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LIBRARY
Michigan State
Unlverslty

 

 

 

This is to certify that the
thesis entitled

Isolation of Salmonella and Escherichia Coli in
Feces of Cull (Market) Dairy Cows at Slaughter

presented by

Ozlem Akpinar

has been accepted towards fulfillment
of the requirements for

 

 

 

 

M.S. degreein LCS
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[)ate

 

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6/01 c:lClRC/DateDue.p66-p.15

 

ISOLATION OF SALMONELLA AND ESCHERICHIA COLI IN FECES
OF CULL (MARKET) DAIRY COWS AT SLAUGHTER

By

Ozlem Akpinar

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Large Animal Clinical Sciences

2001

ABSTRACT

ISOLATION OF SALMONELLA AND ESCHERICHIA COLI IN FECES
OF CULL (MARKET) DAIRY COWS AT SLAUGHTER

By

Ozlem Akpinar

Objectives of this study were to evaluate the prevalence and concentration of
Salmonella and EHEC (Enterohemorrhagic E. coli) in feces of dairy cows at slaughter,
and to evaluate the effects of season on the isolation of pathogens from dairy cows at
slaughter. Samples were collected at slaughter from cattle that had either been shipped
directly or indirectly to slaughter. Fecal samples from 1006 cows were collected in
winter and summer of 1996. Salmonella and E. coli isolates were analyzed with respect
to animal disposition including body condition score, animal health, source of the animal,
and season of the year. Salmonella was isolated from 94 of the 1006 fecal samples.
Twenty-two serotypes were identified with the predominant isolates comprising of S.
typhimurium (22/94), S. senftenberg (17/94), and S. kentucky (8/94). Salmonella was
isolated almost three times more often in the August sampling (70/505 for 13.86%) than
the February sampling (24/501 for 4.79%). Coliform bacteria (Escherichia coli and
Klebsiella) were isolated from 829 of 1006 fecal samples and there was no growth from
127 of 200 frozen samples. Sorbitol-negative E. coli was isolated twice as often in the
summer 39% (199/505) than in the winter 19% (59/301). Of the 265 sorbitol-negative E.
coli, 6 samples were identified as positive (0.023%) to EHEC and 2 were serotype
0157zH7. A total of 47 samples contained only Klebsz'ella spp., 34 samples in winter and

13 samples in summer.

€0pyright by
OZLEM AKPINAR
2001

DEDICATION

It gives me great honor to dedicate this work to my family,
Mustafa G. and Berkcan Akpinar
and my parents Salih and Siiheyla Yildirim.
Thank you for believing in me, your unfailing love, support, and

encouragement throughout my masters program at Michigan State University.

iv

ACKNOWLEDGMENTS

I would especially like to extend sincere thank you to my major advisor, Dr.
Philip M. Sears, for always having an open door and mind, his help initiating this project,
for his patient assistance, wise counsel, and instructional help throughout its duration.
Another heartfelt thanks to my graduate committee members Dr. Paul C. Bartlett, Dr.
Ronald J. Erskine and Dr. Robert E. Holland for their strong support and advice that
enhanced completion of this study. I never would have gotten through this without you.

I would like to thank you Dr. Frederick Derksen chair of the Large Animal
Clinical Science department for excepting me to the program, and all the employees of
the department for giving me an opportunity to do my masters in such a wonderful
university.

I would like to thank you Dr. Fred Troutt for his help and sharing his knowledge
on this project.

I would like to thank you Dr. Barbara E. Straw for her help during collecting the
data working in the laboratory and for the final touches on my thesis.

I would like to thank you my colleague Anantachai Chaiyotwittayakun, laboratory
technician Chris Gage and all the other students for being a wonderful friend, their
support and assistance in the laboratory culturing samples and traveling to slaughter with
us to collect the data.

Finally I would like to thank you my dear husband Dr. Mustafa Akpinar even
with his busy schedule who has put his knowledge in through out this project and help me

out with the house work, my son Berkcan Akpinar (10) who is a great fan of the Spartans,

for being such a nice child and patient during mom was gone early in the morning to
school, my sister Izlem Yildirim for being a great support while studying for her
mechanical engineering undergraduate degree at Kettering University. My dearest parents
Siiheyla and Salih Yildirim even while you were so far away from me but kept your heart

and thoughts always beside me.

vi

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................... ix
LIST OF FIGURES ........................................................................................................ xi
INTRODUCTION ........................................................................................................... 1
CHAPTER 1
REVIEW OF LITERATURE ......................................................................................... 6
Salmonella ............................................................................................................ 6
Isolation and prevalence of Salmonella at slaughter ............................................ 9
Prevalence on farm ..................................................................................... 9
Prevalence on slaughter ............................................................................ 11
Identification and improved method of isolation in the industry
of monitoring ............................................................................................ 14
Pre-enrichment ................................................................................. 15
Enrichment ....................................................................................... 16
Plating media and biochemical screening ........................................ 17
Rapid methods ................................................................................. 19
Nucleic acid-based assays ................................................................ 21
Antibody-based assays ..................................................................... 22
Instrumentation and automated assays ............................................. 24
Methods of control of organism ......................................................................... 25
Organism shedding and control in farm .................................................... 25
Control in contamination and monitoring at slaughter plant .................... 30
Federal and State standards for pathogen reduction and control .............. 35
CHAPTER 2
REVIEW OF LITERATURE ....................................................................................... 41
Escherichia coli ................................................................................................. 41
Escherichia coli 0157:H7 as an important food borne
pathogen in human beings ................................................................................. 4]
Isolation and prevalence of E. coli at slaughter ................................................. 42
Prevalence on farms .................................................................................. 42
Prevalence at slaughter ............................................................................. 44
Identification and improved method of isolation in the industry of
monitoring ................................................................................................. 46
Methods of control of organism ......................................................................... 57
Organism shedding and control on farm ................................................... 57
Source of Escherichia coli 0157:H7 infections in human beings ..................... 59
Epidemiology of E. coli 0157:H7 ..................................................................... 61

vii

CHAPTER 3

PREVALENCE OF E. coli IN DAIRY CATTLE AT SLAUGHTER .......................... 64
Abstract .......................................................................................................... 64
Introduction ........................................................................................................ 64
Sample collection and procedures ..................................................................... 66

Slaughter animal collection ....................................................................... 66
Sample preparation and handling for E. coli ............................................ 67
Media and organisms isolation and detection ........................................... 67
Culturing .......................................................................................... 67
Enzyme immunoassay for the detection of the toxins produced
by Enterohemorrhagic E. coli in culture systems (EIA) .................. 68
Polymerase chain reaction (PCR) .................................................... 68
Statistical analysis ..................................................................................... 68
Results ....................................................................................................... 69
Discussion ................................................................................................. 70
Conclusion ................................................................................................ 71

CHAPTER 4

PREVALENCE OF Salmonella IN DAIRY CATTLE AT SLAUGHTER .................. 74
Abstract .......................................................................................................... 74
Introduction ........................................................................................................ 75
Material and Methods ........................................................................................ 77

Study design at slaughter .......................................................................... 77
Sampling and bacterial culture methods for feces .................................... 78
Statistical analysis ..................................................................................... 79
Results ....................................................................................................... 80
Discussion ................................................................................................. 81
Conclusion ................................................................................................ 83

CHAPTER 5

CONCLUSION .............................................................................................................. 88

REFERENCES .............................................................................................................. 92

viii

LIST OF TABLES

TABLES
1. Bacteria isolated during investigations of 82 outbreaks of food borne disease,

1986 to 1995 ...................................................................................... 3
2. Major source of selected food-home and water-borne pathogens .......................... 4

3. Food vehicles implicated during investigations of 82 outbreaks
of bacterial food borne disease, 1986 to 1995 ................................................ 4

4. F ood-borne disease rates in the United States ................................................ 5

5. The 10 most frequently isolated Salmonella serotypes (July 1995 to June 1996
and July 1996 to June 1997) from human sources at the Centers for Disease Control
and Prevention (CDC), from cattle submissions to the National Veterinary Services
Laboratory (NV SL), and culled dairy cows (Dairy) at 5 non-fed beef slaughter
establishments during winter and summer periods in 1996 ................................. 8

6. Selected rapid methods/materials commercially available for the identification
of Salmonella in foods .......................................................................... 20

7. Classifications and characteristics of Escherichia coli of the

gastrotestinal tract in people .................................................................. 49
8. Identification of coliforms and related organisms .......................................... 52
9. Growth characteristics of Shigella on selective media ..................................... 52
10. Isolation of Escherichia coli from feces of dairy cattle before slaughter ................ 72
1 1. The prevalence of sorbitol-negative E. coli during winter and summer. . . . . . . . . ..73

12. Prevalence of E. coli sorbitol-negative and MacConkey positive

growth of the fresh and frozen samples in winter ........................................... 73
13. Isolation of Salmonella from cull cows at slaughter ........................................ 84
14. Seasonal difference in Salmonella prevalence at slaughter ................................ 85

15. Prevalence of Salmonella in cattle that were assembled prior to
shipment (indirect) or directly shipped to slaughter ........................................ 85

16.Salmonella prevalence in cull dairy cows originating from Michigan, Indiana and
Ohio .............................................................................................. 86

ix

17. Salmonella prevalence related to body condition scores ................................... 87

LIST OF FIGURES

FIGURES
1. Prevalence of Salmonella in dairy calves by season ....................................... 11
2. Critical control points in controlling Salmonella in cattle ................................. 29
3. Generic HACCP for beef slaughter, fabrication and packaging .......................... 40
4. Prevalence of Escherichia coli 0157: in fecal samples from yearling cattle

and cull cows at slaughter in Alberta by season ............................................ 46
5. The molecular pathogenesis of Escherichia coli infections ............................... 50
6. Diagram of Salmonella isolation procedure ................................................. 79

xi

INTRODUCTION

The term ‘food-borne disease’ is any illness that results from ingestion of food.
The epidemiology of food-home disease is changing. New pathogens have emerged, and
some (have spread worldwide. The potential microbiological hazards for food-borne
illness from healthy and cull dairy cows include Salmonella (with special attention to
DT104), Escherichia coli 0157:H7, Campylobacter jejuni, Listeria monocytogenes,
Clostridium perfringens and Staphylococuss aureus. Salmonella spp., and Escherichia
coli 0157:H7 have been the focus of outbreak investigations (Table 1,4).37"“3’97"°9’149
These pathogens cause millions of cases of sporadic illness and death over many states
and nations.37 These reports have increased consumer distrust in the safety of the food

“8'97 The major food vehicles associated with outbreaks were beef, turkey,

supply.
chicken, ice cream, pork, dairy products, and eggsm From 1986 tol995, Salmonella
spp., S enteritidis, and E. coli 0157 :H7 accounted for 48% of food-home diseases (Table
2,3).5.36,l49

Food safety was identified as a priority area in Michigan agriculture for extension
and research. Food safety can be defined in terms of risk of pathogens and risk of drug
residues. Drug residues on farms are often related to the level of clinical disease and
treatment procedures used to manage these cases. Risks of preharvest Salmonella and E.
coli are related to disease incidence on the farm, methods of managing cases and
decisions to cull animals. Culling decisions may be precipitated by disease, but while

cull animals are not likely to be considered an important population on the farm, they can

be an important contributor to antibiotic residues in meat, and a source of pathogens at

slaughter. “’97 Cull dairy cows that are removed from the herd due to health problems
including diarrhea, mastitis and pneumonia may pose a higher risk of contamination at
slaughter. Shedding of pathogens such as Salmonella spp., and Escherichia coli
increases when animals are stressed due to secondary health problems.48’76"53 Although
the reason for culling may present no immediate human health concern, the increase in
fecal shedding of Salmonella spp., E. coli and other pathogens could result in a risk of
meat contamination.“"‘9’7‘5’153 Not all bacteria isolated from cull cows are pathogenic, but
handling practices which influence non-pathogen microorganism shedding in feces, could
also increase pathogens if present in the herd.

As a part of a larger study the Allendale slaughter facility was chosen for
sampling dairy cows. Animals presented for slaughter were evaluated as to the health of
the animal, body condition and any physical abnormalities.48 The effects of cold winter

1148"” were also

and hot summer conditions on shedding of the Salmonella and E. co
tested, the transportation of these animals as a variable was determined in this study.

On July 25, 1996, sweeping reforms on food safety regulations, known as the
final rule on pathogen reduction and Hazard Analysis Critical Control Points (HACCP),
was published by the United States Department of Agriculture (USDA).178 Although
targeted for slaughter and processing plants that handle meat and poultry, the

8 All plants were also being

requirements could have an impact on dairy farmers.17
required to adopt and implement their own HACCP plan, and slaughter plants and plants
that produce raw ground products were required to ensure that the rate of contamination

from Salmonella spp. was below the current national baseline incidence. Beginning

January 27, 1997, the Food Safety and Inspection Service (F SIS) required all slaughter

plants to conduct microbial testing for generic Escherichia coli and to prepare and

implement standard operating procedures for sanitation (SSOP).I78

Table l: Bacteria isolated during investigations of 82
outbreaks of food-bome disease, 1986-1995"”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BACTERIAL AGENTS FREQUENCY (%)
Salmonella sp* 21 (24)
Salmonella enteritidis 14 (16)
Escherichia coli 0157:H7 13 (15)
Shigella sonnei (n=6), S flaneri (2) 8 (9)
Bacillus cereus 5 (6)
Campylobacter botulinum 5 (6)
Clostridium botulinum 5 (6)
Staphylococcus aureus 5 (6)
Listeria monocytogenes 4 (5)
Clostridium perfringens 3 (3)
Escherichia coli 0104:1121 l (1)
Enteroxigenic E coli 1 (l)
Yersinia enterocolitica 1 (1)
Group-A Streptococcus 1 (1)
*Includes S typhimurium (5), S tyhi (4), S heidelberg (2),

S newport (2), S agona (l), S infantis (1), S montivedeo (l),

S oranienburg (1), S poona (1 ), S reading (1), and S stanley (1).

 

 

 

Table 2: Major source of selected food-borne and water-borne pathogens.

36

 

 

 

 

 

 

 

PATHOGENS SOURCE
Raw or undercooked beef, poultry, pork, and
Escherichia coli lamb; cheese; raw or inadequately pasteurized
milk; apple cider; green salads
Raw or undercooked beef, poultry, lamb, eggs,
Salmonella spp. fish, shellfish, and pork; ice cream; raw or
inadequatelypasteurized milk
Raw or undercooked poultry, pork, lamb, and
Campylobacterjejuni beef; raw or inadequately pasteurized milk;
untreated water; fresh mushrooms
Seafood; raw or undercooked beef, pork, poultry,
Listeria monocytogenes lamb, and eggs; fermented sausages; produce
and vegetables; ice cream
Ctyptosporidium spp. Water

 

 

Table 3: Food vehicles implicated during investigations of 82
outbreaks of bacterial food-borne disease, 1986 to 1995.149

 

 

 

 

 

 

 

 

 

 

 

 

 

SOURCE FREQUENCY (%)
Other" 24 (29)

Meat 20 (24)

Eggs 12 (15)
Poultry 7 (9)

Milk 5 (6)

Fish or shellfish 5 (6)

Meat products 4 (5)
Unknown'l' 4 (5)
Poultry products 1 (1)

* Includes reports in which food vehicles is not otherwise listed;
1 Includes reports in which a meal or buffet, not a specific food,

was implicated.

 

 

 

Table 4: Food-borne disease rates in the United States.“’109

 

 

 

 

 

 

 

 

 

Pathogens Cases per Total no. of
100,000 persons cases
Campylobacter 25.4 3,359
Salmonella 15.6 2,069
Shigella 9.6 1 ,272
E. coli 0157:H7 2.9 388
Yersinia 1.1 149
Listeria 0.5 64
Vibrio 0.2 21
Total 55.4 7,322

 

 

 

 

‘ Data are from the F oodNet 1996 final report and are for all sites covered

by FoodNet.

 

CHAPTER 1

SALMONELLA

Salmonellosis in farm livestock and its association with human infection has
attracted a great deal of attention, particularly in recent years. Salmonella reside in the
intestinal tracts of humans and other animals, including birds. While there are over 2,450
recognized serotypes of Salmonella, only 5 or 6 serotypes are involved in the majority of
infections in cattle. These serotypes include S. typhimurium, S. dublin, S. neWport, S.
montevideo, and S. anatum. Recently Salmonella typhimurium DT104 with resistance to
5 antibiotics has been reported as the cause of human infections in the United Kingdom
and United States. The proportion of Salmonella typhimurium isolates that were R-type
ACSSuT increased from 2% in 1980, to 30% in 1999 in the United States.'90

As cattle operations have grown in size and animal density, sahnonellosis has
gained importance as a disease of calves.16 The type and severity of the disease are
influenced by the Salmonella serotype, the infective dose, and the age, immunity, and
health of the calvesm’n’206 Salmonellosis in farm livestock and its association with
human infection has attracted a great deal of attention, particularly in recent years.9
Salmonellosis is an infectious disease that continues to plague human populations in both
developed and developing countries, and is usually transmitted to humans by eating
contaminated foods.182 Salmonella are of major concern to the dairy industry because a
variety of serovars have been incriminated in outbreaks of human sahnonellosis that were

associated with the consumption of dairy products.59 Salmonella infections cause

significant morbidity, mortality, and economic loss and are particularly severe in infants,

elderly, or immunocompromised patients.5 Although incidents of human salmonellosis
are frequently limited to single cases the size of the international list of large food-bome

2 The reported incidence of Salmonella

outbreaks of sahnonellosis is alarming.18
infections in the US. has increased substantially since reporting to the Centers for
Disease Control (CDC) began in 1943.5 Each year in the United States, there are an
estimated 800,000 to 4 million Salmonella infections, and approximately 500 are fatal.98
From 1983 tol987, Salmonella accounted for 28% of .food-bome disease outbreaks and
45% of food-borne disease cases of known etiology in the USS The major food vehicles
associated with these outbreaks were beef, turkey, chicken, ice cream, pork, dairy
products, and eggs although vegetables may become contaminateds’I61

Cull dairy cattle are especially important potential reservoirs for human
salmonellosis because they are the source of much of the hamburger consumed in the
United States about 17%.190 Unlike fed cattle, cull dairy cows may be in poorer body
condition; therefore, meat from these dairy cows often is used for ground beef.190
Undercooked ground beef has been implicated as an important source for Salmonella
infections in humans. 74"”

Between 1994-1995, the serotypes most frequently isolated from cull dairy cattle
were: S. typhimurium, S. dublin, S. kentucky, S. montevideo, S. muenster, S. newport, S.
anatum, and S. cerro.68’162 Primary serotypes isolated from cattle were S. typhimurium
and S. dublin.°8"62 In 1996 a nation wide study has a new list of 10 most isolated

serotypes from cull dairy cattle: S. montevideo, S. muenster, S. kentucky, S. anatum, S.

cerro, S. lille, S. typhimurium, S. mbandaka, S. give, and S. meleagridis (Table 5).

Table 5: The 10 most frequently isolated Salmonella serotypes (July 1995 to June 1996
and July 1996 to June 1997) from human sources at the Centers for Disease
Control and Prevention (CDC), fiom cattle submissions to the National
Veterinary Services Laboratory (NV SL), and culled dairy cows (Dairy) at 5 non-
fed beef slaughter establishments during winter and summer periods in 1996.7“'190

 

 

 

CDC NVSL Dairy'
Rank 1996 1995/1996 1996/1997 1996
l (v; i’andlifgen) (V172) 22mg; ) S Typhimurium S Montevideo
2 s Typhimurium” s Tmhimurium s Dublin s Muenster
3 S Heidelberg S Montevideo S Tmhimurium S Kentucky
4 S Newport m S Montevideo S Anatum
5 S Montevideo S Anatum S Kentucfl Cerro
6 S Javiana S Muenster S Anatum S Lille
7 S Orianenburg S Dublin S Meleagridis S T mkimurium
8 S Hadar S Kentuch S Muenster S Mbandaka
9 S Agona S Give m m
10 S Muenchen S Meleagg‘dis S Menhaden S Meleagn‘dis

 

 

 

Salmonella serotypes that are underlined appear on a NVSL list as well as the Dairy list. Salmonella
serotypes in bold type are connnon to all 3 lists.

‘ If S Typhimurium (var Copenhagen) is included in the count of S Typhimurium, as was done by the CDC,
S Typhimurium would move up to rank 5. b Includes S T yphimurium var Copenhagen.

ISOLATION AND PREVALENCE OF Salmonella AT SLAUGHTER
Prevalence on farm

In England over a three-year period (1969-72) four large calf units were examined
for Salmonella infection. The 4 units had different husbandry and farm management.
The incidence of Salmonella in fecal samples ranged from a low of 0.7% to a high of
11.1%. The dominant serotype was S. dublin.34

In 1985 after detecting a rise in human infection with S. newport in California,
Los Angelos County Department of Health Services conducted a survey on dairies in
Califomia. Of the 75 dairies randomly selected, the median number of cattle on the
dairies were 580 adult cows (range 80 to 2900) and 50 calves less than 2 month old
(range 0 to 300). Salmonella was isolated from at least 1 of 4 sample sites on 12 of 75
dairies (16%).

Calves were the single best source (8 of 12 Salmonella positive dairies) for
isolating Salmonella at dairies where the organism was detected. Four Salmonella
serotypes were isolated: S. newport (6), S. montevideo (3), S. dublin (2) and S.

”3 Wray et al.2'5 examined calves for Salmonella in England and

typhimurium (1).
Wales between September 1985 and April 1986. At least 28 days after their arrival on
farms, 589 animals distributed in 25 groups on 11 farms were examined for excretion of
Salmonella. Salmonella was found in 212 calves. Salmonella were not detected in 7 of
the 25 groups. In the other 18 groups, between 3% and 90% of animals were identified
as excretors. On arrival fi'om various markets, total incidence of Salmonella fecal

excretion was 0.7% and it reached it’s peak around 2 to 3 weeks afier arrival on the

farms. Salmonella typhimurium and S. dublin were the predominant strains isolated.

There was no difference in excretion rates between calves housed singly compared to
calves group-housed in pens.

Between 1991 and 1992 Salmonella prevalence was 2.1% from 6861 preweaned
dairy heifer calf fecal samples from 1063 dairy farms at 28 states in the USA.”140
Salmonella serotypes found in this national survey showed that, S. typhimurium
prevalence was 27.6%, S. dublin prevalence was 10.3% and S. mbandaka prevalence was
8.9%.'40 In European countries, S. dublin and S. typhimurium are found to be the most
common serotypes in cattle. 14°

The number of S. typhimurium DT104 cases in humans and in animals for
England and Wales rose between 1990 and 1996.151 In the United States, the frequency
of S. typhimurium isolation has also increased recently. In cattle DT104 was most
commonly recognized in 2 to 4 week old sick calves suffering from diarrhea. Calves that
were recently purchased and had traveled became sick soon after arrival and experienced
a mortality of 40% to 50%.60 Fecal shedding is persistent after outbreaks of this type of
salmonellosis, and the organism has been recovered from feces for up to 18 months after
infection. A case control study in Great Britain reported that most outbreaks lasted for
less than a week and less than 4% of animals within herds were clinically affected. The
incidence of disease was about 33% in calves, compared with only 4% of adults in
affected herds. Sub-clinical carriage was common and persisted for up to 18 monthsm

In 1991-1992, the US. Department of Agriculture did a project named the

National Dairy Heifer Evaluation Project (NDHEP) which determined Salmonella

prevalence rates across the nation. According to this study Salmonella prevalence was

10

highest in the late summer (July-September) with 36.1 of every 1000 samples testing

positive (Figure 1).

 

40

35

30

25

20

15

10

Prevalence per 1000 Calves

 

 

 

Quarter

Figure]: Prevalence of Salmonella in dairy calves by season. ‘94

Prevalence at slaughter

Rumen fluid is an important potential source of contamination of carcasses at
slaughter and a reservoir of Salmonella for infection of the intestine.34’m5 Grau and
Browlie,84 found that 61 of 170 rumen samples (36%) and 39 (27%) of 146 fecal samples
were positive for Salmonella. The prevalence of Salmonella infection in the rumen and
feces of slaughtered cattle has been shown to increase with increased time between farm

and slaughter and also to be higher in animals which have been fed once during the

11

holding period. ‘9 In an Australian slaughter project, the inspectors observed that animals
that were slaughtered on the first two days of the week, Monday and Tuesday, had a
higher prevalence of Salmonella in both rumen and mesenteric lymph nodes than animals
slaughtered on other days of the week.'“"65 ‘166 The difference was attributed to the cattle
killed on Monday and Tuesday being held, and usually fed, over the weekend, after
traveling considerable distances from their property of origin.165 "66

Salmonella can persist in dairy cows and the surrounding environment for several
years without showing evidence of clinical disease or production inefficiency.79'82 There
is a possibility that during such periods, cows which may have been Salmonella carriers
are routinely culled for slaughter. If the time from farm to slaughter facilities is
prolonged, the stress of transport and fasting prior to slaughter can increase the
prevalence of Salmonella infection among animals.126 Puyalto et al.‘53 have shown that
the prevalence of Salmonella was 8% (6/80) on leaving the farms and this number
reached 25% (20/80) on arrival at the slaughter plant.

Studies84'86’126 show that shedding of Salmonella is affected by rumen pH and
volatile fatty acid level. Acidic pH and increased volatile fatty acid level prevent grth
of enteric bacteria. Moderately elevated rumen pH and decreased concentrations of total
acidity, as would occur in withholding of food, are conditions which foster grth of a
variety of Salmonella serotypes in rumen fluid of cattle at slaughter.

In a study, Gay et al.80 showed that the rate of fecal shedding of Salmonella in

1,289 cull dairy cows marketed in the state of Washington to be approximately 0.5%. In

the same study mesenteric lymph nodes and rumen contents were cultured, a wide variety

12

of serotypes of Salmonella were isolated with a high prevalence (76%) from a population
of 100 cull cows.166

The appearance of a chloramphenicol resistant strain of Salmonella typhimurium
phage type 204 in calves in Great Britain highlighted potential public health risks and
since then chloramphenicol resistant strains of the same organism, thought to have in
some cases been derived from calves, have been isolated from sick humans.187

Though many of the more than 2,450 Salmonella serotypes can infect cattle, most
infections are limited to a few serotypes. Recently, there has been an increase in the
incidence of Salmonella outbreaks in dairy cattle in the Pacific Northwest of the USA.H
Studies revealed that isolates from these outbreaks were S. typhimurium DT104, this was
the first report of this definitive type of Salmonella.11 Salmonella enterica serotype
typhimurium characterized as definitive type 104 (DT104) is now the second most
prevalent Salmonella in human beings and animals in the United Kingdom (England and
Wales) and Europe.""'198 In this studyll investigators suggested that farmers can contract
S. typhimurium DT104 by handling sick cows and calves but they didn’t identify a single
food stuff as responsible for the increasing number of human isolations of S. typhimurium
DT104.'°"198"99

The isolations referred to the Laboratory of Enteric Pathogens increased, 250 in
1990, 2873 in 1994, and 3837 in 1995.‘85 The importance of this increase has been the
epidemic spread of a S. typhimurium R-type ACSSuT strain multiresistant to five
antimicrobial agents (A, arnpicillin; C, chloramphenicol; S, streptomycin, Su,
sulphonamides; T, tetracyclines).185 ”99 A recent report199 from England and Wales

showed that infections caused by this multidrug-resistant typhimurium were associated

l3

with greater morbidity and mortality than other salmonella infections. The DT104 strain
. is also resistant to drying and chemicals, which makes it a substantial potential zoonotic
threat!“ Molecular studies have demonstrated that in multiresistant DT104 all the
resistance genes are located on the chromosome, which is rare phenomenon for S.
typhimurium. ‘85

Unlike S. enteritidis phage type PT 4, which is almost entirely associated with
poultry and poultry products, the epidemiological evidence indicates that multiresistant
DT104 is widely distributed in a variety of different food animals.186 Although most
commonly associated with cattle, the strain has also been isolated fi'om sheep, pigs, goats,
chickens, and turkey, and from a wide range of food products and processed foods in the
United Kingdom.2

Another study'42 has demonstrated a high incidence of multiresistant DT104 in
fresh raw sausages purchased from a range of retail outlets in the United Kingdom. The
strain has also been isolated from farm workers and from domestic pets. It is now
generally accepted that the incidence of Salmonella in farm livestock is related to
husbandry methods and practices and there is a great deal of evidence to indicate that
extensive systems in particular favor the spread of infection and a subsequent increase in

the level of clinical disease.9

IDENTIFICATION AND IMPROVED METHODS OF
ISOLATION IN THE INDUSTRY OF MONITORING
Salmonellae are part of a family of Gram-negative, rod-shaped bacteria known as

Enterobacteriaceae, which occur in the intestinal tract of humans and in warm-blooded

l4

and cold-blooded animals. To date, more than 2,300 serotypes of Salmonella are known
to exist and new serotypes are being discovered each year. Of these recognized
serotypes, only about 100 are routinely isolated from food, animals and man.89 These
facultative anaerobic Gram-negative bacteria produce gas from glucose and utilize citrate
as their sole carbon source through their flagellated rods.”6

The detection of food-home pathogens is complicated because low numbers of
the organism of interest are often present in a complex microbial flora and because of
complex compositions of different foods.152 In food microbiology, the presence of a
single pathogenic organism is considered significant. Therefore, methods and media
must be capable of enabling growth to occur from extremely low initial cell numbers.
Five steps are common to most culture procedures for isolating and identifying
Salmonella in foods. These include (1) pre-enrichment of a food sample in a nutritious,
nonselective broth; (2) selective enrichment in a broth that allows sahnonellae to grow
but suppresses the growth of competing bacteria; (3) isolation of Salmonella by streaking
onto selective plating agar; (4) biochemical characterization of isolates; and (5)

serological confirmation of biochemically screened isolates.

Pro-enrichment

Pre-enrichment is the initial step in which the food sample is enriched in a
nonselective medium to a stable physiological condition so that the bacteria can grow on
the nutrients present in the medium.”161 Sublethal cell damage may have resulted from
thermal processing of food, freezing, thawing, osmotic shock, or prolonged storage of

low-moisture foods at elevated temperature.40 Satisfactory resuscitation and pre-

15

enrichment generally require a nutritious nonselective medium. Generally, pre-
enrichment media are nutritionally complex and may include trypticase soy broth,
nutrient broth, reconstituted nonfat dry milk, or lactose broth. Pre-enrichment requires

incubating cultures at 35-37°C for 16-24 hours.8’131

Enrichment

Selective enrichment is the step in which the sample is further enriched in a
growth-promoting medium containing selective inhibitory reagents. This medium allows
a continued increase of salmonellae while simultaneously restricting proliferation of most
other bacteria."’40 Selectivity of the enrichment process is based on synergism between
inhibitory agents in enrichment media and temperature of incubation."°"5330‘5 Two media
commonly used for selective enrichment are selenite cystine broth and tetrathionate
broth.179 The addition of cystine to selenite broth enhances Salmonella growth. All the
variations of selenite broth are suitable for most serovars, including S. typhi, S. dublin,
and S. choleraesuis. Selectivity of tetrathionate broth depends on the ability to suppress
the growth of coliforrn organisms.""’°’86’131 In addition to these two broths, Rappaport
enrichment broth can be used that includes modifications for Rappaport-Vassiliadis (RV)
enrichment broth, and Rappaport 25 (R25).4°'46"31'153

The temperature of incubation during selective enrichment significantly

influences the successfirl recovery of Salmonella in food. Selective enrichment cultures

are incubated for 16-24 hours at 35-37 or 43°C.4°"792°6

16

Plating media and biochemical screening

Selective plating uses solid selective media that restrict growth of bacteria other
than salmonellae."’179 Several agar media, including bismuth sulfite agar (BSA), brilliant
green (BGA), xylose lysine desoxycholate (XLD), Hektoen enteric (Hek) agars, and
xylose-lysine-tergitol 4 agar (XLT4) are widely used in standard methods for the
isolation of Salmonella in foods.4’4°’46'86’131"79206

The BGA, XLD and Hektoen media are related in bacterial utilization of lactose
and/or sucrose, low selectivity and incidence of numerous false-positive reactions,
whereas the bismuth sulfite agar (BSA), a non-saccharide differential medium, shows
good selectivity against non-salmonellae. Identification of salmonellae on this agar
(BSA) is based on the development of black colonies resulting from the capture of
metabolic H2S gas as the insoluble FeS salt, and frequent appearance of a black halo
around suspect colonies. Xylose-lysine-tergitol 4 (XLT4) is a new media in the 20th
century, it was found to strongly inhibit Proteus, Pseudomonas, Providencia and many
other non-salmonellaem'179 After 20 to 24h at 35 to 37°C, typical (st-positive)
Salmonella colonies on XLT4 media are smooth and creamy in texture and appear black
or black-centered with a yellow (acid) periphery that changes to pink (alkaline) as the
xylose is depleted/“5’13"179 It was concluded that the only genus capable of forming black
colonies within 24h on XLT4 media was Salmonella, allowing easy differentiation from
other organisms. None of the Salmonella plating media are fully selective, recovery of
the widest possible range of Salmonella serovars requires two or more plating media.86"52

Salmonella-like colonies are selected and identified by biochemical tests. Two

differential agars, that is, triple sugar iron agar (TSI) [salmonellae typically produce

17

alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening
of agar)] and lysine iron agar (LIA) [salmonellae typically produce an alkaline (purple)
reaction in the butt, with or without production of HZS], are commonly used in
combination to provide initial biochemical data about the isolates.103 "91 The presence of
glucose and an H28 detection system in TSI facilitates screening of non-glucose-
fermenting organisms such as Pseudomonas spp. and presumptive identification of
Salmonella. The LIA medium is of equal diagnostic value because it screens for the
presence of the lysine decarboxylase enzyme, which is commonly encountered in
Salmonella spp.40 Cultures typical of Salmonella in these media are then tested by
biochemical tests to confirm the isolates. Biochemical tests typically used include urease
(negative), lysine decarboxylase (positive), fermentation of dulcitol (positive), utilization
of sodium malonate (negative), and production of indole (negative). Other tests
occasionally used include fermentation of lactose and sucrose (both negative), Voges-
Proskauer test (negative), and methyl red test (positive)."0

Serotyping is the definitive step in providing a specific identification of the
cultures. 4'86 Cultures are tested by agglutination assays with antisera specific for somatic
(O), flagellar (H), and capsular (Vi) antigens. The heat-stable somatic antigens (O) of the
bacteria are identified first, using the slide agglutination method. Unlike “O” antisera,
“H” antisera are used in tube agglutination tests. If the slide technique is used, the “H”
antisera either must be fi'eed of “O” agglutinins by absorption or must be used in

dilutions sufficiently high that “O” reactions do not occur.62

18

Rapid methods

Rapid detection methods for food samples have been a subject of research since
the early 1980s, and these tests take 4 to 12h to completem Commercial diagnostic
assays for Salmonella may be placed in five general categories: miniaturized biochemical
tests; new media; instrumentation or automated systems; nucleic acid-based assays; and
antibody-based assays."°’66 Although many of these tests are referred to as “rapid
methods”, most of these Salmonella detection systems, regardless of the technology or
assay format, still rely on cultural methods for selective amplification of Salmonella
population in the broth culture. Therefore, pre-, selective-, or postenrichment procedures,
or some combination of them, must be used in conjunction with these “rapid” methods
for sensitivity and specificity.41 Sensitivity of a test refers to the minimum amount of an
organism or other substance that can be detected. Specificity is the ability of a test to
distinguish exactly the component of interest with no other interactions. Most of the
assay systems are screening assays and only provide for the presumptive identification of
salmonellae. Negative results, therefore, are considered definitive, but presumptive
positive results must be confirmed by conventional methods and serology.‘56 Table 6

shows some of the commercial tests that are used for detecting Salmonella spp.

l9

Table 6: Selected rapid methods/materials commercially
available for the identification of Salmonella in foods.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mimi-idly Assay format Manufacturer 1:32:
Miniaturized tests
API 20E Biochemical Analytab Final action
Enterotube II Biochemical Rochggizfinsostics
Enterobacteriaceae Set 11 Biochemical BBL Final action
MICRO-ID Biochemical Organon Teknika Final action
Media
HGMF/EF -18 Selective, differential QA Life Sciences First action
MSRV Selective, difl‘erential Various First action
Oxoid SRT Selective, differential Oxoitlljgglaitslion 0f None
Rambach agar Selective, differential Technogram (France) None
Nucleic Acid-Based
DNAH DNA probe GENE-TRAK First action
Antibody-based
Oxoid Latex beads Oxoid (UK) None
MicroScreen Latex beads Mercia (UK) None
Spectate Latex beads May and Baker (UK) None
Bactigen Latex beads Wampole None
Assurance ELISA, polyclonal BioControl First action
TECRA ELISA, pochlonal Bioenterprises (Australia) First action
Salmonella-Tek ELISA, monoclonal Organon Teknika First action
Salmonella 1-2 Test Immunodiffusion BioControl First action
UNIQUE Dipstick Bioenterprises (Australia) None
PATH-STIK Dipstick rhthggége None
Instrumentation
GNI Biochemical BioMérieux Vitek First action
Biolog Carbonutilization Biolog None
VIDAS ELFA BioMérieux Vitek None
Malthus Conductance Malthus(IIInsKt)r'urnents First action

 

 

 

 

Note: AOAC, Association of Official Analytical Chemists; ELISA, enzyme-linked imrnunosorbent assay;

ELFA, enzyme-linked fluorescent immunoassay“3

20

9,4 l .42. 104

 

Nucleic acid-based assays
DNA Probe. A DNA probe is normally a short sequence of nucleotide bases that will
bind to specific regions of a “target” sequence of nucleotides where the homology
between the target and the DNA probe results in a stable hybridization. When a protein
is the target of detection, as in an immunoassay, there is a risk that the nucleic acid
sequences that coded for the amino acids that make up the protein might be changed or
lost due to stress on microorganisms during food processing. Hybridization assays can
detect the presence of bacterial cells, regardless of the physiological state of the organism
or the status of proteins or lipids in or on the microorganism.213
Colorimetric DNA Hybridization Test. The first-generation DNA hybridization test used
a radioactively labeled probe (HP). The colorimetric assay employs Salmonella specific
DNA probes and a colorimetric (instead of radioisotopic) detection system for the
detection of Salmonella species in food samples following broth culture enrichment.
According to the studies, the GENE-TRAK Salmonella Assay appeared to be an
effective screening procedure for rapid detection of salmonellae in meat and poultry
products.163 "79 The major advantage of the colorimetric DNA probe assay is that large
numbers of samples can be screened fairly rapidly for salmonellae.“S3 ’179 However, all
DNA probe-positive samples should be confirmed by culture.
Polvmegag Chain Reaction (PCR). Genetically based, non-cultural, primer-mediated
enzymatic amplification of target-DNA, called PCR, has been applied successfully for
the detection of a large number of pathogens, including Salmonella.I79 The PCR method
can specifically amplify a single copy to one million-fold of a gene or DNA segment

unique to a target microbial pathogen. After amplification, the DNA segment can be

21

readily detected by DNA-DNA hybridization.179 Using PCR-based probes and
recombinant DNA hybridizations to detect pathogenic organisms has many advantages
over classical culture techniques. Amplification of DNA sequences unique to an
organism by PCR improves the speed and sensitivity at which organisms can be detected.
PCR has been used to identify several bacterial species including Salmonella serovars

from food and clinical samples.”’38

Antibody-based assays

The antibody-based assays are the largest group of commercial tests for detecting
Salmonella. They can be classified into the following categories on the basis of their
assay formats, including latex agglutination, enzyme-linked immunosorbent assay
(ELISA), immunodiffusion, and dipstick.
Latex Agglutination Assay: These agglutination assays use latex particles coupled with
polyvalent antisera to various Salmonella antigens. The latex particles with bound
Salmonella-specific antibodies amplify agglutination reactions and allow visual
identification of positive samples.42

The Oxoid Salmonella latex test, uses Salmonella antibodies that are specific both

for somatic and flagellar antigens."6 Positive results are confirmed by the reference

culture method. In the MicroScreen® test, reagents of this latex agglutination kit respond

to Salmonella flagellar antigens.42 A study with isolated bacterial strains, including‘24
Salmonella strains, showed high specificity as well as high sensitivity for this test (both

96%).123

22

Enzyme-Linked Immunisorbent Assays (ELISAs): Several ELISA’s have been
developed, using both polyclonal antibodies and monoclonal antibodies that will detect
most Salmonella serotypes. These assays and others have been developed subsequently
in kit form and are available commercially. Kit assays require enrichment steps to
resuscitate injured cells and to selectively amplify salmonellae. All ELISA kits are
designed in a “sandwich” or “capture format”, that is, antibody-coated polystyrene wells
are used to capture salmonellae antigen, and a second antibody to Salmonella conjugated
with an enzyme is added to form an antibody-antigen-antibody (sandwich) complex. The
sandwich complex is then determined by a colorimetric enzyme substrate, and the results
are recorded either visually or with a spectrophotometer.

Most of the Salmonella ELISA kits use alkaline phosphatase or horseradish
peroxidase enzyme conjugates with a colorimetric substrate system.66 Polyclonal enzyme
immunoassay (EIA) (Assurance) for Salmonella is configured in a microwell plate
format. It is designed for the rapid detection of motile and non-motile Salmonella.
Sensitivity is enhanced through the addition of another antibody that immunochemically
links the bound Salmonella antigens with the enzyme conjugate. The colorimetric,
monoclonal enyzme immunoassay (Salmonella-Tek) is a microtiter plate format, which
was reported to be a promising test for the detection of Salmonella antigens with very
low cross-reaction of the anti-Salmonella antibodies.57’196 Detection of Salmonella
antigens is based on EIA using specific monoclonal antibodies. This method is designed
for detection of Salmonella in all foods. The test is not confirmatory because monoclonal

antibodies used in this test may cross-react with a small percentage of non-Salmonella.57

23

lmmunodiffusion: The Salmonella 1-2 Test is the only commercial assay for Salmonella
in foods that uses the immunodiffusion format. It is a screening method for motile
Salmonella in foods. One study reported a high false-negative rate for the 1-2 Test when
the unit was inoculated from pre-enrichment broth (and suggested that better productivity
would likely be obtained with a modified enrichment protocol that included selective
enrichment before inoculation of the unit.39 Another study reported that use of
tetrathionate brilliant green agar broth enrichment step following pre-enrichment
enhanced the reliability of the 1-2 Test.139

Dipstick Assays: TECRA UNIQUE® Salmonella assay system uses an antibody-coated
dipstick after pre-enrichment to selectively capture salmonellae. Because competing
bacteria are not picked up by the dipstick, the UNIQUE system coupled with TECRA
ELISA should produce in fewer false-positive reactions. Sensitivity is reported
equivalent to that of standard culture methods. PATH-STICK is another assay that
makes simultaneous use of Salmonella antibody, conjugated with enzyme, and a

membrane-tipped dipstick bound with another antibody to Salmonella.66

Instrumentation and automated assays

Several automated and semi-automated systems using different technologies have
been developed for Salmonella identification. The Vitek AutoMicrobic System with the
Gram-Negative (GNI) Card uses a computer, optical reader, and test kits, with disposable
plastic cards that have wells containing different biochemical substrates. Once a test kit
has been inoculated with a suspension of the sample organism and has been loaded into

the system, no additional biochemical reagents need to be added. A final report is printed

24

automatically for each test kit at the end of its cycle, which is 4h for Salmonella.l '4 The
GNI system correctly detected 96.7% of Salmonella species in food samples.114 Other
automated identification systems include the Biological Identification System, which
measures the ability of the bacteria to oxidize 95 different carbon sources in order to
generate identification and metabolic information, and VIDAS (V itek Immuno
Diagnostic Assay System), which uses the Enzyme-Linked Fluorescent ImmunoAssay

(ELFA).8"”"

METHODS OF CONTROL OF ORGANISM
Organism shedding and control in farm

Microbiological food safety is an important issue in beef products for human
consumption. Cattle producers are implementers of management practices to reduce risk
and are supportive of research for improvement. When cattle leave the farm or feed-lot
for slaughter they will carry within their intestinal tracts and on their hooves and hides a
large population of microorganisms. Under feed-lot conditions the hide may become
heavily contaminated with feces. The percentage of animals carrying sahnonellae in their
intestinal tracts varies between different herds and at different times of the year (fall-
winter-spring-summer).

Control measures for bovine salmonellosis have been well documented. In
general, there are 3 main control points; (1) rodents and birds, which bring in Salmonella
from outside sources or which act to maintain infection on premises as a vector into cattle
feed, (2) contaminated feed sources, especially high moisture commodities in which

Salmonella readily multiply after contamination by birds, rodents, or equipment, and (3)

25

infected cattle, either asymptomatic carrier cattle or ill and recovering animals, which
magnify the number of Salmonella in the farm environment. '4'132"59’2'4

Birds, rats and mice are frequently infected with Salmonella, particularly
Salmonella typhimurium. Mice and rats may also be infected with S. dublin and should
be eradicated as a part of the dairy control program. When feeds are contaminated by
rodents and birds, multiplication of Salmonella in areas of high moisture occurs.132 Fecal
shedding of sahnonellae by infected cattle is the main source of infection in calves, which
are infected by the oral route.214 Reduction of Salmonella in feeds is possible by use of
organic acids.13 Elimination of Salmonella from feeds may require high temperature
pelleting or irradiation together with dehydration to reduce moisture content below 5%
and proper handling to prevent wetting and recontamination.13

If farm waste or sewage sludge is applied to pasture, then it should have been
stored for at least 4 weeks before application and there should be an interval of at least 4

weeks between application and grazing.132

Animals should not graze pastures which
have been flooded. '

Whether the number of Salmonella in feces and the immediate surroundings is
sufficient to cause clinical disease is not known, but it is also possible that close grazing
during the late autumn may result in an increased infection rate. Clegg et al.31 mentioned
that disease couldn’t be produced in calves allowed to graze on grass which had been
sprayed with slurry containing S. dublin. During late summer, reliance on grass of
deteriorating nutritional value may have precipitated the clinical infection.

In addition, septicemic infected calves shed salmonellae in nasal secretions and

saliva, which can contaminate feeding equipment and farm personnel. Many calves are

26

infected by direct contact with their dams or from the calving environment during the
first 24 hours of life and up to 2 month of age by S. dublin, similarly calves infected with
S. typhimurium tend to be infected from one to 35 days old. During this time low
numbers of organisms can establish an infection because the abomasum lacks protective
acidity and there are no competing flora in the gut.

Carrier animals are important in transmission. One asymptomatic carrier cow can
shed over 10 billion Salmonella dublin per day in feces and milk.173 A combination of
routine serology and bacterial culture of milk and feces from suspect animals can help
identify persistently infected cattle, which can then be culled. Good husbandry and
hygiene practices, such as housing calves individually and keeping the calving areas
clean, will reduce the calves’ exposure to salmonellae and other pathogens.

In 1996, the United States (particularly in western states) recognized a new and
apparently more virulent phage type of S. typhimurium, DT104. Investigations in the
United States have found associations between typhimurium DT104 infections in humans
and the consumption of unpasteurized dairy products and direct contact with livestock.
Salmonella typhimurium phage type (PT) or definitive type (DT) 104 is a virulent
pathogen for humans and animals, particularly cattle. It has been isolated increasingly
from humans and animals in the United Kingdom and several other European countries
and, more recently, in the United States and Canada.

Farm families are particularly at risk of acquiring the infection by contact with
infected animals or by drinking unpasteurized milk. Salmonella typhimurium DT104
infections in cattle may be prevented by purchasing replacement stock directly, rather

than via livestock dealers, by maintaining a 4-week quarantine period for purchased

27

cattle, by housing sick animals in dedicated isolation areas, and by preventing wild birds

from having access to feed for cattle.

60,61

To prevent the contamination and spreading of Salmonella infection in the dairy,

it is important to have good sanitary conditions and to minimize the contact of ill or

carrier animals and their feces with the other healthy animals (Figure 2).31 Some of the

other methods can be summarized as:

l.

14,120,129,l32,214

Because Salmonella infections are less likely to be found in calves individually
penned as compared to calves housed in a group pen, individual housing systems are
recommended for young calves.

Only strong, healthy calves should be purchased as replacement stock.

Purchased replacement stock should be serotested, cultured and quarantined.

Sick cows and calves should be isolated.

Avoid wet areas, provide dry areas such as free stalls for loafing, and clean and
disinfect calf pens and maternity areas between calves.

Rendering trucks and other vehicles which may be contaminated or carry infectious
material shouldn’t be allowed on the farm near animals or feed. Front-end loaders
used for dead animals or manure shouldn’t be used for feed.

Do not use routine prophylactic antibiotics, as this promotes bacterial resistance and
may harm cattle gut flora, predisposing to salmonella infection.

If there is clinical salmonellosis in the farm, vaccinate cows with a killed Salmonella
bacteria specific for the serotype isolated. Killed vaccine also can cause side effects
and side effects can increase with hot weather and administration with other

vaccinations such as Escherichia coli bacterins and Brucella abortus live vaccine.

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CONTROL IN CONTAMINATION AND MONITORING
AT SLAUGHTER PLANTS

Between the farm and the slaughter floor, the microbiological status of cattle can
change. Food deprivation (high concentration of volatile fatty acids-VFA and low pH)
and intermittent feeding that some herds undergo when they travel long distances to
slaughter make such animals very sensitive to salmonellae and the prevalence of infection
with Salmonella may increase markedlny’M”166

Grau et al.84, have shown that Salmonella may grow in the rumen of such animals
and it was shown that large numbers of Salmonella may be present in the mesenteric
lymph nodes as well as in the gut contents. Not only can the intestinal tract of these
cattle become highly contaminated with salmonellae but these animals in turn
contaminate the environment through which they pass; such as trucks, railway wagons,
sale-yards and holding areas.84216 In these environments, hides and hooves are also
contaminated. Even in clean areas, sahnonellae shed in the feces of a few animals can
get on the hooves, legs and, when cattle lie down, larger areas of hide.

Puyalto et al.153 between April 1994 and May 1995, documented the increase in
hair contamination by salmonellae in cattle between the farm and slaughter plant.
Samples from animals and environment in which they were stationed were collected.
Hair samples as well as the environmental samples were the most frequently
contaminated (26% to 69%). Contamination of the hair had a frequency of 25%, during
the time when the cattle were transported to slaughter.

At slaughter animal goes through a couple of steps, which gives an opportunity

for food-home pathogens to contaminate the products and the environment. These steps

30

are hide removal, evisceration, boning, chilling or freezing of boneless products, and
temperature control in transport to the export market. While some of the same principles
apply to both control of microbial contamination in a beef slaughter and to other meat
species, there are a number of differences. There is considerably more vertical
integration in the poultry and pig industries with a greater possibility of the one owner
controlling handling from birth of the animal to final sale of the packaged meat. The skin
is left on pig and poultry carcasses, and a heat treatment is applied to their surface tissues.
The much larger size of a beef carcass, compared to poultry and sheep, increases the time
required to chill it, and so influences the pattern of microbial growth. At the completion
of slaughter and dressing, beef carcasses tend to carry a smaller load of microbial
contaminants than is found on sheep, pig, and poultry carcasses.

There are multiple factors responsible for reductions in Salmonella prevalence
within slaughter plants. Size, congestion, and maintenance of a slaughter plant, the
number of animals slaughtered per day, the flow of carcasses through the plant, control of
each step in the sanitary dressing procedure, sanitation of facilities and equipment, and
personal hygiene are factors to be considered to reduce pathogens on carcasses. If all
these measures are not possible, at least not shortly, consideration should be given to
eliminating pathogens on the meat afler slaughter procedures. The interventions
available to plants to reduce pathogens on carcasses include; lowering water activity,
reducing surface pH, using enzyme inhibitors, cooling (refiigeration or freezing),
applying lactic fermentation, irradiation, and treating with organic acids, chlorine and hot

water, sodium chloride, or sorbate.

31

Lowering water activig; surface drying of carcasses reduces the water activity
and inhibits microbial growth.

Reduction of surfge pH and tgatment with organigcids; a low pH ranging 4.0-
4.5 inhibits the growth of both spoilage and pathogenic micro-organisms.22 This pH
reduction has often been achieved by treating meat with organic acids such as acetic or
lactic acids. These acids cause a transient drop in surface pH and affect the micro-
organisms thereon. Spray treatrnent with a solution containing 2% lactic acid and 20%
sodium chloride produced a shelf-life of 28h in wrapped and 36b in unwrapped carcasses
stored in ambient temperaturem

fitment with chlorinL and hot Wm; although there is a real decline in numbers
of bacteria afler chlorination it does not have a significant effect on shelf-life. Hot water
treatment appears to be more successful provided the surface reaches a high enough
(60°C) temperature for a sufficient period.'76 There will be a slight discoloration of the
meat, which may be regained during the holding period.

Sodium chloride trgrtment: sodium chloride lowers the water activity and inhibits
microbial grth and also it is used to flavor and preserve a variety of meats.

SorbaLte treatment; the primary inhibitory action of sorbate is against yeasts and
molds. Sorbate inhibits many bacteria including Salmonella, Escherichia,

77 Sorbate treatments are used for controlling

Staphylococcus, and Clostridium.1
microbial growth in beef carcasses held at a temperature of 15°C. Chemical dips
containing potassium sorbate substantially reduce the counts of bacteria on unchilled beef

and on beef stored at 30°C and 20°C and extend the shelf-life up to 32h at 30°C and 68h

at 2°C.”5

32

Enzme inhibitors; administration of epinephrine controls the post-mortem

7 It was

autolysis of meat by inhibiting catheptic activity at ambient temperature.”
suggested that this process could be useful for long-term storage and for more efficient
meat distribution at ambient temperature. Anti-autolytic activity of urea was
demonstrated in meat kept at ambient temperature.156

Co_olir_rg; refiigeration is the most commonly used method for carcasses
immediately afler slaughter, during transport and storage and for packed meat. At
refrigeration temperature (4°C) the self-life of properly packaged retail meat is 72h, after
which some discoloration can be expected to appear, while the shelf-life of ground meat
is only one day.22 Carcass chilling rooms are normally operated in the temperature range
of -2°C to -4°C (28-25F) with relative humidity of 88-92%.22 The faster the air
movement, the more rapid is the cooling. Accelerated cooling is achieved by using
extremely low temperatures (-15°C to -35°C) or by spraying with or immersion in
cryogenic liquids. Liquid nitrogen is the ideal cryogenic agent.”

firming; is an effective method of storing cuts of large carcasses, whole small
carcasses, and retail cuts in a fresh state for extended periods. Marketing of frozen meat
is unsuccessful due to the appearance of the product. Frozen meat will not give the
appearance of fresh meat due to the ice crystal formation on the meat surface. The
recommended storage temperature for frozen meat is -18°C (0F). Freezing must be rapid.
Rapid freezing produces smaller ice crystals on the surface of meat and damage to the

meat tissues is very much less.22 Thawing the meat is also important which is the reverse

of freezing. While the meat is thawing the watery drip occurs which contains proteins,

33

vitamins, and minerals. The less damage that occurs to the tissues during freezing and
frozen storage, the less drip loss during thawing.22

L_actic fermentation; meat preservation is attributed to the combined effect of
several substances (lactic acid, volatile acids such as acetic acid, antibiotics and
bacteriocins) produced by lactic acid bacteria (LAB) though lactic acid plays a vital
role.137 It is a simple, low-tech and inexpensive method that can be practiced at ambient
temperatures.

Irradiation; has good potential in the elimination of pathogenic and spoilage
microorganisms from carcasses, cuts and minced meat and in the preservation of meat. It
has emerged as a cost-effective method and finds a place in developing countries. WHO
clarified in 1980 the medical acceptability of irradiated foods and said “no health hazard
results from consuming any food irradiated up to a dose of one megarad (1Mrad)."7
Irradiation reduces microbial levels and pathogenic microorganisms and eliminates
parasites like Trichinella spiralis.47 The USA permitted irradiation in pork and poultry.22
The UK has permitted irradiation only in poultry. Several other countries have also
permitted irradiation in meat, fish, and poultry.

Packaging; packaging protects the meat fi'om moisture loss, contamination by
microorganisms, changes in color and physical damage. Packaging fresh meat varies
from simple wrapping to advanced systems like vacuum packaging (VP) and modified
atmosphere packaging (MAP).22 Carcasses and large size meat cuts are wrapped in
simple polyethylene films to protect them from contamination during handling. Fresh
retail meat cuts are packed in pouches (polyethylene or polyvinyl chloride). These

pouches allow oxygen transmission which maintains the bright red color of meat and

34

reduces the moisture loss.”’56 Shelf—life of these meat cuts varies between 3 and 5 days
at 4C. Vacuum packaging provides at least three weeks shelf-life for the product under
adequate refiigeration but the product looks dark. When the package is opened it regains
its bright red color because of exposure to air (oxygen). In modified atmosphere
packaging (MAP) three principal gases are used; 10% carbon dioxide (inhibits bacterial
and mold growth), 85% nitrogen (inhibits the oxidation of fats and mold growth) and 5%
oxygen (prevents anaerobic spoilage)?”6 The expected shelf-life of fresh meat in MAP

is ten days.22

FEDERAL AND STATE STANDARDS FOR

PATHOGEN REDUCTION AND CONTROL
Ensuring the safety of food is an enormously complex task. Hazards can arise at
every stage of the food production process: from the farm to the processing facility, in
transportation and storage, in food service and retail establishments, and in the homes of

7 During each of these steps along the way, measures must be taken to

consruners.9
prevent or minimize hazards. On July 25, 1996, the US. Department of Agriculture
(USDA), Food Safety Inspection Service (F SIS), adopted Pathogen Reduction, Hazard
Analysis and Critical Control Point Systems (HACCP) to improve food safety for meat
and poultry.193 HACCP system requires a detailed analysis of the whole process from the
farm through to slaughter.

The hazards are scored according to the magnitude of risk to the consumer and a

judgement is then made as to the necessary control points needed to eliminate or

minimize the hazards. Once the critical control points (CCP) are in place, a monitoring

35

system to ensure that the CCP are working should be maintained. This kind of system
requires the cooperation and motivation of everyone involved in the chain and
independent auditing to ensure that problems are not overlooked.

Although the HACCP system is intended as a means of eliminating or minimizing
microbial hazards, other hazards such as residues, contaminants and parasitic infestations
are all open to the same approach. The purpose of the pathogen reduction and HACCP
regulation is to improve food safety. However, the regulation will also improve
industry’s ability to compete in international markets. The HACCP regulation is
consistent with the General Agreement on Tariffs and Trade (GATT), which requires
countries to ensure that their sanitary or phytosanitary measures are based on science and
risk assessment principles. The combination of performance standards and HACCP
enables the United States to objectively demonstrate that the level of protection the US.
system provides is science-based, addresses likely hazards, and is equivalent to foreign
requirements.“97

The new rules apply to both slaughter and processing plants that handle meat and
poultry, but the requirements could have an impact on dairy farmers. The new rule
includes the following four major topics:

W- Every plant must adopt and carry out its own HACCP plan that systematically
addresses all significant hazards associated with its products.192
Men reduction performance sfldirris for Salmonella- All slaughter plants and plants

producing ground products must ensure that their Salmonella contamination rate is below

the current national baseline prevalence. This regulatory performance standard for a

36

pathogen on raw meat and poultry will ensure progress in reducing pathogenic
bacteria.192
Mandatory Escherichia coli testing in slaughter pl_a_n_t§- Every slaughter plant must
regularly test carcasses for E. coli to verify the effectiveness of the plant’s procedures for
preventing and reducing fecal contamination. E. coli is the best microbial indicator of

fecal contamination currently avaliable.192

Sanitation standard operating procedures (SSOP)- As the foundation for HACCP, every
plant must adopt and carry out a written plan for meeting its sanitation responsibilities.
Effective sanitation in slaughter and processing plants is essential to prevent adulteration
of meat and poultry products.192
HACCP is a system that identifies potential food safety risks, prevents or corrects
them, records actions, and verifies that it worked. HACCP is a systematic approach to
controlling potential hazards in post-harvest food production. HACCP tries to identify
problems before they occur and then establishes control measures that are critical for
maximizing food safety at each stage in food processing and production.” The principles
of HACCP implementation for food production processes have been identified by the
National Advisory Committee on Microbiological Criteria for Foods (NACMCF) of the
Food Safety and Inspection Service (FSIS) of the US. Department of Agriculture.183
These principles31’36'178’183 are:
Conduct an analysis of potential hazards- Plants determine the food safety hazards
reasonably likely to occur and identify the preventive measures the plant can apply to

control these hazards. A food safety hazard is any biological, chemical, or physical

property that may cause a food to be unsafe for human consumption.

37

Determine critical control points for the targeted hazard and hazards- A critical
control point (CCP) is a point, step, or procedure in a food process at which control can
be applied and, as a result, a food safety hazard can be prevented, eliminated, or reduced
to an acceptable level.

Establish critical limits for each CCP- Each CCP will have preventive measures that
must be properly controlled to assure prevention, reduction to a tolerable level, or
elimination of hazards. Each preventive measure has critical limits associated with it that
serve as boundaries of safety for each critical control point.

Establish critical control point monitoring requirements- Monitoring activities are
necessary to ensure that the process is under control at each critical control point. F SIS is
requiring that each monitoring procedure and its frequency be listed in the HACCP plan.
Establish corrective actions for each critical operation when the control data
indicate that the operation is out of control- These actions are to be taken when
monitoring indicates a deviation from an established critical limit. The final rule requires
a plant’s HACCP plan to identify the corrective actions to be taken if a critical limit is not
met. Corrective actions are intended to ensure that no product injurious to health or
otherwise adulterated as a result of the deviation enters commerce.

Establish record keeping procedures- The HACCP regulation requires that all plants
maintain certain documents, including its hazard analysis and written HACCP plan, and
records documenting the monitoring of critical control points critical limits, verification
activities, and the handling of processing deviations.

Establish a system of verification to document that the HACCP program is being

followed- Validation ensures that the critical control points and associated critical limits

38

are adequate and sufficient to control likely hazards. Plants will be required to validate
their own HACCP plans. FSIS will not approve HACCP plans in advance but will
review them for conformance with the final rule.31 Verification ensures the HACCP plan
is acceptably. Verification procedures may include such activities as review of HACCP
plans, CCP records, critical limits, and microbial sampling and analysis. FSIS is
requiring verification tasks to be performed by plant personnel and varied by FSIS
inspectors. Both FSIS and industry will undertake microbial testing as one of several
verification activities.

The Pathogen Reduction and HACCP systems regulation requires testing for
Salmonella and E. coli. Fecal contamination from the gastrointestinal tract, hide, and
feathers are primary means for contamination of livestock and poultry carcasses with
enteric zoonotic pathogens.”97 Major sources of carcass contamination during slaughter
include rupture of the intestine or crop during evisceration, contact of the hide or feathers
with muscle of the same or adjacent carcasses, and airborne spread of materials during
hide pulling or feather removal (Figure 3)."9’155 ’205

The sample collection procedures required for Salmonella and Escherichia coli
testing are the same. Cattle and swine carcasses must be sampled at the end of the
slaughter process in the cooler. A sampling sponge is used to swab a 10cm by 10cm area
at three sites on beef carcasses (flank, brisket, and rump) and three sites on pork carcasses
(belly, jowl, and ham). Poultry carcasses must be sampled after the chill tank at the end

of the drip line or the last readily accessible point prior to packaging or cut up. Carcass

sampling for poultry carcasses is a nondestructive whole bird rinse.

39

Microbiological Contamination During Slaughter ‘3

Cattle receiving
and handling
V
Stunning
V
Decontamination

V
Bleeding

V
Head and shank removal
V
Skinning 0 CCP l
V
Trim rail
V

Post-skinning wash ‘
V CCP 2

 

Bacterial rinse

 

V
0 Viscera handling Evisceration 0 CCP 3
V
Splitting e
V
Final wash CCP 4
‘7
Chill CCP 5
V

Fabrication O

Primals

I___ Vacuum

Refrigerate Freeze Grind 0
Package

Trimmings

 

 

 

 

. Package/label CCP 7 Refrigerate

Freeze

Further processing I

Storage

 

 

 

 

Distribution, retailing, end use

 

CCP6

Figure 3: Generic HACCP for beef slaughter, fabrication and packaging. Potential site of minor
contamination( O ); potential site of major contamination (0) (NAC, 1993).

0= Pens ; o = Holding

40

 

CHAPTER 2

ESCHERICHIA COLI
Escherichia coli 0157:H7 as an important food-home pathogen in human beings

Esherichia coli 0157:H7 was first isolated in 1975 from a California woman with
grossly bloody diarrhea.160 The 0157:H7 designates a serotype of the E. coli bacteria
that was first identified as a cause of human illness in 1982 when 47 persons in Michigan
and Oregon developed bloody diarrhea after eating hamburgers which were sold by a
national fast-food chain.37’87’16° Since 1982, more than 100 outbreaks of EHEC 0157
have been documented, and of those outbreaks, 52% have been linked to food derived
from cattle.70

Dairy cattle, especially young animals, have been implicated as a principal
reservoir of E. coli 0157:H7, with undercooked ground beef and raw milk being the
major vehicles of food-home outbreaks.88'2m’203 The public was generally unaware of E.
coli 0157’s existence until a decade later, when more than 500 laboratory-confirmed
infections occurred in four western States, also as a result of hamburger consumption.1
Since then, several outbreaks of E. coli 0157:H7 infection in the North America, Canada,
United Kingdom, Japan have been reported. Because they cause bloody diarrhea and
produce potent toxins, serotype 0157:H7, 026:H11 and several others are classified as
Enterohemorrhagic E. coli (EHEC). Escherichia coli 0157:H7 is the EHEC serotype
most often associated with human disease episodes and the EHEC most studied in food-

producing animals.160

41

A single fast-food chain with restaurants in California, Idaho, Nevada, and
Washington in late 1992 and early 1993 was associated with the largest outbreak
involving ground beef. This western state outbreak resulted in more than 500 confirmed
cases and four deaths.45

Between 1982 and 1995, Esherichia coli 0157:H7 was implicated in 75 outbreaks

5

involving 2,562 individuals.‘1 The traceback studies support epidemiological data that

link Esherichia coli 0157:H7 with a bovine reservoir.76’95’203

ISOLATION AND PREVALENCE OF E. coli AT SLAUGHTER
Prevalence on farm
It is well known that Shiga Toxin-E. coli (Stx-E. coli) is commonly isolated from
feces of clinically normal as well as diarrheatic cattle.78"°‘ There is no direct evidence

that E. coli 0157:H7 is an animal pathogen?”

There is great variation in E. coli
0157:H7 prevalence in dairy cattle on farms. These variations are due to location of
farms, herd management of the farms, age of the animals, seasonal effects, and isolation
techniques. The prevalence of 0157:H7 E. coli in cattle appears to be low, although the
prevalence of other serotypes of Stx E. coli that are potential human pathogens are much
greater. The prevalence of E. coli 0157:H7 in the United States ranges from less than
1% to 61%.58,125.217

Verotoxin-producing E. coli in cattle was frequently detected on farm, but those
isolates were comprised mostly of serotypes that have not been associated with human

54,88,133

disease. In Australian dairy herds, verotoxin producing Escherichia coli were

42

isolated from 16.7% of fecal samples from cattle.“ Of those isolates, only 11.2% were
serotype 0157:H7 (prevalence of 0157:H7 was 1.8%)“

In a survey of dairy herds in 14 different states from February to May 1993, E.
coli 0157:H7 was isolated from 6 of 399 calves (1.5 %) that were between 24h old and
weaning fi'om 13 of 263 calves (4.9%) that were between weaning and 4 months.203 In
another survey of previously positive herds in the 11 states from June to August 1993, E.
coli 0157:H7 was isolated fiom 5 of 171 calves (2.9%) that were between 24h old and
weaning and from 7 of 132 calves (5.3%), that were between weaning and 4
months.2°3’2”

Esherichia coli 0157:H7 was isolated from 10 of 3,570 dairy cattle in the state of
Washington (0.3%).90 Another survey in 1991 and 1992, examined preweaned dairy
calves in 28 states throughout the United States for E. coli 0157:H7, and found that 0.4%
(25 of 6,894) of the calves and 1.8% (19 of 1,068) of the herds tested positive.”217 In
another study of nine farms that may have been sources of meat involved in an outbreak
of hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic
purpura, five of the farms (55.5%) and 7 of 315 heifers (2.2%) tested positive for E. coli
01573173023"3

Although it is difficult to directly compare the results of previous studies because
of different sampling and testing procedures employed, as well as missing information on
the shedding of E. coli 0157:H7, data from published reports suggests that the prevalence
of E. coli 0157:H7 in cattle ranges from 0.3 to 0.7% and the prevalence in cattle herds

ranges from 1.8 to 16%.90’96 In a study of 70 Wisconsin dairy farms, 5 of the farms (herd

prevalence, 7.1 :I: 4.5%) and 10 of 560 weaned calves (animal prevalence, 1.8%) tested

43

positive for E. coli 0157:H7.63 In this study, they sampled the weaned calves because in
previous studies workers found that weaned calves are more likely to shed E. coli
0157:H7 than cattle in other age groups.76’9°’2 ‘2

Hancock et al.90 found a prevalence of less than 1% (0.28%) in over 3,500 fecal
samples obtained fiom dairy cattle of various ages and a prevalence of 8.3% (5/60) of
herds tested. E. coli 0157:H7 was isolated from 2 of 1,273 lactating and 1 of 477 non-
lactating dairy cows, and within the positive herds, E. coli 0157:H7 was found in 1.7%
(2/ 120) of lactating cows and 2.6% (1/39) of non-lactating cows.

In other studies the prevalence of E. coli 0157:H7 in beef and dairy cattle on

farms ranged from 0% to 68%, and the herd prevalence ranged from 1.8% to

100%.48,l68,210,212

Prevalence at slaughter

Prevalence of E. coli in feces, hides and carcasses at and during processing may
have been underestimated in the in the past, because of a lack of highly sensitive and
specific methods for isolation of EHEC 0157 from those elements.70

Studies”58 have been completed to determine the prevalence of EHEC 0157 in
cattle feces and on carcasses during slaughter processes. From cattle presented for
slaughter in the United Kingdom, 0.83% of 6,495 bovine fecal samples in South
Yorkshire found 4% of rectal swabs positive for EHEC 0157. A study done at meat
processing plants in Midwestern United States looked at the frequency of
enterohemorrhagic E. coli 0157:H7 in feces and on hides within groups of fed cattle

from single lots that were going to slaughter, as well as investigating carcass

44

contamination."9 From 29 lots that were sampled, 38% had positive hide samples, and
72% had at least one E. coli 0157 :H7 positive fecal sample. As a result the prevalence in
feces and on hides was 28% (91 of 327) and 11% (38 of 355). There was a significant
correlation between the prevalence of E. coli on hides and feces, and the frequency of
carcass contamination indicating that control of E. coli 0157 in live animal would have
some potential for reducing rates of carcass contamination.49 The carcasses were
sampled at three points during slaughter: pre-evisceration, post-evisceration, and post-
processing after the carcasses had been placed in the cooler. 29,58’158 Of 30 sampled lots,
E. coli 0157 was isolated from 87% pro-evisceration, from 57% post-evisceration (before
antimicrobial intervention), and from 17% of post-processing samples.58 The prevalence
of E. coli 0157:H7 at the three sampling points were as follows; 43% (148 of 341), 18%
(59 of 332), and 2% (6 of 330). The decrease in carcass prevalence suggests that sanitary
procedures of slaughter were effective in reducing bacterial load.58

More recent studies have isolated E. coli 0157 from 3.6% and 13.4% of beef
cattle, and 3.9% and 16.1% of dairy cattle at slaughter.” 48"”

Seasonal variation in E. coli 0157:H7 excretion by cattle was demonstrated in
several epidemiological studies. Shedding of E. coli 0157:H7 at slaughter was
significantly higher in summer than any of the other seasons. The prevalence of E. coli

0157:H7 in fecal samples at slaughter was 19.7% in the summer and 0.7% in the winter

(Figure 4).48

45

Prevalence of E.coli 0157:H7 in cattle by season

25'

 

Dec-Feb March-May June-Aug Sept-Nov

Figure 4: Prevalence of Escherichia coli 0157:H7 in fecal samples from
yearling cattle and cull cows at slaughter in Alberta by season."8

IDENTIFICATION AND IMPROVED METHODS OF
ISOLATION IN THE INDUSTRY OF MONITORING
Esherichia coli, a member of the family Enterobacteriaceae is considered to be a

part of the normal microflora of the intestinal tract of humans and most warm-blooded
animals. They are Gram-negative, straight rods, (1.1 - 1.5pm x 2.0 - 6.0um) that are
oxidase negatives Organisms of this species are generally lactose ferrnenters, but
sometimes lactose fermentation is delayed.131

The enterohemorrhagic E. coli prototype, E. coli 0157:H7, like all E. coli, is

typical of the species (the 0 refers to the somatic antigen, and H to the flagellar antigen),

46

with the exception of sorbitol fermentation and B-glucuronidase activity.52"°9'2°' About
93% of E. coli isolates of human origin ferment sorbitol within 24h; however, E. coli
0157:H7 does not.201 Additionally, 93% of E. coli strains posses the enzyme [3-
glucuronidase that is the basis for a rapid fluorogenic assay for E. coli.67 This assay uses
4-methylumbelliferyl B-D-glucuronide (MUG) as an indicator which is hydrolyzed to a
fluorogenic product by the enzyme B—glucuronidase. B-glucuronidase activity is not
phenotypically expressed by these organisms.“109

E. coli grow rapidly between 30-42°C, with generation times ranging from 0.49h
at 37°C to 0.64h at 42°C.52 The organism grows poorly at 44-45°C and does not grow
within 48h at 10 or 45.5°C.52"54 The organism can survive well in ground beef during
frozen storage at -20°C. There is no major change in populations of E. coli 0157:H7, in
ground beef frozen at —80°C and held at -20°C for up to 9 months.52 The organism is not
unusually heat resistant (e.g. cooking ground beef to well-done to kill typical strains of
Salmonella will also kill E. coli 0157:H7).209

Escherichia coli usually remains confined within the intestinal lumen of mammals
as a harmless saprophyte, but in the debilitated or immunosuppressed host or in the
immunologically normal host with disruption of critical anatomical barriers, normal
intestinal strains of E. coli are major causes of opportunistic infections. These strains
cause diarrhea and other symptoms in humans and warm-blooded animals by producing
different toxins. Researchers1 ‘2" ”"60 have demonstrated that toxins produced by strains
of E. coli 0157:H7 are cytotoxic for vero (African green monkey kidney) cells. Infection
with verotoxigenic strains of E. coli in people can cause diarrhea, hemorrhagic colitis,

hemolytic uremic syndrome, renal failure and death. Verotoxins are heat-labile proteins

47

that produce an irreversible cytopathic effect in vero cells.”2’”9 Verotoxins have two
subunits; an A (active) subunit and several B (binding) subunits. The B-subunit of the
verotoxins binds to a receptor on the surface of a cell. The A-subunit is then internalized
in the cell and cleaves to an active fragment, and then inhibits cellular protein synthesis.
Because of their close homology to Shiga-toxin, VT] and VT2 are often referred to as
Stx-I and Stx-II. Escherichia coli strains that produce verotoxins or Shiga-toxins have
been referred to as verotoxigenic E. coli (VTEC), Stx-producing E. coli, and

enterohemorrhagic E. coli (EHEC) (Table 7, Figure 5).

48

Table 7: Classifications and characteristics of Escherichia coli

of the gastrointestinal tract in people.

37,109,148

 

CLASSIFICATIONS

CHARACTERISTICS

 

Enteropathogenic E. coli (EPEC)

Appears to destroy microvilli without further
invasion; bundle-forming pili mediating
localized adherence; type III secretion system
mediating attaching and effacing lesions. Only
a minority of these organisms produce
verotoxins. Human are the main reservoir.

 

Enteroinvasive E. coli (EIEC)

Invades and proliferates within epithelial cells
and causes cell death Human are the main
reservoir.

 

Enterotoxigenic E. coli (ETEC)

Penetrates the mucous layer of the proximal
small intestine. The organism adheres to
mucosal cells and produces heat-stable or heat-
labile enterotoxins. This type frequently causes
traveler’s diarrhea. Human are the main
reservoir.

 

Enterohemorrhagic E. coli (EHEC):
E. coli 0157:H7 and E. coli 0126:Hll

This is the most important group of E. coli in
terms of food-bome disease. Shiga-toxins; type
III secretion system mediating attaching and
effacing lesions. Three principal syndromes
have been linked to E. coli 0157:H7;
hemorrhagic colitis, hemolytic uremic
syndrome (HUS), and thrombotic
thrombocytopenic purpura (TTP). Its main
animal reservoir is the rumens and intestines of
cattle and sheep.

 

Enteroaggregative E. coli (EAEC)

The EaggEC strains have the ability to attach to
tissue culture cells in an aggregative manner.
These strains are associated with persistent
diarrhea in young children. They resemble
ETEC strains in that the bacteria adhere to the

intestinal mucosa and cause non-bloody
diarrhea without invading or causing
inflammation. They produce a heat-labile-

plasmid-encoded toxin (EnteroAggregative ST
or EAST).

 

Diffusely adherent E. coli (DAEC)

F imbrial and afrrnbrial adhesins; elongation of
microvilli. Diarrhea in older children.

 

Uropathogenic E. coli (UPEC)

Virulence factors P and other
hemolysin. Urinary tract infections.

fimbriae;

 

E. coli that cause neonatal meningitis (NMEC)

Virulence factors capsules; S-firnbriae; cellular
invasion. Sepsis and meningitis in neonates and
infants. Transmission person-to-person.

 

 

Verotoxigenic E. coli (VTEC)

 

Strains of E. coli produce heat-labile toxins that
are cytotoxic for vero cells in an invitro assay.

 

49

 

EPEC Initial adherence by EHEC

bundle-forming pilus

   
   
   

 

 

 

 

 

 

Intimate
attachment of
bacteria

fl” Actin 33“

Intimate \ condensation
attachment Actin and
Of bacteria condensation microvillous

and microvillous efi‘acement

effacement

Delivery of shiga toxin
ETEC EAEC

Adherence via coloization
factor antigens

a .

 
   

mucus
biofilm

 

I

delivery of cytotoxin

Delivery of LT or ST
entererotoxins

 

 

 

 

 

 

EIEC DAEC

elongated

membrane ¢ nricrovilli
ruffling

invasion ©
normal cytoplasm

phagosomal w
rupture

 

 

Lateral

intracellular spread to

movement adjacent
cell

 

 

 

 

 

Figure 5: The molecular pathogenesis of Escherichia coli infections.21

50

For detection of E. coli 0157:H7 , fecal and rumen content suspensions are placed
on the primer media, MacConkey (MAC) and sorbitol MacConkey agar (SMAC).
Lactose positive organisms (coliforms) are identified on MacConkey agar, as colonies
that appear in 24h; are flat, pink to red and 2-4mm in size. Colonies have a characteristic
pink halo of precipitated bile salts surrounding them. These colonies are transferred to
sorbitol MacConkey agar and incubated at 37°C overnight. Many screening methods
used for the isolation of EHEC from food products and stool specimens utilize sorbitol
MacConkey agar (SMAC) as the primary isolation medium.”’23’64 Sorbitol MacConkey
medium is designed to detect only this serotype; E. coli 0157:H7. Since E. coli 0157:H7
does not ferment sorbitol within 24h and does not posses B—glucuronidase activity the
pale white colonies will appear on the medium next day. Colonies that are sorbitol-
negative are selected and confirmed as E. coli 0157:H7 by morphology, biochemical,
serological and vero cell cytotoxicity assays. Some of the biochemical tests used include;
Triple Sugar Iron Agar (TSI), motility test, Indol test, Ornithine test, Simmons’ citrate
agar, Oxidase test, and Urease test to differentiate E. coli from Salmonella, Klebsiella,
Proteeae, Shigella, and Serratia (Table 8,9). Escherichia coli can be serogrouped by
slide, tube, or latex agglutination to detect the 0157 antigen. Serotyping can be
completed by looking for immobilization in H7 antisera-containing motility media;

however, some strains may be nonmotile (H‘).

51

Table 8: Identification of coliforms and related organisms.62

Strong Weak
Lactose(+) Lactose (+) Lactose (-)
Klebsiella' Enterobacter' Citrobacter Proteus' Serratia Pseudomonas

red

oges-proskauer

rease
ysine +/_

d

+/- = most strains positive;

-/+ = most strains negative;

(I = different biochemical types;

* = some species of the genus will show different reactions for some of the tests.

 

Table 9: Growth characteristics of Shigella on selective media.55

 

Organism Medium Colony Appearance

 

MacConkey Lactose fermentor; flat, pink colonies
surrounded by darker pink region
(indicates sorbitol fermentors, non-
sorbitol ferrnentors form colorless

 

 

 

 

 

 

E. coli colonies)

Hektoen Yellow

XLD Yellow

MacConkey Colorless (lactose non-fermentors)
Salmonella

Hektoen Green

XLD Red with black center

MacConkey Colorless (lactose non-fermentor)
Shigella Hektoen Green

XLD Colorless

 

XLD: Xylose-lysine-desoxycholate agar

52

Although the inclusion of pre-enrichment incubations and immunomagnetic
separation (IMS) and additional selective subculturing or secondary enrichment
incubations have been reported to increase the detection rate of E. coli 0157:H7 from
foods and fecal specimens, these methods are dependent on isolating individual colonies
from selective and/or indicator media and then characterizing them using immunological
and biochemical/fermentation reactions.7'28’39’72’110 In the use of immunomagnetic (IMS)
method, 0157-specific antibodies attached to superparamagnetic polystyrene beads (anti-
E. coli 0157 Dynabeads; Dynal, Inc., Lake Success, N.Y.) are added to an enriched
sample and subsequently subjected to magnetic separation. E. coli 0157 organisms, if
present in the enrichment culture, are removed with the magnetic beads.”’109 The
sediment containing the bacterium-coated beads is streaked to selective plating medium
such as SMAC or CT-SMAC (potassium tellurite and cefixime added to SMAC).109 IMS
increased the detection rate of 0157 by 65% in a sampling of specimens associated with
several outbreaks and was superior to molecular methods such as polymerase chain
reaction (PCR), cytotoxicity assays, and direct plating.109 IMS has the advantage of
being simpler and easier to perform than similarly sensitive methods, such as PCR
assays, and leads to the isolation of the targeted organism.”109 However, it will select
only for E. coli 0157 and not for other serogroups of STEC (Stx-producing E. coli),
unless the beads are coated with antisera for the specific serogroup to be isolated.'09

Antibody-based methods for the immunologic detection of STEC include colony
immunoblot assays and antibody capture or toxin receptor-mediated EIAs.
Immunological assays are used to determine if the 0157 somatic and H7 flagellar

antigens are present, while the biochemical/fennentation reactions determine in classical

53

taxonomic fashion the genus and species of the isolate. Combined with the initial
replication steps in the isolation process, the current E. coli 0157:H7 identification
process takes 5 or more days to complete.105 "46"70 This adds considerably to the cost
required to determine whether a sample contains E. coli 0157:H7 and is a limiting factor
in doing a large number of E. coli 0157:H7 tests. A commercial EIA kit (Premier
EHEC; Meridian Diagnostics, Inc., Cincinnati, Ohio) for detecting Stx in suspensions of
stool or fecal enrichment cultures has recently become avaliable.55 "09'190 Another
commercially available product is a latex agglutination kit (V TEC-RPLA; Denka Seiken
Co., Tokyo, Japan), which detects both Stx-I and Stx-H from culture supernatants.55"°9
These commercial methods are easy to perform and do not require specialized equipment
and laboratory skills, unlike cell culture and DNA-based methods.5°’55’6"’"’9’1 '3’ ‘97

Nucleic acid-based assays for detection of E. coli 0157; colony hybridization
assays with DNA probes for Stx genes, the 60-Mda plasmid present in E. coli 0157, and
the eae gene have been used to detect and characterize STEC.105 "09'1“"70 Rapid methods
for identifying E. coli 0157:H7 in foods or fecal specimens have been directed at
immunological or genetic targets. Antigenic targets have included the E. coli somatic
(0157) or flagellar (H7) antigens, two low-molecular-weight antigens, and the virulence-
associated Stx types I and 11.50'64’1'3’197 However, these assays are occasionally unable to
distinguish certain other E. coli strains fi'om E. coli 0157:H7 strains and/or toxigenic
from nontoxigenic E. coli 0157:H7 strains.'°5""6’170

Most E. coli 0157:H7 carry a 60-megadalton plasmid. This plasmid is required

for expression of fimbrial adhesion and adherence to Henle 407 intestinal cells.“ Toth et

al.189 developed a direct ELISA for detection of E. coli 0157:H7 and other Stx-producing

54

E. coli.189 The assay is based on the presence of two proteins of 82 and 92 kDa in E. coli
which are associated with the 60-MDa plasmid commonly carried by VTEC. Hence, this
ELISA is a specific test for detecting 60-MDa plasmid-associated proteins. The method
works well in pure cultures.128

A monoclonal antibody (MAb) 4E8C12 specific for enterohemorrhagic E. coli of
serotypes 0157:H7 and 026:H11 was produced and characterized by Padhye and
Doyle.144 Using this MAb, a rapid and sensitive procedure was developed for the
detection of E. coli 0157:H7 from food in less than 20h.144 The procedure involves
enrichment of a food sample in a selective enrichment broth for 16-18h at 37°C with
agitation. Enrichment culture is applied to a sandwich-enzyme-linked immunosorbent
assay (ELISA) procedure that has a polyclonal antibody specific for E. coli 0157 antigen
as the capture antibody and the MAb 4E8C12 as the detection antibody.‘“"89 In addition
to being highly specific, sensitive, and rapid, this procedure is easy to perform and is
amenable to use by laboratories performing routine microbiological testing. Also no
cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica,
Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae,
Campylobacter jejun i, Serratia marcescens, Citrobacter spp., Enterobacter cloacae,
Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than
serotype 0157:H7 (including strains of serotype 0157 but not H7). 109"“

A polymerase chain reaction (PCR) procedure for rapid and specific detection of
verotoxin genes in E. coli was developed by Pollard et al.150 PCR is an in vitro method
for amplifying specific nucleic acid sequences by repeating cycles of DNA synthesis over

a period of hours. Polymerase chain reaction-based detection procedures have been used

55

to identify E. coli 0157:H7 and have targeted the Stx-I and Stx-H genes, the
enterohemorrhagic E. coli (EHEC) uidA gene, and a portion of a 60-megadalton plasmid.
With this procedure, DNA is amplified to increase the level of target DNA when VTEC
are present in very low numbers. A variety of PCR amplification methods have been

developed for detecting and characterizing STEC. Recently, Ganon et al.75

reported the
development of two multiplex PCR assays; one provides identification of E. coli
0157:H7, other STEC strains, and potential enteropathogenic E. coli (EPEC) isolates; the
other provides identification of only E. coli 0157:H7 and other STEC strains. In the
former assay, these organisms are detected by targeting Stx-I, Stx-H, a region of the eae
gene that is conserved between STEC and EPEC, and H7 antigen-specific sequences in
the flagellin gene, fll'C.5°’6"'75"°9’1‘3’197 The polymerase chain reaction technique is both
sensitive and specific, hence it may be useful for rapidly screening clinical specimens for
VTEC. But PCR can suffer fi'om common problems such as contamination, the presence
of inhibitors of the polymerase enzyme, and undesirable reaction conditions that
influence laboratory assay detection limits, assay sensitivity, and assay specificity, which
can lead to false-negative or false-positive test results.188

Although PCR can amplify DNA molecules thousands-fold, the specifically
amplified product must be detected in order to prove its presence, and a variety of
methods have been developed for this purpose. The most commonly used research
technique, gel electrophoresis, does not show the specificity of the PCR and lacks

sensitivity. Southern blots or dot blot hybridizations with probes demonstrate the

specificity of the PCR, but they require multi-step processing and add considerable time

56

and expense to the detection process. Neither of these PCR detection processes is

conducive to rapid, high-throughput, automated PCR detection schemes.loo

METHODS OF CONTROL OF ORGANISM

Organism shedding and control on farm

Dairy cattle are the main reservoir of E. coli 0157:H7. Also a wide variety of
animals including sheep, and deer may carry the organism. In the farm environment,
weaned calves have the highest rate of E. coli 0157 shedding. Calves are exposed to E.
coli during the first week of life and the infection rate after this exposure is generally
steady, until weaning time around 8 weeks of age.32’63'76"°9'2” The pre-weaning period
may play an important role in determining the farm prevalence of E. coli 0157:H7. The
prevalence of E. coli 0157 in calves less than 8 weeks old was 1.4% and in calves 8
weeks or older was 4.8%.76 Calves were three times more likely to shed E. coli 0157
afier weaning than before weaning.76'212

Farm management in the pre-weaning period is very important in controlling the
overall rate of E. coli 0157:H7 infection in herd. Farms in which calves are kept in
groups from birth to weaning or grouped before weaning have higher rates of E. coli
0157:H7 shedding.”76 Crowding may create stress in calves that are already susceptible
to infections. Stress and nutritional deprivation increases the shedding of E. coli
0157:H7.76 Close contact with other calves and their by-products facilitates infection by

the fecal-oral route. It is natural for calves to lick and suckle each other in the pre-

weaning period.76

57

Another control point that influences E. coli 0157:H7 shedding involves the
feeding practices used on the farm. Sharing nipples and bottles between calves without
rinsing or cleaning may contribute to spread of infection among calves. Also the use of
open pails instead of nipple bottles for feeding calves is associated with increased rates of
E. coli 0157:H7 shedding?”

Composition of the feed can affect shedding of E. coli 0157.91 Some studies
report conflicting results such as the inclusion of cottonseed which was reported by
Garber et al.76 to have a negative relationship with E. coli 0157 shedding and yet Herriot
et al.99 could not find any association between cottonseed feeding and E. coli shedding by
calves (even though cottonseed is toxic for baby calves) in their 1977 study. However,
they did find positive relationships between feeding corn silage, grain screenings and
ionophores, and E. coli 0157:H7 shedding. Corn silage may provide a moist
environment suitable for bacterial growth of E. coli 0157. Also ionophores in the cow
ration create an environment that favors the development of Gram-negative intestinal
flora. Ionophores alter the ratio of proprionic acid and acetic acid in the rumen but their
effects again are controversial.”99

Hancock et al.l 1.123 found that previous irrigation of grazing land with fecal slurry
is a positive risk factor for carriage of E. coli 0157 in a dairy herd. If fecal slurry is used
on grazing land some months should elapse between spreading of slurry and grazing of
animals.

In a survey""3 conducted to determine the prevalence and to identify the sources of

E. coli 0157:H7 isolates on Wisconsin dairy farms, only animal drinking water was

identified as a non-fecal source of E. coli 0157:H7 within the farm environment.

58

Because water can serve as a reservoir for E. coli, farm managers should take extra
precaution to protect water sources from fecal contamination.

Vaccination is another control option on the farm against E. coli 0157:H7 as in
the case of other Gram-negative bacteria.91 The purpose of vaccination would be to
reduce the susceptibility of cattle to colonization with E. coli 0157:H7 or to decrease the

”"09 This would presumably require targeting an

duration of such colonization.
adherence or other surface antigen with a mucosal immune response.109 Currently, it is
not clear if this is theoretically feasible, and a number of practical problems would need
to be addressed even if effective immunization could be demonstrated under controlled
conditions. Fimbrial vaccines are used especially by parenteral administration to
pregnant cows to protect neonatal calves by increasing antibody in colostrum and
milk.9"1°9"34

Another control point involves “niche engineering”. Modifying the environment
to make an ecosystem less susceptible to sustaining a particular agent has been called
niche engineering.109 For E. coli 0157:H7 on cattle farms, the best candidates for niche
engineering are related to feed and water trough management. Preventing multiplication
of E. coli 0157:H7 in moist cattle feeds would decrease exposure doses.91’122 Frequent

cleaning and appropriate sanitation of water troughs can reduce replication and

maintenance of E. coli 0157:H7 in sediments.9|

SOURCE OF Escherichia coli 0157:H7 INFECTIONS IN HUMAN BEINGS
The hemolytic uremic syndrome which is caused by 0157:H7 was first described

in 1955.78 Zoonotic transmission of 0157:H7 is thought to occur because epidemiologic

59

studies have found associations of HC and HUS with ingestion of ground beef and

18,125.201203

consumption of raw milk, although outbreaks also have been associated with

O 0

flesh-pressed apple cider,1 unchlorinated drinking water,18 and person-to-person
transmission.88’147 Among the 8 outbreaks with an identified food vehicle in the US, 6
were traced to ground beef and 2 to roast beefmg’l‘mm

Beef, beef products and untreated milk have been suggested as possible sources of
VTEC (verotoxin-producing E. coli) infection for man.5 8'63 ’125 “60 In May-June 1992 there
were 5 cases of VTEC 0157 human infection in the Sheffield area of England, 3 of phage
type 2 and 2 of phage type 8 that may have been associated with consumption of beef
originating from a South Yorkshire slaughter plant. E. coli 0157 was subsequently
isolated from the rectal contents of 84 (4%) of 2103 cattle at the slaughter plant and it
was suspected that they were the source of this organism. Seventy-eight (93%) of the 84
isolates were VT(+) and were of the same phage type, toxin and plasmid profile as strains
implicated in human disease in the same area!” A comparison of human and bovine E.
coli 0157:H7 isolates by toxin genotype, plasmid profile, and bacteriophage A-restriction
fragment length polymorphism profile has linked 5 sporadic human cases to bovine
originm’m

Other than fecal shedding, E. coli 0157 has been isolated from beef carcasses
(from excised meat and from the surface of carcasses) in slaughter plants and these
isolated strains have been linked to human cases in the UK.26

An outbreak of VTEC 0157:H7 infection among 60 children and 14 adults who

had visited a dairy farm resulted in 48 cases of diarrhea and 3 of the affected people

developed HUS. A significant association was found between infection and the

60

consumption of unpasteurized milk. VTEC 0157:H7 was isolated from a fecal sample
from one animal on the farm. All these findings provide direct evidence that cattle may
be one of the main reservoirs of VTEC 0157:H7 bacteria that are associated with human

disease. '8

EPIDEMIOLOGY OF Escherichia coli 0157:H7

After HC and HUS outbreaks, in the course of investigation to identify the source
of infection, E. coli 0157:H7 was isolated from 5 (5.9%) fecal samples from 85 heifers
and calves and none of 141 adult cows on two dairy farms in Wisconsin. Also in another
HUS outbreak a related survey in dairy farms in Washington State revealed that 2.2% of
315 heifers and calves and none of 224 adult cows had E. coli 0157:H7 in their feces.202
Culture surveys not related to outbreaks were also conducted. The rate of E. coli
0157:H7 isolation has been 15% or less in most surveys, with generally higher rates up
to 5% in heifers and calves.

Escherichia coli 0157:H7 has been isolated only rarely from animals with
diarrhea, and it was not known whether the E. coli was the cause of the diarrhea or
not.87"°° Therefore, no animal illnesses have been conclusively attributed to E. coli
0157:H7. Other EHEC serotypes, however, have been isolated fiom calves with bloody
diarrhea.51 The lack of observable illness in food-producing animals that are shedding E.
coli 0157:H7 in their feces, makes it more difficult to identify carrier animals so that they
can be removed from the food chain”,172

In herds, prevalence of fecal shedding of E. coli depends on; age of the cattle, pre-

or post-weaning status of calves (calves older than 3 weeks do not develop attaching-and-

61

effacing A/E lesions with E. coli 0157:H7, and a large inoculum is required to infect
adult cattle), ecology of environment, effects of fasting-dietary, stress and
seasons.”’48’93 94.109 The highest prevalence of fecal shedding is seen at 8 weeks of age,
although calves are less likely to be E. coli positive before weaning than after weaning,
the pre-weaning period may play an important role in establishing and maintaining E. coli
shedding on the farm. Herds in which calves are housed in groups from birth to weaning
or in which previously individually housed calves are grouped before weaning are more
likely to include calves that shed E. collisgm’204

One explanation can be related to stress from crowding and competition
triggering shedding of organisms. A second possibility may be that multiple calf
facilities can concentrate the bacteria that are shed. Sharing nipples and bottles among
calves without rinsing or washing also may serve to spread infection from one calf to
another.”76

Certain feeding practices were negatively associated with E. coli shedding. None
of the 6 herds in which clover hay was fed, were positive for E. coli. Additionally, 7 of 8
herds in which heifers were pastured on clover were negative for E. coli. Feeding whole
cottonseed to heifers prior to first calving also was negatively associated with carriage of
E. coli, with all of 7 herds were negative of E. coli in which this feeding practice was
reported.76 Shedding of E. coli was not associated with any of the signs of illness
evaluated (poor condition, dehydration, diarrhea). These results were consistent with
those of other studies27203 in which E. coli was found in healthy cattle.

Escherichia coli can not grow in the rumen under normal conditions because of

rumen pH and volatile fatty acid (VFA) concentrations.195 However, during times of

62

food deprivations or other nutritional stress, inhibitory conditions of the rumen are
eliminated as pH rises and VFA concentration declinesm’204 Such conditions allow
enteric bacteria to survive and even multiply in the rumen (especially when feeding is
briefly resumed after feed deprivation). Thus, rumen contents can become a reservoir of
enteric pathogens. For that reason any stress factor, transportation from farms to
slaughter plant, food deprivation, or illnesses, can cause an increase in E. coli shedding.

Prevalence studies should be done in slaughter plants as well as on farms.'57304

63

CHAPTER 3

PREVALENCE OF Escherichia coli IN DAIRY CATTLE AT SLAUGHTER

ABSTRACT

The objectives of this study were to evaluate the prevalence and concentration of
enterohemorrhagic Escherichia coli (EHEC) in feces of dairy cows at slaughter, and to
evaluate the effects of winter and summer on the isolation of pathogens from dairy cows
at slaughter. Samples were collected at slaughter from cattle that had either been shipped
directly to slaughter or that had been sold through auction markets. Fecal samples from
1006 cows were collected in two seasons, winter and summer of 1996 to study E. coli
shedding patterns. Escherichia coli isolated were analyzed with respect to animal
disposition including body condition score, animal health, source of the animal, and
season of the year. Escherichia coli was isolated from 829 of 1006 fecal samples.
Sorbitol-negative E. coli was isolated twice as often (199/496) in summer 40%, compared
to 22.6% (59/260) in winter. 0f the 265 sorbitol-negative E. coli, six samples were
positive (0.022%) by ELISA to EHEC and two were serotype 0157:H7. Forty-five fecal

samples were positive only for Klebsiella sp; 34 in winter and 13 in summer.

INTRODUCTION
This project was undertaken to evaluate the effects of: 1) season, 2) body
condition score (BCS), 3) health (lameness, respiratory, non-ambulatory), and 4) route of

shipping to slaughter, on shedding of E. coli in feces at slaughter. Earlier reports

investigated seasonal effect on fecal shedding of E. coli. Two studies reported the peak
prevalence of fecal shedding of E. coli was most common in summer and early fall?"92
Other studies reported the highest occurrence of E. coli in the feces of cattle in the
summer.48’93’94’95

Transmission of pathogens between animals during marketing, transport and
waiting prior to slaughter is a possibility. Studies preformed by Brownie and Grau19 in
1967, working with cattle and Grau et al.85 in 1969 working with sheep, found evidence
suggesting that transmission of E. coli between animals can occur at markets and during
transport, especially if the time from farm to slaughter is prolonged. Another study137
suggeSted that cattle that were fasted prior to slaughter were more susceptible to
colonization with E. coli 0157:H7. However, a Canadian study48 did not show fasting as
an important risk factor for the increased fecal shedding of E. coli 0157:H7 in slaughter
cattle under existing commercial transport.

The differences in prevalence estimates may be due to the factors listed above and
may be affected by the time of year that the study was conducted and the geographical
location of the study. In other studies‘“”174 fewer Escherichia coli 0157:H7 organisms
were shed by feedlot cattle near the end of the feeding period than by newly arrived
cattle. Moreover, there is less shedding of the organisms in cattle of slaughter age than in

younger cattle. The prevalence of E. coli 0157:H7 in feedlot cattle is similar to that in

range cattle.

65

SAMPLE COLLECTION AND PROCEDURES
Slaughter animal collection

A total of 1006 fecal samples were collected. One group of 501 samples was
collected during 6 days in February and a group of 505 samples was collected during 6
days in August. All samples were collected from cattle delivered to the same slaughter
facility in Allendale, MI. Fecal samples were collected from the cecal-colon juncture of
each study cow on the viscera table at slaughter. For each sample, a single-edged razor
was used to make a linear incision and cecal-colon contents were collected and stored
immediately on ice in sterile plastic bags. The specimen was approximately divided in
half and each half placed in a separate sterile plastic bag which was labeled with the
sample number, date, market tag number and location code. Samples were held on ice
prior to being transported to the laboratory within 24h. From each cow one sample that
was to be examined for Salmonella spp. was shipped directly on ice to USDA-Animal
Health Laboratories (Ames, Iowa) by overnight Federal Express mail. The other samples
which was held on ice examined for E. coli within 24h at the MSU laboratory.

Prior to slaughter each cow was examined for breed, body condition, ambulatory
score (L= obvious lame, NR= moves easily), hide score (C= clean, D= dirty), udder
condition (NR= normal, UP= problem), respiratory problems (NR= normal, RRP=
problem), vulva problems (NR= normal P= problem), eyes (NR= normal, 0D=
defective), gastrointestinal problems (NR= normal, D= diarrhea), lumps (Y= yes, NR=

normal). The same person performed the examination on each cow.

66

Sample preparation and handling for Escherichia coli

Upon arrival at the laboratory, 1.0g of feces was diluted in 9ml of peptone broth.
This mixture was vortexed to assure a uniform dilution. Fecal debris was removed by
straining the solution through sterile gauze. Fecal samples (1.0g) were serially (1:10)
diluted in 9ml of peptone broth (0.85% PBS to 10'7 CFU/g.). Because of the heavy
concentration of microorganisms 7 dilutions were made to facilitate counting. The
dilutions were prepared by adding 0.2m] of strained fecal solution to a tube containing
1.8m1 peptone broth and mixing thoroughly by vortex and serially transferring 0.2ml of
the resulting mixture intol.8ml for seven dilutions (10", 102, 103, 10”, 10's, 10'6, 107).
The diluted samples were stored overnight at 4°C and plated on agar the next day. In the

winter, 200/501 fecal samples were stored at -20°C for 5-7 days to accommodate the

laboratory’s culture capacity. Results between frozen and fresh samples were compared.

Media and organism isolation and detection

Culturing. All sorbitol-negative and EHEC positive E. coli samples were further
screened with 0157 antiserum. E. coli that were positive for the 0157 serotype were
further tested with H7 monoclonal antibody. A 0.1ml sample from each of the seven
dilutions was plated on MacConkey medium to isolate lactose-fermenting
microorganisms (coliforms) and on Sorbitol-MacConkey medium to identify sorbitol
non-fermenting colonies. Plates were read afier 18h of aerobic incubation at 32°C. The
SMAC medium is designed to detect only the serotype 0157:H7 by not fermenting the

37

sorbitol within 24 hours. Each sorbitol negative colony (colorless) and lactose

fermenting colony (pink colonies) was subcultured to MacConkey agar and triple sugar

67

iron slants at 37°C for 18h. Escherichia coli isolates were confirmed biochemically
using urease, Simons’ citrate, oxidase, and indol.
Enzyme immunoassay for the detection of the toxins produced by Enterohemorrhagic
E. coli in culture systems (EIA): All sorbitol-negative E. coli isolates were subcultured
and tested for the presence of Stx using a commercial EHEC kit (PremierR, Meridian
Diagnostic).50 The EHEC (Enterohemorrhagic E. coli ) test utilizes monoclonal anti-Stx
capture antibody absorbed to microwells. Diluted samples were added to the wells and
incubated at room temperature, washed and enzyme conjugated anti-IgG polyclonal
antibody applied. The presence of Stx-produces a reactive antibody-enzyme complex
that can be interpreted visually.
Polymerase chain reaction (PCR): A random selection of fecal samples flom winter
and summer collection periods were cultured on MacConkey’s agar and 25 colonies
selected to determine the prevalence of E. coli attaching and effacing gene type A (eaeA)
and Stx gene strains. PCR was performed on all lactose fermenting isolates for
amplification of both Stx I and Stx II. Isolates were serotyped against E. coli 0157 and
H7 monoclonal antibody.
Statistical analysis

Prevalence of sorbitol-negative E. coli was compared to season, slaughter
using chi-square (X2). Prevalence of E. coli and sorbitol-negative E. coli was compared
to fresh versus flozen sampling for the winter samples. The P—value of significance was
set at 0.05 with a degree of fleedom at 1 to compare season. The degree of fleedom was

2 when comparing flesh versus frozen samples.

68

RESULTS

Coliform bacteria were isolated flom 829 of 1006 fecal samples cultured on
MacConkeys agar (Table 10) during summer and winter periods. Two hundred samples
were flozen in the winter and examined after being held at ~20°C for 5-6 days. In the
winter sampling, frozen samples yielded a lower recovery of E. coli than flesh samples
with 260 flom 301 (86.4%) for flozen samples as compared to 73 flom 200 (36.5%).
During the summer sampling period, all fecal samples were processed immediately and
plated within 24 hours. Of the 829 coliforrn bacteria positive cultures, 265 were sorbitol
negative of which 6 were positive on the EHEC Elisa test and 2 positive for serotype
0157 (Table 10). The prevalence of coliforrn bacteria flom flesh samples was compared
between seasons with 260/301 (86.4%) isolated in the winter and 496/505 (98.2%) in the
summer (Table 10). Of the 829 colifonn bacteria positive cultures, 265 were sorbitol
negative of which 6 were positive on the EHEC Elisa test and 2 positive for serotype
0157 (Table 10). There were twice as many sorbitol-negative E. coli isolated in summer
with 199/505 (39%) compared to 59/301 (19%) in winter, but EHEC positive were the
same (2-3%) flom these isolates (Table 11). A total of 47 samples were Klebsiella spp.
positive (34 samples in winter and 13 samples in summer, Table 10).

Of the 501 samples, the E. coli prevalence for the flesh processed samples on
sorbitol MacConkey-negative agar was 19.79% (59/301; Table 12) compared to flozen
samples 3.5% (7/200; Table 12). Using flesh samples greatly increased the isolation of
sorbitol-negative E. coli with a prevalence of 19% (59/301) and an OR=6.72, 95%
confidence limits 2.88 -—l6.48 (P<0.0001) (Table 12). Both (2) E. coli 0157 serotype

isolates were identified flom flesh samples. Also the grth of flesh samples on

69

MacConkey agar was significantly greater, with a prevalence of 86% (260/301) and an

OR=11.03, 95% confident limits 6.97 —17.52 (P<0.0001) (Table 12).

DISCUSSION

In this study, two E. coli 0157:H7 serotypes were isolated flom 1006 fecal
samples (0.2%). One of them was isolated in the summer and the other in winter. In
other surveillance studies flom North America and England the prevalence of E. coli
0157:H7 was 0.7% and 19.7% for winter and summer respectively.

In the previous studies large variances were noticed in the E. coli 0157:H7
prevalence due to methods of handling samples and different isolation and identification
techniques. The first step in isolation of 0157:H7 is based on non-fermentation of
sorbitol and lack of B-glucronidase activity which is tested on sorbitol MacConkey agar
(SMAC).

In our study, 265 of 829 E. coli cultures were SMAC and B-glucoronidase
negative suggestive of the presence of 0157. However, only 6 of these sorbitol negative
cultures were found to be EHEC positive by using the EHEC Elisa that detects Stx I and
H. This monoclonal anti Stx antibody test requires culturing of stool samples within two
hours or if not possible placing the stool samples in transport media immediately and
culttning within two to three days if they were stored at 2-8°C. In our study we were not
able to place specimens into the transport media within two hours. This may have
affected the detection of EHEC positive serotypes. Although all EHEC-positive
serotypes were isolated flom flesh samples, we can not make any assumption that flesh

samples might yield better EHEC isolation.

70

Our study also detected a seasonal effect on sorbitol-negative E. coli shedding.
Sorbitol-negative E. coli isolation was greater in the summer than in winter (199/505,
39% in summer compared to 59/301, 19% in winter). Enterobacteria infections in
humans as well as animals may peak in summer months.58’99"°(’°’190 Several studies have
shown seasonal differences in prevalence of bacterial shedding in cattle.58 In a study
done in Washington State, E. coli 0157:H7 isolation in cattle fecal samples was highest
during the period from June to September.” Donkersgoed et al.48 found that the

prevalence of E. coli 0157:H7 was significantly higher in the summer than winter.

CONCLUSION

We investigated the prevalence of sorbitol-negative E. coli shedding in live cull
(market) dairy cows presented for slaughter. According to our results the prevalence of
sorbitol-negative E. coli was higher in summer than winter. The shedding of sorbitol-
negative E. coli was not associated with the body condition scores (BCS) at slaughter.
Similarily, the source of cattle by direct or indirect shipment did not influence the
shedding of sorbitol-negative E. coli at slaughter. Overall we were only able to isolate
two E. coli 0157:H7 serotypes, one isolate in each season (winter and summer). These
findings will be. important considerations for researchers designing future studies to

reduce shedding of pathogens at slaughter.

71

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72

 

Table 11: The prevalence of sorbitol-negative E. coli during winter and summer.

 

 

 

 

WINTER SUMMER TOTAL
Sorbitol Negative
E. coli*
Positive 59 199‘I 258
Negative 242 306 548
TOTAL 301 505 806

 

 

 

 

“ OR = 0.37, 95% confidence limits 0.26 — 0.53; P < 0.001

* Only fresh samples

Table 12: Prevalence of E. coli sorbitol-negative and MacConkey positive

growth of the fresh and frozen samples in winter.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FRESH FROZEN TOTAL
MacConkey agar
E. coli - Klebsiella
Positive 2608 73 333
Negative 41 127 168
TOTAL 301 200 501
Sorbitol-Negative
E. coli
Positive 59b 7 66
Negative 242 193 435
TOTAL 301 200 501

 

8 OR = 11.03, 95% confidence limits 6.97 — 17.52; P < 0.001
b OR = 6.72, 95% confidence limits 2.88 -— 16.48; P < 0.001

73

 

 

CHAPTER 4

PREVALENCE OF Salmonella IN DAIRY CATTLE AT SLAUGHTER

ABSTRACT

The objectives of this study were to evaluate the prevalence and shedding of
pathogens in feces of cull dairy cows at slaughter and also to evaluate the effects of
winter and summer on the isolation of pathogens fi'om cull dairy cows at slaughter.
Samples were collected at slaughter fi'om cattle sold direct or through auction markets.
Fecal samples from 501 cows were collected in winter of 1996 and 505 samples in
summer of 1996 to study Salmonella spp. shedding patterns. Salmonella spp isolates were
analyzed against animal disposition including body condition score, animal health,
source of the animal, and season of the year. Salmonella was isolated from 94 of the
1006 fecal samples. Twenty-two serotypes were identified. The predominant isolates
comprising of S. typhimurium (22/94), S. senftenberg (17/94), and S. kentucky (8/94).

Season had a major effect on the prevalence of Salmonella. Salmonella
prevalence was significantly higher (P< 0.0001) in the summer (13.86%) than in winter
(4.79%). Both body condition scores and transportation (direct versus indirect) were
significantly associated with prevalence in the summer. Thin animals having a BCS s
2.0 in the summer (P = 0.033) had a significantly higher prevalence of Salmonella.
Fecal samples from cull cow sent directly to slaughter were less likely (13.7%) to culture-
positive for Salmonella from fecal samples than animals that were sent though auction

markets (25.1%) in the summer (P = 0.018).

74

INTRODUCTION

This study was undertaken to evaluate the prevalence and shedding of pathogens
in feces of cull dairy cows at slaughter and evaluate the effect of body condition and
direct / indirect shipping to slaughter on prevalence of Salmonella at slaughter.

The distribution of Salmonella along the gastrointestinal tract and in associated
with lymph nodes was studied in 100 sheep and 100 cattle at slaughter plant in a study
conducted in United Kingdom. The carriage rate of Salmonella spp. was 77% at cattle
and 43% at sheep.164 In another study from Australia, livers from normal slaughter cattle
were examined for surface contamination by Salmonella spp. immediately after
evisceration and again after inspection. Salmonella spp. was isolated from 32% at
evisceration and 82% afier inspection.

Numbers of Salmonella present were low at evisceration, and rose after
inspection. The sources of the Salmonella spp. were probably the contents of the
gastrointestinal tract and the mesenteric lymph nodes, both of which may show high

65 It was

prevalence of infection in cattle which have been held before slaughter.l
concluded that contamination of viscera during handling and inspection at the slaughter
plant occurred fi'equently.

Anderson et al.214, found that 0.5% of calves in the market were infected with
Salmonella. An infection rate of 0.6% was found in calves if animals were kept in the

premises few hours before slaughter. Infection rate was further increased if animals were

slaughtered after staying 2-5 days in the premises.

75

Cattle are commonly exposed to Salmonella in feed and a variety of stresses can
increase susceptibility to colonization.91 Grau et al.84 also found that the incidence of
Salmonella in the bovine rumen was greater the longer the period between the farm and
the abattoir. Also they found that persistence of Salmonella inoculated into the rumen of
cattle were depend on the pre and post inoculation feeding of the cattle. Well-fed cattle
before Salmonella inoculation eliminated Salmonella rapidly but starvation 2-3 days prior
to inoculation increased intestinal prevalence of Salmonella and a further increase was
seen when normal feeding was restarted.86

Frost et al.‘55 examined aspects of Salmonella infection in cattle at slaughter.
Three groups of cattle (15 in each group) taken from the sale yard, were transported to
feedlots near slaughter. Animals were slaughtered in 2 days, 18 days and 80 days
respectively and rumen contents, and mesenteric lymph nodes were examined for
Salmonella infection. The first and second group (2 and 18 days) showed 7/15 and 15/ 15
Salmonella infection but there was no Salmonella isolation on third group. It was clear
transportation, starvation and re-feeding was increasing the Salmonella shedding.

The hide is thought to be the immediate source of most bacterial contamination of
carcasses.58 During transportation, close contact between animals may cause hide
contamination. Puyalto et al.153 found that hair contamination increased from 8% at farm
to 25% at slaughter after transportation.

However, these surveys may underestimate E. coli 0157:H7 prevalence at time
of slaughter because stress, health condition, feeding frequency, and fasting, can

influence populations of E. coli in the gut. Salmonella spp. and E. coli are poorly

adapted to rumen and hind gut fermentation due to their sensitivity to the low pH and

76

‘57 In the rumen of

high volatile fatty acid [VF A] concentrations of these compartments.
well-fed animals, growth inhibition is greatest at pH<6.5 and [VFA]>100mM.'9’86
During periods of fasting, however, these factors of inhibition diminish as ruminal [VFA]
decline (<50mM) and pH values exceed 7025’130 Under these conditions the rumen may
be a potential reservoir of enteric pathogens instead of an obstacle to their growth.“166

For that reason, the prevalence of Salmonella spp. and E. coli 0157:H7 was
evaluated in cull dairy cows at the slaughter plant.

To evaluate the effects of season on isolation of pathogens from cull dairy cows at
slaughter, samples were taken in the winter and in the summer.

According to the CDC (Center for Disease Control) from 1973-1987 Salmonella
outbreaks have mostly occurred in July and August warm months of the year.40 In 1991-
1992, the US. Department of Agriculture conducted a study called the National Dairy
Heifer Evaluation Project (NDHEP) which has determined Salmonella prevalence rate

194

according to season. More Salmonella positive calves were found in late summer with

194

prevalence of 36.1 of every 1,000 samples. The prevalence was lowest 12.3 per 1,000

in the winter period. ‘94

MATERIAL AND METHODS

Study design at slaughter
' A USDA inspected slaughter plant located in Allendale, MI was used for
surveillance because cull cows arrive for slaughter as the result of direct sale and through
auction markets. Animals presented for slaughter were evaluated as to the health of the

animal, the body condition, and any physical abnormalities. The study was conducted in

77

February, 1996 and August, 1996 to compare the shedding of bacteria in cull cows during
winter and summer. The slaughter plant was visited for 3 to 5 days during each period
until approximately 500 cull dairy cows were sampled. Since cull dairy cows were only a
portion of all the cattle processed daily at the slaughter plant all dairy cows were sampled
on each day until the desired number for each sampling period was obtained. The dairy
cows in this study came from auction markets and directly from local farms. Each cow
was identified with a back tag number, which enabled us to trace the origin of these cattle

and viscera through the slaughter process.

Sampling and bacterial culture methods for feces

Fecal samples were collected from the cecal-colon juncture of each study cow on
the viscera table at slaughtered. For each sample, a single-edged razor was used to make
a linear incision and cecal-colon contents were collected and stored immediately on ice in
sterile plastic bags. At the end of each day, all samples were shipped by overnight
delivery to the Diagnostic Bacteriology Laboratory, National Veterinary Service
Laboratory, USDA-APHIS (NV SL) in Ames, Iowa for isolation of Salmonella.

One gram of feces was removed and inoculated into a sterile culture tube with
10ml of tetrathionate broth and incubated at 37°C for 48hours79’l3 1"“ (Figure 6). After
incubation, the tube was vortexed and a 0.1m] sample was pipetted into a tube with 10ml
Rappaport-Vissiliadis R10 broth and also streaked onto Brilliant Green Agar plates with
Novobiocin (BGN).79 The Rappaport-Vissiliadis R10 tube and Brilliant Green Agar with
Novobiocin were incubated at 37°C for l8-24h.‘“5’79 After incubation, the tube was

vortexed and a streak sample was taken and plated to xylose-lysine—tergitol-4 (XLT-4)

78

agar and to another Brilliant Green Agar with Novobiocin plate.46’13' The plates were
streaked for colony isolation and incubated at 37°C for 18-28h. At least three suspected
Salmonella colonies were picked from each plate and transfered to XLT-4 agar and BGN
agar for identification (black in color on XLT-4 agar and red on BGN agar).13' Each
colony was transferred to separate tubes containing 5ml of triple sugar iron agar (TSI)
and lysine iron agar (LIA) slants and incubated at 37°C for 18-24h for biochemical
profiles.80 The XLT-4 and BGN plates that were presumptive negative for Salmonella
colonies were held at room temperature for an additional 18-24h and rechecked for
suspected colonies. Suspect colonies (three colonies) were picked and differentiated by
use of LIA and T81 agar slants.46‘62"3 '

All isolates were serotyped for both Salmonella O-antigen and H-antigen

determination for verification and complete serotyping.

 

1.0 gram 1.0 gram
24 hours BG Pick 2 suspects - - - Identify
Rappaport — 48 hours BG Pick 2 suspects - - - Identify
Broth

jaw“. IA

Figure 6: Diagram of Salmonella isolation procedure.

 

 

 

\ 72 hours BG Pick 2 suspects - - - Identify

 

Statistical Analysis

Prevalence of Salmonella was compared to season, body condition and
transportation route to slaughter using chi-square (X2). The P—value of significance was

set at 0.05 with a degree of freedom at 1 to compare season, transportation, and body

79

condition score. Because cattle came from Michigan, Ohio and Indiana the degree of

freedom was 2 when comparing state of origin.

RESULTS

Of the 1006 samples, 94 (9.34%) were positive for Salmonella (Table 13). Of the
summer samples, 70 of 505 (13.86%,) were positive for Salmonella with 16 different
serotype isolated. Salmonella typhimurium (22), S. seftenberg (17), and S. kentucky (8)
were the most frequently isolated serotypes (Table 13). Of the samples collected in the
winter, 24 of the 501 (4.79%) were Salmonella spp. positive. There were 12 different
serotypes of Salmonella isolated during winter, Salmonella enteritidis phage type (4), S.
kentucky (4), S. muenster (4), and S. Iilie (4) were the most frequently isolated serotypes
(Table 13). Salmonella typhimurium was not isolated in any winter samples.

Salmonella isolation was significantly higher, (OR=0.31, 95% confidence limits
0.19 - 0.52, P < 0.001), in summer than winter (Table 14).

Of the 640 cull cows that could be tracked from place of origin, 243 cattle were
sent to slaughter via auction markets. Salmonella was detected in 17 of the samples
(17/243; 6.9%) during winter. Of the 102 cattle that were sold directly to slaughter from
the farm, Salmonella was detected in 7/102 samples (6.8%). The difference was not
significant. However, of the 179 cattle brought to slaughter from auction markets during
the summer, Salmonella was detected from 45/179 samples (25%), as compared to
16/116 (13.8%) from the cattle sent directly to the slaughter. This difference was
statistically significant with an OR=2.10, 95% confident limits 1.08 -— 4.13, P = 0.0187,

(Table 15).

80

Overall, cattle from out-of-state did not differ from cull cows shipped from
Michigan. The only exception was cull cows shipped from Indiana in the winter (P =
0.0094; Table 16).

The body condition scores of the 974 cull cows were recorded for correlation with
Salmonella prevalence. Salmonella isolation in the cull cows with a BCS S 2.0 (thin
cows) was compared to Salmonella isolation in the cull cows with a BCS > 2.0.
Salmonella was isolated more ofien from thin cows in the summer with a prevalence of
18% (45/250) with an OR=1.94, 95% confidence limits 1.11 — 3.39, P = 0.012, (Table
17). Cows with BCS s 2.0 were twice as likely to shed Salmonella than cows with

BCS > 2.0 (Relative Risk=l .8).

DISCUSSION

In our survey, cull cows sent to slaughter in the summer were three times more
likely to be shedding Salmonella than in the winter. Salmonella typhimurium was the
most common isolate in the summer. But S. typhimurium was not isolated in winter.
Other frequently isolated Salmonella serotypes in summer were S. senfienberg and S.
kentucky, S. kentucky, S. meanster, and S. lille were most often isolated in the winter.
Other surveys also determined Salmonella typhimurium as the most common serotype in
the Northeast United States (USDA, 1994).

Cull cows coming through auction markets were three times more likely to shed
Salmonella than those sent directly to slaughter. In winter, Salmonella prevalence was
higher in the cows coming from auction markets than cows coming directly from the

farm to slaughter.

81

Previous studies determined starvation and stress during transportation increases
Salmonella shedding.65’84’214 Thus introducing animals originating from different farms
to auction markets causes cross contamination between the animals and increase
shedding especially if there is a delay before slaughter.

According to this survey, the comparison between states may lack significance
because of the low number of samples collected from states other than Michigan.
Isolation was numerically higher for out-of-state cull cows and a large sample size might
have concluded different results. However, this maybe related to the time in transit and
indirectly shipping to slaughter.

Although our data set was limited, body condition of the cull cows were an affect
on Salmonella shedding. Thin cows (BCS S 2.0) were twice as likely to shed
Salmonella in summer as heavier cows. The thin cull cow may be more susceptible to
the effects of transportation, starvation, and stress than better conditioned cows. Summer
heat may add to this stress as seen in this study.

Although animals were examined at slaughter for health conditions (lameness,
hide score, udder condition, respiratory problems, vulva discharge, eyes, fecal
consistency, and lumps and lesions) none of these health factors were shown to be a
factor in the shedding of Salmonella.

The facts of high level of Salmonella isolation on cattle feces at slaughter require
more attention for preslaughter management of animals. Further Studies of control points
on transportation conditions and managing holding facilities should be conducted in

prospective, controlled manner. Duration of preslaughter period needs further study.

82

CONCLUSION

To make significant improvements in food safety, measures should be taken at all
points in the farrn-to-table chain including production, transportation, slaughter
processing, storage, retail and food preparation. In our present study season, body
condition scores and the source of cattle by direct or indirect shipment can affect the
prevalence of salmonellae at slaughter. Because of these factors slaughter cattle will, at
times, carry food-home pathogens that can be transferred to carcass and meat. While
care taken during the slaughter operations can minimize the transfer of food-borne
pathogens from animal to meat, it cannot entirely prevent it. Furthermore, it can be
predicted that the above factors will affect the shedding of Salmonella at slaughter. More
controlled studies are needed to understand the precise roles of each of the factors in the

shedding of Salmonella to be able to reduce the risks of food-borne diseases.

83

Table 13: Isolation of Salmonella fiom cull cows at slaughter.

 

 

 

 

 

 

 

Salmonella
Samples
Total Winter Summer
Total 1006 501 505
Salmonella Isolation 94 (9.34%) 24 (4.79%) 70 (13.86%)
Serotypes
S. typhimurium 22
S. sefienberg 17
S. kentucky
S. muenster
S. mbandaka
S. lille

S. enteritidis phage type 8
S. cerro

S. dublin

S. oranienburg

S. uganda

S. braenderup

S. havana

S. java

S. thompson

S. agona

S. montevideo

S. ohlo

S. schwarzengrund
S. panama

S. enteritidis"

S. bovismorblflcan

 

Huunuaunuuuuuuuauaxa;

 

unacccOQ-‘uchct-ibbéb‘hac

 

coca—uuuuuceuwNe—aaanSS

 

* phage untypable

84

 

Table 14: Seasonal difference in Salmonella prevalence at slaughter.

 

 

 

 

 

WINTER SUMMER TOTAL
Salmonella (+) 24 70" 94
Salmonella (-) 477 435 912
TOTAL 501 505 1006

 

 

 

 

3 OR = 0.31, 95% confidence limits 0.19 — 0.52; P < 0.001

Table 15: Prevalence of Salmonella in cattle that were assembled prior to

shipment (indirect) or directly shipped to slaughter.

 

 

 

 

 

 

 

 

 

 

 

 

INDIRECT DIRECT TOTAL
WINTER
Salmonella (+) 17 7 24
Salmonella (-) 226 95 321
TOTAL 243 (6.9%) 102 (6.8%) 345 (6.9%)
SUMMER
Salmonella (+) 45 16“ 61
Salmonella (-) 134 100 234
TOTAL 179 (25.1%) 116 (13.7%) 295 (20.6%)
SUM TOTAL 62/422 23/218 85/640

 

 

 

 

a OR = 2.10, 95% confidence limits 1.08 — 4.13; P = 0.018

85

 

 

Table 16: Salmonella prevalence in cull dairy cows originating from

Michigan, Indiana and Ohio.

 

 

 

 

 

 

 

 

 

 

 

 

 

MICHIGAN INDIANA OHIO TOTAL
WINTER
Salmonella (+) 14 8a 2 24
Salmonella (-) 354 55 54 463
TOTAL 368 (3.8%) 63 (12.6%) 56 (3.5%) 487 (4.9%)
SUMMER
Salmonella (+) 46 7 9 62
Salmonella (-) 274 58 74 406
TOTAL 320 (14.3%) 65 (10.7%) 83 (10.8%) 468 (13.2%)
SUM TOTAL 60/688 15/ 128 11/139 86/955

 

 

 

 

 

“ Chi-square = 9.33; P = 0.0094

86

 

Table 17: Salmonella prevalence related to body condition scores.

 

 

 

 

 

 

 

 

 

 

 

 

BCS s 2.0 BCS >2.0 TOTAL
WINTER
Salmonella (+) 9 15 24
Salmonella (-) 137 317 454
Total 146 (6.1%) 332 (4.5%) 478 (5%)
SUMMER
Salmonella (+) 45a 25 70
Salmonella (-) 205 201 426
TOTAL 250 (18%) 246 (10%) 496 (14.1%)
SUM TOTAL 54/396 40/578 94/974

 

 

 

 

3 OR = 1.94, 95% confidence limits 1.11 - 3.39; P = 0.012

87

 

CHAPTER 5

CONCLUSION

Cattle-associated pathogens like enterohemorrhagic E. coli and Salmonella
infections pose serious challenges for beef markets and constitute emerging threats to
public health. Recent reports indicate that dairy cows account for about 8% of US.
domestic beef production, 25% of US. non-fed beef available for consumption in the
US, and about 18% of US. ground beef. Producers remove the majority of cull dairy
cows for reproductive problems, udder or mastitis problems, poor production unrelated to
disease, or lameness or injury. These reasons for culling are not usually related to ill
health or systemic disease, which might preclude their wholesomeness as a human food
source which makes cull dairy cows a likely source of food-bome microbiological
hazards. For that reason we have recorded the health conditions along with body
conditions for each cow that was slaughtered. As a result we found out that health
conditions did not affect the shedding of either E. coli or Salmonella whereas body
condition influenced the shedding of Salmonella at the slaughter plant during the
summer. This finding may raise a question of “why body condition did not affect E. coli
shedding”? One possibility may be the limited number of fecal samples being sampled at
the time. We also examined fresh sampling versus frozen sampling. Using fresh samples
significantly affected the prevalence of not only E. coli but probably also 0157:H7. We
only found 2 positive samples of 0157:H7 both from fresh samples and from each
season. In the case of Salmonella fecal samples were all processed fresh.

It has been estimated that approximately 17% of the nation’s ground beef may

come from cull dairy cows.190 Nearly 77% of cows intended for beef slaughter are sent

88

to markets, auctions, and sale barns, while 22% are sent straight to slaughter facilities.
This information indicates a relatively high amount of transportation involved in the
movement of cull dairy cows to slaughter plants. Increased transportation poses risks of
nutritional and environmental stresses, exposure to disease pathogens either from other
cattle by contact or from feed deprivation that cattle go through during transportation.
Transportation was another variable of our study. We observed that cows shipped
indirectly to slaughter had a higher prevalence of Salmonella than cows shipped directly
to the slaughter plant. This result also agreed with the previous studies. 48’58’65’84’2” We
were unable to detect an influence on E. coli shedding due to transportation.

We also observed a seasonal effect on the shedding of both E. coli and Salmonella
at the slaughter plant. Shedding of both was higher in the summer than winter as seen in
the other studies.48

Practices that have been tentatively, but not consistently, associated with the fecal
prevalence of E. coli 0157:H7 and Salmonella include herd size, grouping, weaning,
manure management, equipment sanitation (including water troughs), feed composition,
feed additives (by products, ionophores), and parenteral antibiotics.48’77’99

In acknowledging that variable densities of microbial pathogens in gastrointestinal
contents are likely to have a significant effect on subsequent contamination levels of beef
carcasses, consideration of preharvest controls must be included in any farm-to-plate
safety strategy. The management of food-borne pathogens will become part of an
integrated program to enhance food safety, which includes the producer, the packer, the

distributors, retailers and the consumer. Hazard Analysis and Critical Control Points

(HACCP) type prevention programs, using scientifically based critical management

89

points, will help further reduce risk.”4 Caution must be exercised when making direct

comparisons among prevalence estimates of bacteria from various studies. There are

differences in culture techniques, including a large variability in the sensitivity of tests.

Additionally, the number, frequency and timing of sampling (on-farm, and at slaughter);

the handling, transport and storage of samples; the type and age of cattle; the type of

sample (fecal pat, fecal swab, weight of fresh feces); the season of sampling; the unit of

analysis (individual, herd, process lot); and the serotype of bacteria may affect prevalence

estimates.48

repeated and updated periodically as appropriate, to ensure they are aware:

At the farm level there should be an education/awareness program for farm workers,
148

of the existence, potential prevalence and nature of E. coli 0157:H7 and Salmonella;
of the potential for the spread of infection on farms, notably from fecal material, and
of the consequent need for scrupulous personal hygiene;

of the need for care in the use of untreated slurry or manure; and

of the absolute requirement for the presentation of animals in an appropriate, clean
condition for slaughter.

At the slaughter plant: ‘48
Good practices in slaughter procedures must be identified and promoted by Industry
with the help and support of government departments, particularly in the areas of the
presentation of clean cattle and of hide and intestine removal.

Slaughter workers should be trained in good hygiene practice during slaughter and

enforcement should concentrate on slaughter and subsequent handling of carcasses.

90

o The Hazard Analysis and Critical Control Point (HACCP) system should be
enshrined in the legislation governing slaughter plants and the transportation of
carcasses and meat. Meanwhile, enforcers and the trade should ensure that HACCP
principles are observed.

Further consideration should be given, involving the industry and consumer interests,
to the potential use and benefits of end-process treatments such as steam
pasteurization.

Finally it is clear that for many food-bome pathogens, the gastro-intestinal tract of
clinically normal cattle is an important reservoir of infection for human beings. These
organisms find ready access to the food chain at processing due to the inevitable transfer
of bovine fecal flora onto carcasses. New opportunities are being sought to improve the
microbial safety of beef products by applying interventions in both the ‘preharvest’ and
‘postharvest’ periods.106 Preharvest control measures are those that can be implemented
while cattle are on the farm, during marketing and transport, and while waiting at
slaughter. Such measures have the appeal of not placing total reliance on the hygienic
practices of processors, food handlers and consumers. They are also consistent with the
belief that, where possible, control should be exercised at all possible points within the
food chain.106

But still, as long as you cook the hamburgers really good keep eating those

delicious ‘Big Macs’.

91

10.

ll.

12.

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