Wegener, H.C., and D.L. Baggesen. 1996. Investigation of an outbreak of human salmonellosis caused by Salmonella enterica ssp. enterica serovar Infantis by use of pulsed field gel electrophoresis. Int. J. Food Micro. 32: 125-1 3 1.

White, C.E., and R.J. Argauer. 1970. Fluorescence analysis: A practical approach. Marcel Dekker Inc. New York.

WHO Expert Comrnittee. 1988. Salmonellosis control.: The role of animal and product hygiene. Tech. Report Series 774, Geneva: World Heait h O rganization.

Wierup, M., Wahlstrom, and D. Engstrom. 1992. Experience of a 10-year use of cornpetitive exclusion treatment as part of the Salmonella control programme in Sweden. Int. J. Food. Micro. 1 S:S87-29 1.

Wilson, S.G., S. Chan, M. Deroo, M. Vera-Garcia, A. Johnson, D. Lane, and D.N. Halbert. 1990. Development of a colorirnetric, second generation nucleic acid hybridization method for detection of Salmonella Ui foods and a cornparison with conventional culture procedure. J. Food Sci. 5511394-1398-

Wolber, P.K. and R-L. Green. 1990. New rapid method for the detection of Salmonella in foods. Trends in Food Sci. and Tech. 1:80-82.

Worthy, W. 1990. Bacteria assay exploits ice nucleation research. Chem. & Eng. News. 68:23-25.

Wright, D. J., P. A. Chapman, and C.A. Siddons. 1994. Immunomagnetic separation as a sensitive method for isolating Escherichia coli 0157:H7 from food samples. Epiderniol. Infect. 1 13:3 1-39.

Yu, H., and J.G. Bruno. 1996. Imrnunomagnetic-eïectrochemilurninescent detection of Escherichia coli 0 157:H7 and Sulmonella typhimz~rizim in foods and environmental water samples. Appl. Environ. Micro. 621587492.

Yu, H., and P.J. Stopa. 1996. Application of an immunomagnetic assay system for detection of virulent bactena in biological sarnples, p. 297-306. Ln J.M. Van Emon, C.L. Gerlach, and J.C. Johnson (ed.), Environmental imrnunochemical methods: Perspectives and applications. Amencan Chernical Society.