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LIBRARY
Michigan State
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This is to certify that the

thesis entitled

Effects of Monensin and Sodium Chlorate on
Salmonella Infantis Colonization in Broilers.

presented by

Geetha S. Kumar

has been accepted towards fulfillment
of the requirements for

Master's , Animal Science
degree 1n

 

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lt ajor professor

Date /Q/2 g/ag

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6/01 c:/CIRC/DateDue.p65-p.15

 

EFFECTS OF MONENSIN AND SODIUM CHLORATE ON SALMONELLA
INFANTIS COLONIZATION IN BROILERS
BY

Geetha S. Kumar

A THESIS
Submitted to
Michigan State University

In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE

Department of Animal Science

2002

ABSTRACT
Effects of monensin and sodium chlorate on Salmonella lnfantis
colonization in broilers
By
Geetha S. Kumar
Two hundred and ten Salmonella-free broilers were raised on feed with or
without monensin. AII chicks were orally infected on day 3 with S. lnfantis (1O8
CFU / bird). Caecal colonization by S. lnfantis was evaluated on days 7, 10, 14,
21, 28, 35, and 42. Both groups had similar caecal concentrations of S. lnfantis,
indicating that monensin was not an effective means of reducing S. lnfantis
colonization. Next, 150 Salmonella-free broilers were divided into four groups,
three of which received a commercial sodium chlorate solution days 10, 20, and
38 for four consecutive days with the last group given no sodium chlorate
treatment. All chicks were orally infected on day 3 with S. lnfantis (108 CFU I
bird). Caecal colonization and liver invasion by S. lnfantis were assessed on
days 7, 10, 14, 17, 20, 24, 27, 30, 38, and 42 with fecal swab samples also
analyzed on days 11, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25, 26, 28, 29, 39, 40,
and 41. Birds receiving sodium chlorate on days 10 and 20 yielded significantly
lower caecal populations of S. lnfantis up to 7 days after treatment (P<0.05)
compared to the untreated control group. No significant reduction of Salmonella
was observed in birds treated with sodium chlorate on day 38 and in the liver
invasion. However fecal shedding of S. lnfantis decreased 2.0 to 3.5 Iogs three

to four days after treatment with sodium chlorate on days 10 and 20.

Loving/y dedicated to my Guruji, Sri Sri Ravi Sankar, parents, Radha & Sana],

brother, Madhu, and dearest son, Aksha y

iii

ACKNOWLEDGEMENTS

I thank my major professor, Dr. Sam.K.Varghese, for providing me the
oppurtunity, resources and help in many other ways, to pursue this degree
program at Michigan State University.

I extend my sincere gratitude and appreciation to my research advisor, Dr.
Elliot Ryser, for his guidance, support, encouragement and endless patience
throughout this research and in writing the thesis.

I thank Dr. Fulton and Dr. Bursian for agreeing to be my committee
members, for their encouraging attitude and for all the help and support they
have provided throughout the course of this research.

Special thanks to Angelo Napolitano, the Farm manager at MSU Poultry
Teaching & Research Center, and Carol Stevens at ULAR for helping me with
the resources necessary for the successful completion of this project.

Thanks to the administrative staff Jackie, Faye, Jeaniva for their efficiency
and gracefulness in taking care of all the administrative details related to my
degree program here at MSU.

My sincere thanks and appreciation to all my friends and lab mates for
being there when I needed them most and for providing me with the support and
encouragement throughout the course of my stay here at MSU.

None of this would have been possible if it were not for the help and
support of my parents and my brother. I would also like to thank my son for
exhibiting remarkable patience and understanding for one so young throughout

this period which has been anything but easy for him.

iv

TABLE OF CONTENTS

LIST OF TABLES

CHAPTER 1: INTRODUCTION

CHAPTER 2: LITERATURE REVIEW

2.1.
2.2.
2.3.

2.4.

2.5.
2.6.

Food safety
Foodbome bacterial pathogens in poultry
Salmonellosis
Salmonella in poultry
Salmonella enterica serovar lnfantis
Control of Salmonella in poultry
2.6.1. Pre-harvest control measures
2.6.1.1. Poultry feed additives
2.6.1.2. Antibiotic resistance and Salmonella
2.6.1.3. Competitive exclusion
2.6.1.4. Sodium chlorate
2.6.1.5. Other pre-harvest control measures
2.6.2. Post-harvest control measures
2.6.2.1. Acids
2.6.2.2. Trisodium phosphate
2.6.2.3. Chlorine
2.6.2.4. Chlorine dioxide
2.6.2.5. Ozone

2.6.2.6. Ultraviolet radiation

Ah

16
21
22
23
24
26
29
30
31
32
32

33

53

if

35

2.6.2.7. Electricity
2.6.2.8. Irradiation
2.6.2.9. Other post-harvest control measures
CHAPTER 3: EFFECTS OF MONENSIN AND SODIUM CHLORATE ON
SALMONELLA INFANTIS COLONIZATION IN BROILERS
3.1. Abstract
3.2. Introduction
3.3. Materials and Methods
3.3.1. Effect of monensin on S.lnfantis in broilers
3.3.2. Control of S.lnfantis using sodium chlorate
3.3.3. Salmonella lnfantis culture
3.3.4. Chick hatching and housing
3.3.5. Treatment with monesin and sampling
3.3.6. Treatment with sodium chlorate and sampling
3.3.7. Microbiological analysis
3.3.8. Statistical analysis
3.4. Results
3.5. Discussion
CHAPTER 4: CONCLUSIONS AND FUTURE RESEARCH
APPENDIX
REFERENCES

vi

35
36

36

38
39
40
4o
41
41
42
43

43

45
46
58

62

67

Table 1

Table 2

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5
Table 3.5

LIST OF TABLES

Foodbome illness associated with poultry and

poultry products

Class I recalls of poultry products contaminated with

Salmonella in the United Sates

Mean caecal populations of S. lnfantis (CFU/g) in birds

Raised on a diet with or without monensin

Mean S. lnfantis caecal colonization using sodium chlorate

on days 10, 20, and 38 (log CFU/g)

Invasion of S. lnfantis into the liver using

sodium chlorate

Mean fecal shedding of S. lnfantis (CFU/g) using

sodium chlorate...

Confirmation results on S. lnfantis selected isolates...

Body weights of broilers using sodium chlorate

as recorded on day 42

vii

...11

17

.47

50

52

56

57

LIST OF FIGURES
Figure 1 Main points in a modern processing plant............... 20

Figure 3.1 S. lnfantis caecal colonization of broilers with or without

Monensrn 48
Figure 3.1 S. lnfantis caecal colonization using sodium chlorate... 51
Figure 3.1 S. lnfantis fecal shedding using sodium chlorate ................. 55

viii

CHAPTER 1
INTRODUCTION

Salmonella, one of the leading causes of foodbome illness in the United
States, is responsible for an estimated 1.4 million cases and 553 deaths annually
with associated costs of $2.4 billion (Mead et al., 1999). According to the Center
for Science in the Public Interest (CSPI, 2001), poultry products rank among the
top five food vehicles responsible for outbreaks of foodbome illness. The other
four vehicles listed were seafood, eggs, produce and beef. The United States
Department of Agriculture - Food Safety and Inspection Service (USDA-FSIS)
issued the Pathogen Reduction / Hazard Analysis and Critical Control Peint
program final rule in July of 1996 to reduce the incidence of foodbome pathogens
in raw meat and poultry products. According to this program, Salmonella
performance standards, which are product specific, were determined from
nationwide microbial baseline data. Based on performance standards, the
maximum allowable Salmonella contamination rate for broilers was fixed at 20%.
After implementation of HACCP, more than 80% of samples met the Salmonella
performance standards from 1998 to 2000 (Rose et al., 2002) with FSIS
expecting this trend to continue so that the targeted incidence rate of 6.8 cases
of salmonellosis l 100,000 persons is achieved by the year 2010.

Efforts to meet the 2010 projection for salmonellosis should be aimed at
reducing the prevalence of Salmonella in poultry. Broilers are widely recognized

as reservoirs for Salmonella, which colonizes and proliferates in the

gastrointestinal tract of these birds. The primary source of Salmonella in finished
poultry products is the infected gastrointestinal tract of birds. Thus, control
measures aimed at reducing Salmonella colonization in the gastrointestinal tracts
of birds will be very rewarding to the poultry industry.

One of the age-old methods to reduce Salmonella colonization in birds is
the use of antibiotic feed additives. Antibiotics are added at sub-therapeutic
concentrations to animal feed to prevent diseases as well as to improve growth
rate and weight gain. Antibiotics such as chlortetracycline, neomycin and
oxytetracycline have proven to be effective against Salmonella and have been
commonly used as poultry feed additives (Quarles et al., 1977; Williams, 1985).
The practice of feeding animals sub-therapeutic levels of antibiotics often leads
to selective pressures that result in the emergence of new, antibiotic-resistant
strains of Salmonella and other pathogens in the food chain (Dupont and Steele,
1987; Holmberg, 1987; D’Aoust 1989). Foodbome infections are becoming
increasingly difficult to treat with the emergence of multi-antibiotic resistant
bacteria such as Salmonella Typhimurium DT 104 in the food chain (Wall et al.,
1994).

Given the increasing problem of antibiotic resistance, the poultry industry
has developed alternative measures such as competitive exclusion to control
Salmonella. Competitive exclusion cultures, obtained from mucosal wall
scrapings of pathogen-free adult birds, have reportedly reduced Salmonella
colonization (Blankenship et al., 1993; Bailey et al., 2000). However, some

commercially available competitive exclusion products have proven to be

ineffective in protecting chicks against Salmonella infections (Stavric et al.,
1992). A new approach gaining popularity is the feeding of sodium chlorate to
food-producing animals to reduce Salmonella colonization. Bactericidal activity
of sodium chlorate has been demonstrated against several important foodbome
pathogens in the Family Enterobacten'aceae including Salmonella and
Escherichia coli O157:H7 (Anderson et al., 2000). Both of these organisms
possess respiratory nitrate reductase activity which can bring about the
anaerobic reduction of nitrate to nitrite as well as intracellular reduction of
chlorate to chlorite. The resulting chlorite is cytotoxic and is lethal to those
bacteria possessing the nitrate reductase enzyme which includes Salmonella. In
one such study, Salmonella populations in the meca of pigs decreased by Mo
logs following oral administration of a sodium chlorate solution (Anderson et al.,
2001).

The goal of this study was to investigate two treatments for broilers, which
may help reduce colonization by Salmonella, and thereby reduce the incidence of
Salmonella on broiler carcasses. For this purpose, effects of the ionophore
antibiotic feed additive monesin and sodium chlorate on Salmonella colonization

and fecal shedding in broilers were investigated.

CHAPTER 2

LITERATURE REVIEW

2.1. FOOD SAFETY

Food safety issues are becoming a major concern worldwide due to the
increasing incidence of foodborne disease. In the United States, an estimated 76
million cases of foodbome illness occur annually, resulting in 325,000
hospitalizations and 5,000 deaths costing approximately 9.2 billion dollars (Mead,
et al., 1999). Public health authorities believe that most cases of foodborne
illness are unreported since the symptoms are often mild and do not require
medical attention. Hence, it is very difficult to obtain accurate data on the true
incidence of foodborne illness.

Norwalk virus now accounts for most cases of foodbome illness
(23,000,000) followed by the bacterial pathogens Campy/obacter (2,453,926) and
Salmonella (1,412,498) (Mead, et al., 1999). Since 1985, several bacterial
pathogens including Listeria monocytogenes, Escherichia coli 0157:H7 and
antibiotic-resistant strains of Salmonella have emerged as serious threats to the
food supply. According to the Center for Science in the Public Interest (CSPI),
poultry ranks fifth among the top five single-food vehicles in outbreaks of
foodbome illness reported from 1990 to 2001 (CSPI, 2001). The other vehicles
listed in the order of rank were seafood, eggs, produce and beef. Hence, the
microbiological quality of raw poultry and poultry products has become a major

public health issue.

The risks associated with consumption of contaminated food continue to
change. The current rise in foodbome illness may be due to a wide range of
factors including rapid urbanization in developing countries, movement of people
as well as live animals and food products across borders, changes in food
handling practices, changes in raw material sources, failure to follow standard
hygienic procedures, development of ready-to-eat foods with extended
refrigerated shelf life and emergence of new pathogens as well as antibiotic
resistance and mutations among existing pathogens. In addition, changes in
demographics are playing a major role with the very young and the aged as well
as pregnant and immunocompromised individuals being more susceptible to
foodbome illness than the general population (Gerba et al., 1996). Control of
bacterial pathogens has been further complicated by new modes of bacterial
transmission and resistance to food processing/preservation strategies. While
most foodbome illnesses produce temporary symptoms, salmonellosis, one of
the leading bacterial foodbome illness, can lead to more serious complications
such as reactive arthritis or Reiter’s syndrome - a long-term chronic illness
characterized by joint pain, eye irritation and painful urination (Frenzen et al.,
1999)

In the United States, six federal agencies have primary responsibility for
food safety: two agencies under the Department of Health and Human Services -
the Food and Drug Administration (FDA) and the Centers for Disease Control
and Prevention (CDC); three agencies under the USDA - the Food Safety and

Inspection Service (FSIS), the Agricultural Research Service (ARS) and the

Cooperative State Research, Education and Extension Service (CSREES); and
the Environmental Protection Agency (EPA). While the primary role of the CDC
in food safety is to identify and track foodbome disease outbreaks through
FoodNet and PulseNet, the CDC also supports disease surveillance, research,
preventative efforts and training related to foodbome diseases. USDA-FSIS is
the agency responsible for ensuring the safety, wholesomeness and accurate
labeling of meat and poultry products. According to the Federal Meat Inspection
Act and Poultry Products Act, the sale or transportation of adulterated or
misbranded meat and poultry products is prohibited. The Food, Drug and
Cosmetic Act of 1938 states that a food may be adulterated and therefore unfit
for human consumption if it contains harmful substances (e.g. pathogenic
bacteria or microbial toxins). FSIS is primarily concerned with the presence of
bacterial pathogens and their toxins in foods, as these are responsible for
moderate to severe illness and/or death.

FSIS issued the Pathogen Reduction / Hazard Analysis and Critical
Control Points (PR 1 HACCP) rule on July 25, 1996 (Federal register, 1996) to
enhance the safety of meat and poultry products. According to this rule, all
federally regulated livestock, poultry slaughter and processing facilities were
required to develop HACCP programs. These facilities were also required to
meet pathogen reduction performance standards for Salmonella (fixed at 20%
according to baseline data collected by the USDA) and initiate microbial testing
programs to ensure compliance with the targets. FSIS is also testing for

Salmonella on raw meat and poultry products to determine whether these

performance standards are being met. FSIS reported a substantial decline in the
incidence of Salmonella from the baseline performance standards from 1998 to
2000 after the implementation of HACCP programs (Rose et al., 2002). Only
10.8% of broiler carcasses tested at the processing plants were positive for
Salmonella with this reduction attributed to the implementation of HACCP (FSIS-
USDA, 2002). Along with this reduction, CDC reports a 15% decline in the
incidence rate of Salmonella infections in humans during 1996-2001 (MMWR,
2002). Despite all these reports of reduction, Salmonella still remains the leading
bacterial foodbome pathogen with an incidence rate of 13.8 cases/100,000
people (MMWR, 2002). Most recently, the national health objectives target for
Salmonella was fixed at 6.8 cases per 100,000 persons and it is expected to‘be
achieved by the year 2010 (MMWR, 2002). Since the primary source of
Salmonella contamination in poultry processing environments is the
gastrointestinal tracts of incoming birds (Bailey, 1993), efforts aimed at reducing
Salmonella colonization within the intestinal tracts should prove very rewarding

for the poultry industry.

2.2. FOODBORNE BACTERIAL PATHOGENS IN POULTRY

The most important bacterial pathogens associated with poultry and
poultry products include Salmonella, Campy/obacter and Listeria monocytogenes
with the first two organisms being the two primary causes of bacterial foodborne
gastroenteritis (Todd, 1980; Archer and Kvenberg, 1985; Bryan and Doyle, 1995;

Zhao et al., 2001). Of the 76 million estimated cases of foodbome illnesses,

nearly 2.4 million cases are caused by Campylobacter species with an additional
1.4 million cases caused by non-typhoidal Salmonella (Mead et al., 1999).
Salmonella usually causes foodbome outbreaks involving large numbers of
people. Campylobacter, on the other hand causes more numbers of outbreaks
with smaller numbers of people involved in each outbreak.

Regular production of poultry meat became possible due to the
development of fast-growing broiler chickens, selective breeding programs,
vaccines and antibiotics to control diseases, improved nutrition, and automation
and integration of different production units in the poultry industry. The demand
for chicken products, as well as per capita consumption of poultry meat, has
risen dramatically with per capita consumption having increased from 35 lbs in
1970 to 70 lbs in 1997 (USDA, 1998).

With increased consumption of poultry, poultry-associated foodbome
outbreaks have also increased. CDC reported that foodbome outbreaks due to
contaminated chicken and turkey accounted for 1.5%, 1.9% and 2.4% of all
foodbome outbreaks in the United States during 1995, 1996 and 1997,
respectively (CDC, 2000), with Salmonella and Campylobacter being the most
important bacterial pathogens associated with poultry and poultry products

worldwide (Bryan and Doyle, 1995).

2.3. SALMONELLOSIS
Salmonellosis is of primary concern in the United States and can be

acquired by ingesting undercooked or recontaminated poultry or by cross-

contamination of other foods through improper handling of raw poultry (Bryan
and Doyle, 1995). It is estimated that the presence of Salmonella in poultry
products results in an estimated 409,624 cases of human illnesses, 160 deaths
and associated costs of $ 6.96 million, all of which are extremely detrimental to
the poultry industry (Mead et al., 1999; CSPI, 2001).

Salmonella, first recognized as a foodbome pathogen in the 1880’s
(Marth, 1969), has now emerged as a major cause of foodbome gastroenteritis
worldwide. Salmonellosis can be caused by any one of over 2000 serovars of
Salmonella. The serovars involved in salmonellosis can vary geographically but
most frequently include S. Enteritidis, S. Typhimurium, S. Agona, S. NeWport, S.
Heidelberg, S. lnfantis, S. Saintpaul, S. Hadar and S. Weltevreden. While some
of these serovars maintain their dominant role in foodbome infections, others
emerge and decrease over time (Uyttendaele et al., 1998).

Salmonella, one of the most frequently reported foodbome pathogens in
the United States and other developed countries, is a common contaminant of
fresh poultry (Bean and Griffin, 1990; FSIS-USDA, 2002). Active surveillance for
salmonellosis started in 1955 with annual reports of laboratory isolations from
humans in the United States continuing to increase as a result of FoodNet.
However, most cases of salmonellosis are not reported due to mild symptoms
that often go unnoticed.

Typical clinical syndromes produced by non-typhoid Salmonella include
gastroenteritis, septicaemia and focal infections of the eye, joints, lungs, kidneys,

heart and brain that may follow untreated or prolonged septicaemia (D’Aoust,

1994). Gastroenteritis is characterized by abdominal pain, diarrhea and fever
and chills with nausea. Vomiting and headache occur less frequently. The onset
time for salmonellosis usually ranges from 7 to 72 hours with clinical signs most
often appearing 12 to 36 h after exposure. The duration of diarrhea is usually
three to five days but can persist for up to two weeks. In about half of all cases,
the organism will be shed for two to four weeks with fecal shedding normally
ceasing after two to three months. Salmonella infections can also lead to
secondary complications such as reactive arthritis or Reiter’s syndrome - a long-
term chronic illness characterized by joint pain, eye irritation, and painful
urination. The most severe infections typically occur in infants, the elderly and
immunocompromised individuals. Salmonellosis has been steadily increasing-as
a public health problem over the last 40 years in the United States. Reports
estimate the number of all Salmonella food borne illness cases as 1.4 million
annually with 553 deaths and associated costs of $2.4 billion (ERS, USDA,
2002)

At least 25 poultry-related outbreaks of salmonellosis have been
documented since 1982 (Table 1). One such outbreak associated with ,
consumption of poultry giblets occurred at a Maine restaurant (MMWR, 1984).
One hundred and twelve culture-confirmed cases involving Salmonella enten'tidis
serotype Enteritidis were identified after diners consumed giblet gravy prepared

from refrigerated uncooked giblets over the Thanksgiving weekend.

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A similar outbreak traced to liver pate occurred in Maine in October of 1983 at
another restaurant belonging to the same restaurant chain as the previous
outbreak (MMWR, 1984). The implicated liver pate was prepared from frozen
chicken livers that had been defrosted over a period of 4 days in a refrigerator
before use. Positive cultures from seven individuals who dined at the restaurant
confirmed the causative agent as Salmonella Heidelberg.

In 1985, a turkey-associated salmonellosis outbreak was identified among
children and staff at a Georgia elementary school (MMWR, 1985). From May 10
to May 16, an estimated 351 children and staff at the school developed febrile
gastroenteritis with 23 children being hospitalized. Salmonella Enteritidis was
isolated from more than 100 children. Based on a case-control study, illness Was
associated with consumption of an ovemight-refrigerated turkey salad during
school lunch.

Eggs are considered to be an important source of Salmonella. According
to the CSPI eggs on an average contribute to 21 % of foodbome illness
outbreaks annually (CSPI, 2001). Salmonella Enteritidis is an important
pathogen of the layer industry due to its ability to infect hens and ultimately
contaminate egg contents (Seo et al., 2000). An outbreak traced to consumption
of a nationally distributed ice cream product (Schwan’s ice cream) occurred in
Minnesota during 1994 (MMWR, 1994). A total of 80 confirmed cases involving
Salmonella Enteritidis were reported to the Minnesota Department of Health from
September 19 to October 10 with 73 % of case-patients reporting consumption of

Schwan's ice cream within 5 days of onset of illness. Cases were characterized

13

by diarrhea, abdominal cramps and fever. The company voluntarily stopped
distribution and production at its plant in Marshall, MN following investigation. 8.
Enteritidis was isolated from samples of ice cream from households of ill
persons.

In June 1995, another outbreak associated with consumption of baked
eggs occurred in an Indiana nursing home (MMWR, 1996). Seventy individuals,
including the residents and staff, were infected with three residents dying from
complications. Thirty-nine cases of Salmonella Enteritidis associated with the
consumption of baked eggs were confirmed by stool cultures.

During November of 1995, another outbreak was traced to a Thanksgiving
dinner at a private home in Nevada (MMWR, 1996). All seven individuals Who
consumed turkey and stuffing developed abdominal cramps, vomiting and
diarrhea. Two people were hospitalized and a third person died. Stool cultures
from these three individuals yielded Salmonella Enteritidis phage type 13a.

During April and May of 1995, the Idaho Department of Health and
Welfare and the Washington Department of Health identified three and nine
isolates of Salmonella Montevideo, respectively. In April and May of 1996, a total
of 11 isolates of S. Montevideo were reported in Washington. A case was
defined as a culture-confirmed S. Montevideo infection in an Idaho or
Washington resident with onset of illness during April and May of 1995 or 1996.
A total of 23 cases were identified. Handling of chicks was implicated as a health
risk, especially for children, as most case-patients were children aged two years

or less. An isolate from a 14-month-old child was cultured from blood indicating

14

invasive disease. No common hatchery or sources of feed were identified. But
S. Montevideo was isolated from fecal samples obtained from two chicks handled
by geographically separated patients.

From March to June 1996, the Oregon State Public Health Laboratory also
identified 16 cases of S. Montevideo (MMWR, 1997). A case-control study
involving 11 of the 16 cases revealed previous handling of live poultry (chicks,
hens, or roosters). S. Montevideo was isolated from two chicks obtained from
one of the case-patients.

A similar outbreak of salmonellosis associated with handling live chicks
and ducklings was reported in 1999 (MMWR, 2000). In April, the Missouri
Department of Health discovered a cluster of Salmonella serotype Typhimurium
infections with identical PFGE (Pulsed Field Gel Electrophoresis) patterns. A
total of 40 cases were identified with symptoms of fever, bloody diarrhea,
stomach cramps and vomiting. The Michigan Department of Community Health
was notified of an increase in Salmonella serotype lnfantis infections in May of
1999. From April to July of 1999, a total of 21 cases were reported with these
victims also exhibiting diarrhea, fever and vomiting. The young chicks and
ducklings associated with the outbreak (88%) were traced to a hatchery. S.
lnfantis was recovered from environmental samples and from birds of the
hatchery, which exhibited the same PFGE pattern as the patient isolates from the
outbreak.

In the poultry industry, Salmonella infections have a major impact on

productivity of poultry through mortality, reduced feed efficiency and reduced

15

weight gain. Salmonella in poultry products alone causes 409,624 cases of
foodborne salmonellosis, resulting in an economic loss of $ 6.96 million along
with heavy losses to the industry through plant clean-ups and product recalls

(Table 2) in cases of outbreaks (Mead et al., 1999; CSPI, 2001).

2.4. SALMONELLA IN POULTRY

Salmonellae in poultry are a major source of human infection (Bryan,
1980). This group of bacteria is ubiquitous and has been isolated from various
points during poultry production including the breeder flock, newly-hatched
chicks, grower birds, feed/feed ingredients, water, processing equipment and the
finished raw product (Lahellec et al., 1986; Todd, 1980). Poultry usually achire
salmonellae from the feed and feed ingredients, water, litter and the environment.
After the birds are infected, Salmonella is conveyed to the processing plant
through fecal material on their feet, skin and feathers or through the contents in
their gastrointestinal tracts with the carcasses thereby becoming contaminated.
Numerous studies have shown that broilers entering processing plants are highly
contaminated with salmonellae being firmly attached to poultry skin (Kotula and
Pandya, 1995; Lillard, 1989‘; Lillard, 1989b). Broilers are widely recognized
reservoirs for Salmonella infections in humans due to the ability of the organism
to colonize and proliferate in the gastrointestinal tract of the birds and
subsequently survive on the caris during processing (Todd, 1980; Cason et

al.1997)

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Colonization of chickens with Salmonella depends on many factors such
as the age of the bird, survival of the organism through the gastric barrier,
competing bacteria in the intestinal tract and the effect of antimicrobial feed
additives. Milner and Shaffer (1952) were first to report that newly-hatched
chicks lack a complex gastrointestinal flora and hence are prone to colonization
by enteric bacteria. They also showed that day-old chicks could be infected by
ingesting fewer than 5 cells of Salmonella with older birds gradually becoming
more resistant to infection. Thus, resistance to colonization increases with age
as the normal gut flora becomes established.

Hatchenes frequently serve as reservoirs for Salmonella with
contamination from the hatchery reportedly an important source of Salmonella in
newly-hatched out chicks, which are most susceptible .to intestinal colonization.
A single Salmonella-contaminated egg can reportedly contaminate other eggs
and chicks in the hatching cabinet (Bailey et al., 1994). Another important source
of Salmonella is the feed. Erwin (1955) reportedly recovered viable salmonellae
from commercial poultry feed and since then the role of poultry feed and feed
ingredients in dissemination of Salmonella has received widespread attention.
Jones et al (1991) and MacKenzie and Bains (1976) also isolated Salmonella
from feed and feed ingredients. Morris et al (1969) showed the presence of
Salmonella in both feed samples and breeder flocks. During grow-out, the farm
environment in which broilers are raised is another primary source of

contamination (Blankenship et al., 1993) with the importance of flies in the

18

spread of Salmonella also having been documented by Olsen and Hammack
(2000) and Bailey et al., (2001).

The contamination rate of broiler carcasses in processing plants and retail
outlets also has been studied worldwide with 5% to 100% of such carcasses
harboring salmonellae (Todd, 1980; Jemgklinchan et al., 1994; Plummer at al.,
1995; Rusul et al., 1996; Carraminana et al., 1997; Uyttendaele et al., 1999;
Sackey et al., 2001; Beli et al., 2001; Dominguez et al., 2002). Fecal matter
often found on the breasts of caged birds during transportation is considered to
be an important source of Salmonella in processing plants with broiler carcasses
also being cross-contaminated with salmonellae and other bacteria during
processing (Lillard, 1990). Cross-contamination of the carcass may occur at
many points of contact during poultry processing (Figure 1), particularly during
immersion, scalding and chilling. Thus, since the spread of Salmonella through
integrated broiler operations is complex, multiple control measures need to be
implemented at different processing points to control the entry and spread of

Salmonella into broiler operations.

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20

2.5. SALMONELLA ENTERICA SEROVAR INFANTIS

Salmonella enterica serovar lnfantis, hereafter S.lnfantis, is one of the
more commonly isolated serovars of Salmonella from humans and is an
important pathogen in the broiler industry. Salmonella lnfantis belongs to the
Paratyphoid group of Salmonella, which includes salmonellae that frequently
infect or colonize the gut of many warm- and cold-blooded animals including
humans. Wheeler and Borman (1943) were first to isolate S.lnfantis from a four-
month-old girl in the United States. Levy et al., (1975) described an outbreak of
salmonellosis in Middleton, MN in which S.lnfantis was one of three serovars
identified. In this outbreak 125 of 173 (72.25%) individuals who attended a picnic
and/or smorgasbord prepared by a bar/restaurant developed symptoms of
diarrhea, abdominal cramps, chills, fever, nausea, vomiting and headache.
Potato salad and chicken dressing were implicated as vehicles for the outbreak.
The economic impact of this 1973 outbreak estimated as $ 28,733.

S.lnfantis was the cause of another outbreak in Finland involving railway
and airline passengers in 1986 (Hattaka, 1992). This outbreak was traced to an
employee who was an asymptomatic carrier of S.lnfantis who had contaminated
breakfast I egg sandwiches during preparation.

Kohl and Farley (2000) described a 1998 outbreak of salmonellosis that
occurred among 63 wedding participants. A commercially cooked rice-dressing
mix product contaminated with S.lnfantis was identified as the source of infection.
The rice-dressing mix contained pork, pork liver and chicken gizzards in

combination with spices and other flavorings. Investigations showed that the

21

product had likely become contaminated at the plant through contact with a pump
in the production line. Consequently, on September 22, 1998, the USDA issued
a Class I recall for all products produced before September 15, 1998.

S.lnfantis is frequently isolated from chicken carcasses and derived
products (Lammerding et al., 1988) and has also been recovered from insects in
poultry houses (Jones et al., 1991). According to McBride et al., (1980),
S.lnfantis was the predominant serovar isolated from one poultry processing
plant. They observed that Salmonella contamination was most common before
scalding compared to post-evisceration and post-chilling sites. More recently,
Uyttendaele et al., (1998) reported an isolation rate of 6.6% for S.lnfantis in
chicken carcasses and derived products during a four-year study of Salmonella
in poultry carcasses and their products sold in Belgium. Bailey et al. (2001) have
also recovered S.lnfantis from different points in the production chain including
the carcass rinse, transport coop, paper pads used in transportation trays, litter,

fly strips, and caecal droppings.

2.6. CONTROL OF SALMONELLA IN POULTRY

Salmonellae are ubiquitous bacteria with broilers representing a highly
important reservoir of salmonellae in the human diet. The problem of human
salmonellosis cannot be overcome until Salmonella infections in birds are under
control. In an integrated poultry operation, control of Salmonella is often very
complicated as there are many potential sources of contamination such as the

birds, feed, water, rodents, flies, insects, farm, transportation, and the processing

22

plant environment. Much research has been conducted to eliminate salmonellae
from the various steps in poultry production and processing.

Salmonellae colonize the intestinal tract of chickens with the crop and the
caecum being the main sites of colonization (Hinton et al., 1990; Soerjadi et al.,
1981). Minimizing cross-contamination of the dressed carcass with intestinal
contents of birds during processing helps improve the microbiological quality of
poultry meat (Bailey, 1993). Therefore, reduction of Salmonella in broilers during
the grow-out period is a very important issue. Commonly used control measures
in broiler production include addition of antimicrobials at sub-therapeutic levels to
poultry feed and the use of competitive exclusion products. However, other
control measures such as vaccination, feeding of probiotics, feeding ‘ of
mannanoligosaccharides, and addition of buffered propionic acid also have been
investigated (Izat et al., 1990; Methner et al., 1999; Spring et al., 2000; Tellez et

aL,2001)

2.6.1. PRE-HARVEST CONTROL MEASURES

Salmonella control is very difficult in integrated poultry operations as there
are many sources of contamination such as feed, water, hatchery, farm
environment and processing environment. The gastrointestinal tract of poultry is
one of the primary sources of contamination during processing (Bailey, 1993). To
reduce the contamination of Salmonella on processed carcasses, Salmonella-

free birds must be delivered to the processing plants. Salmonella in the intestinal

23

tract of birds can be reduced or eliminated by pre-harvest control measures,

some of which are described below.

2.6.1.1. POULTRY FEED ADDITIVES

The poultry industry employs different preventative measures to
control economically important diseases such as coccidiosis and different
bacterial infections that negatively impact the performance birds. Addition of
antimicrobials and anticoccidiostats is frequently used as a preventative
measure. As their full potential for disease control was discovered, the demand
for antibiotic feed supplements increased. Supplementation of feed with
antibiotics may occur for any of the following purposes (1) to improve the grthh
rate / weight gain of animals (2) disease prophylaxis and (3) disease treatment.
Antibiotics mixed at low levels in poultry, cattle, swine and sheep rations
reportedly cause increased weight gain and a decrease in shedding rates of
certain pathogenic organisms including Clostn'dium and Salmonella (Engberg et
al., 2000; Bolder et al., 1999).

In broiler production, asymptomatic Salmonella infections are an important
problem, which cannot be solved completely by strict hygienic practices or by the
use of antimicrobials in feed. Antimicrobials added to feed have been used both
in the treatment and prevention of Salmonella infections in chickens. In one
study, addition of chlortetracycline at sub-therapeutic levels to feed reduced
mortality in chicks infected with S.Typhimurium (Quarles et al., 1977). However,

while Nivas et al. (1976) showed that chlortetracycline decreased fecal shedding

24

of S.Typhimurium in turkey poults, higher isolation rates were obtained from the
caecal junction and liver. In other work, \Mlliams (1985) showed a lower
incidence of salmonellosis in chickens fed a combination of neomycin and
oxytetracycline. However, antimicrobials that were effective against S.lnfantis in
vitro were ineffective in treating in vivo intestinal infections (Seuna and Nurmi.,
1979). Virginiamycin, another antibiotic effective against primarily gram-positive
organisms, had no effect on shedding and antibiotic resistance patterns of
S.Typhimurium when added to poultry feed (Abou-Youssef et al., 1982).

Over the last five years, the practice of supplementing feed with sub-
therapeutic levels of antibiotics has become highly controversial, largely due to
the rapid emergence in multi-antibiotic resistant serovars of Salmonella and other
foodbome pathogens. Antimicrobials tend to destroy the normal bacterial flora of
the intestine and can thereby lead to enhanced colonization by opportunistic
pathogens (Rantala, 1974). Seuna et al., (1980) reported the reappearance of
salmonellae in poultry when treatment with antibiotics such as neomycin,
oxytetracycline, polymyxin B, trimethoprim, and sulfadiazine alone or in various
combinations was stopped. They also observed more infected chickens when
the antibiotic treatment was stopped. While ionophore antibiotics are widely
used as poultry feed additives to control coccidiosis, they are also considered to
have growth promoting properties (Elwinger et al., 1998). According to Engberg
et al. (2000), supplementation of feed with ionophore antibiotics such as zinc
bacitracin and salinomycin, either alone or in combination, significantly reduced

natural populations of Clostn'dium perfn'ngens and Lactobacillus salivan'us in the

25

intestines of broilers. It was suggested that the mode of action for these feed
additives is mainly related to either the inhibition of certain toxin-producing
intestinal bacteria or those bacteria that compete with the host for easily available
nutrients (Coates et al., 1963). Monensin, one of the most commonly used
ionophore antibiotics in poultry production, is mainly used as a coccidiostat.
Hence, not much research has been conducted on the effects of monensin on
Salmonella infections in poultry. Studies done by Holmberg et al (1984) showed
that chickens experimentally infected with S.lnfantis had higher caecal counts
when fed diets containing avoparcin and monensin. Although sub-therapeutic
use of broad spectrum antibiotics can decrease shedding of Salmonella, the

effects of coccidiostatic antibiotics such as monensin have yet to be studied.

2.6.1.2. ANTIBIOTIC RESISTANCE AND SALMONELLA

The widespread occurrence of Salmonella species in the natural
environment coupled with intense husbandry practices applied to the broiler
industry have increased the prevalence of salmonellae in the food chain to the
point where Salmonella is one of the leading causes of bacterial foodbome
illness (D’Aoust 1989). The administration of sub-therapeutic doses of antibiotics
as feed additives to domestic livestock is practiced worldwide. Such doses of
antibiotics are lower than those required for treating infections in farm animals
but are sufficiently high to suppress the growth of normal intestinal microflora.
This agricultural practice leads to the development of selective pressures which

in turn can lead to the emergence and spread of resistant salmonellae in

26

products for human consumption (Holmberg, 1987; Dupont and Steele, 1987;
Threlfall et al., 2000; Usera et al., 2002). Poultry is often found to be a major
reservoir of resistant salmonellae (Rajashekara et al., 2000). Foods harboring
resistant strains present an increased level of risk to humans as they can cause
systemic infections that often result in serious complications.

Emergence of multi-antibiotic resistant bacteria has become an
increasing problem worldwide with infections from such organisms becoming
increasingly difficult to treat. The widespread prophylactic use of antibiotics in
human and veterinary medicine, combined with sub-therapeutic use in animal
feed has led to the emergence of antibiotic resistant strains of Salmonella. Many
strains isolated from poultry, especially those fed with antibiotics, exhibit multiple
resistances to antibiotics such as ampicillin, chloramphenicol, streptomycin,
sulfonamides and tetracycline. These strains can then progress through the food
chain to humans via poultry and poultry products (Arvanitidou et al., 1998).

In 1981, approximately 2% of the normal “wild type” bacterial population
was resistant to any given antibiotic (Novick et al., 1981). However, when
bacterial populations from animals are regularly exposed to antibiotics, up to 10%
of the population can acquire resistance. These resistant bacteria are often shed
in feces, where they share extrachromosomal antibiotic resistance plasmids (r-
plasmids) or transposons with native bacteria and may be spread to other
animals. In addition, antibiotics that accumulate in animal tissues can lead to
further resistance in humans when the product is ingested (Siegel at al., 1974).

Genes coding for resistance to several antibiotics can be lomted within the same

27

transposon or plasmid. Aarestrup et al. (2001) showed that the use of a single
antibiotic as a treatment also could select for resistance to other antibiotics when
the genes are located in the same mobile genetic element (“linked resistance”).

The emergence of antimicrobial-resistant salmonellae is a problem not
solely confined to the United States, where it has been increasing. In 1979, 16%
of the Salmonella isolates were found to be resistant to at least one antimicrobial
agent, whereas in 1989 and 1990, 29% and 37% of all Salmonella isolates were
resistant to at least one antimicrobial agent (Riley et al., 1984; MacDonald et al.,
1987; Lee et al., 1994). The United States experienced its first outbreak of
multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage
type 104 (Typhimurium DT 104) in 1996 (MMWR April 11, 1997). This outbreak
occurred in Nebraska during October 1996 among school children.
Investigations identified the cause as chocolate milk poured from cartons during
lunch at school. Stool cultures from affected children yielded S.Typhimurium R-
type ACSSuT and phage typing confirmed all isolates as DT 104.

Threlfall et al. (1994) reported that the DT 104 isolates from the United
Kingdom contained chromosomally integrated genes conferring resistance to
ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R-type
ACSSuT). Multidrug resistance is associated with the presence of two integrons
found in the chromosomal DNA of DT 104 R-type ACSSuT isolates (Threlfall et
al., 1994; Sandvang et al., 1998; Briggs et al., 1999). Additional resistance to
trimethoprim and ciprofloxacin also has been reported in some DT 104 strains

(Threlfall et al., 1997). Though primarily a cattle pathogen, multidrug resistant

28

strains of DT 104 have been recovered from other forms of domestic livestock
including swine and poultry (Besser et al., 1997; Rajashekara et al., 2000).
Infected animals and consumption of contaminated chicken, pork sausage and
meat paste have been identified as potential sources of DT 104 infection in

England and Wales (Wall et al., 1994).

2.6.1.3. COMPETITIVE EXCLUSION

Competitive exclusion (CE), a widely used method for reducing
fecal shedding of Salmonella in poultry, is a technique whereby newly hatched
chicks (day-old) are exposed to the gastrointestinal flora of adult birds.
Colonization of the gastrointestinal tract with normal gut flora from healthy birds
discourages colonization by salmonellae. The benefits derived from competitive
exclusion may be due to either the production of short-chain volatile fatty acids in
the caecum (Bailey et al., 1988; Corrier et al., 1990; Nisbet et al., 1996) or
competition for Salmonella attachment sites on the intestinal mucosa (Soerjadi et
al., 1981a; Hinton et al., 1990; Nisbet et al., 1993). Several researchers including
Blanchfield et al., (1982) and Stavric et al., (1985) further assessed the
effectiveness and practical applications of competitive exclusion treatments.
Blankenship et al., (1993) and Bailey et al., (2000) found that a two-step
treatment of broiler chicks with a mucosal competitive exclusion culture (MCE)
containing intestinal mucosal scrapings of pathogen-free adult broiler chicks was
effective in reducing Salmonella colonization. Treatment consisted of spraying

the chicks in the hatchery with the MCE culture after which the culture was

29

ingested in the drinking water. Competitive exclusion products alone or in
combination with fructooligosaccharide (Fukata et al., 1999) or vaccination
(Methner et al., 1999) to reduce bacterial colonization in poultry particularly of
salmonellae were also studied. Feeding low doses of fructooligosaccharide in
combination with CE treatment decreased Salmonella colonization in chicks.
Although the competitive exclusion treatment effectively reduced Salmonella,
other challenge studies have discounted the use of commercially available CE

cultures to protect against infection by Salmonella (Stavric et al., 1992).

2.6.1.4. SODIUM CHLORATE

The need to develop more potent antimicrobial agents against
Salmonella has increased in recent years due to the increasing number of
foodbome outbreaks and the continuing emergence of multi-antibiotic resistant
strains. One such alternative approach that is gaining much attention is the
feeding of sodium chlorate to domestic livestock to reduce intestinal colonization
by Salmonella and Escherichia coli O157:H7.

In the gastrointestinal tract of animals, nitrate can be reduced to nitrite
anaerobically by some bacteria that possess the intracellular enzyme nitrate
reductase (Alaboudi, 1982). As is true for most members of the Family
Enterobacten’aceae, salmonellae possess respiratory nitrate reductase activity.
This enzyme can catalyze the intracellular reduction of chlorate to cytotoxic
chlorite which is lethal to those bacteria possessing respiratory nitrate reductase

activity (Stewart, 1988). In contrast, much of the normal gastrointestinal flora

3O

does not possess respiratory nitrate reductase and is therefore not affected. In
vivo studies have shown that sodium chlorate is bactericidal to E.coli 0157: H7
and Salmonella Typhimurium DT 104 with the beneficial gut microflora remaining
unaffected (Anderson et al., 2000). Caecal populations of Salmonella were
significantly reduced in weaned pigs treated orally with a sodium chlorate
solution (Anderson et al., 2001). Similarly oral administration of sodium chlorate
was found to reduce gut concentrations of E. coli 0157:H7 in experimentally
infected pigs and wild-type E. coli concentrations in uninoculated pigs (Anderson

etal,2001)

2.6.1.5. OTHER PRE-HARVEST CONTROL MEASURES

Feeding of complex sugars such as mannose or lactose to birds
also can inhibit intestinal colonization by salmonellae. Supplementation of feed
with mannanoligosaccharide reportedly decreased S.Typhimurium populations
about 25-fold in chicks (Spring et al., 2000). Consumption of lactose-
supplemented feed throughout the growing period of broilers also significantly
reduced the number of salmonellae in the caeca (Corrier et al., 1990).

Another control measure gaining much attention is the feeding of
probiotics. Avian Lactobacillus species were shown to prevent S.Typhimurium
from adhering to the crop mucosa and also reduce caecal colonization (Fuller,
1977; Soerjadi et al., 1981”). Studies by Tellez et al., (2001) demonstrated that
the treatment of chicks with the avian-specific probiotic Avian Pac Plus®

significantly decreased caecal colonization and organ invasion by S.Enteritidis.

31

Feeding of a buffered propionic acid product (53.5% propionic acid, 9.5%
ammonium hydroxide, 11.5% 1, 2-propanediol, and 25.5% water) in drinking
water to broiler chickens also significantly reduced the number of salmonellae on
post chill carcasses ( lzat et al., 1989; lzat et al., 1990). Another study by Byrd et
al., (2001) showed that the use of 0.5% lactic acid in drinking water during pre-
transport feed withdrawal time (8hrs) caused a significant reduction in the
incidence of Salmonella in the crop and thereby a reduction in the pre-chill

carcass rinse.

2.6.2. POST HARVEST CONTROL MEASURES

Decontaminating the final raw product is one of the most effective
approaches in reducing poultry-home salmonellosis. The ideal method of
decontamination should not change appearance, smell, taste or nutritional
properties; should not leave residues and should be environment-friendly as well
as cheap and easy to use (Corry et al., 1995). Some of the decontamination

techniques used in poultry processing plants are described below.

2.6.2.1 . ACIDS:

The undissociated forms of lactic, acetic, and other acids are widely
recognized for their antimicrobial activity (Mountney and O’Halley 1965; Stem et
al., 1985; Colberg and lzat 1988). Lactic acid was shown to extend the shelf-life
of processed broilers; however, concentrations of organic acids that effectively

decontaminated poultry carcasses generally caused unfavorable sensory

32

changes (lzat et al., 1989). Addition of 1% acetic acid to poultry scald water
caused instantaneous bacterial death and reduced cross contamination (Okrend
et al., 1986). The effect of acids in combination with other compounds was also
studied. Washing chicken wings with a solution containing 0.5% lactic acid and
0.05% sodium benzoate was found to effectively reduce the number of
Salmonella and thus enhance product safety and shelf-life (Hwang and Beuchat,

1 995).

2.6.2.2. TRISODIUM PHOSPHATE:

Trisodium phosphate is an antimicrobial compound approved for
use in poultry processing facilities by the USDA-FSIS. When used at a.
concentration of 8%, this compound had a pH>12 and was active against gram-
negative bacteria on the skin of chickens (Corry et al., 1995). One such product
marketed as “Avgard®” is used as a dip immediately after water-chilling or before
air-chilling (Corry et al., 1995). Whyte et al. (2001) also reported significant

microbial reductions on broiler carcasses using trisodium phosphate.

2.6.2.3. CHLORINE

Another antimicrobial agent commonly used in poultry processing is
chlorine. Used for chilling carcass, chlorine is converted to its active form
hypochlorous acid (pH 6.5 — 8.0) in water, which destroys pathogenic organisms

on poultry carcasses. Addition of chlorine to poultry chill water was shown to

33

reduce cross-contamination by Salmonella in chill tanks (James et al., 1992;

Morrison et al., 1985).

2.6.2.4. CHLORINE DIOXIDE

Another form of chlorine known as chlorine dioxide that has been
approved by the FDA in 1995 (USDA, 1995) is being used by many poultry
processors as a disinfectant in chiller water. Chlorine dioxide, a gas produced as
a result of the reaction between hydrochloric acid and sodium chlorite can be
incorporated in de-ionized water. Chlorine dioxide acts on the cell membrane of
bacteria resulting in a loss of membrane permeability and thus producing non-
specific oxidative damage to the cell membrane (Berg et al., 1986). Chlorine-
dioxide was found to reduce microbial populations in poultry chiller water (Tsai et
al., 1992; Lillard, 1979). It has also been shown to destroy a wide variety of
microorganisms including spores resistant to chlorine treatment (Richardson et
al., 1994). Other advantages of this compound include its effectiveness over a
wide range of pH, and in the presence of high levels of organic matter and slow

dissociation in water compared to chlorine (White, 1972).

2.6.2.5. OZONE:

Ozone is prepared by passing gaseous oxygen or dry air through a
high voltage electrical field, which is then dissolved in water. The ozonated water
attacks the bacterial membrane and certain amino acids, disrupting the normal

cellular activity and killing the microorganism. Ozone has been tested for

34

disinfecting poultry chiller water, poultry carcasses (Sheldon and Brown, 1986),
and eggs, as well as the hatchery and hatching eggs (Whistler and Sheldon,
1989). When carcasses were chilled in water containing ozone at 3.0 to 4.5 ppm
for 45 minutes, Sheldon and Brown (1986) reported that microbial counts
decreased by more than 2 logs. Yang and Chen (1979) also reported similar

results using ozone at a concentration of 37.7 mg / liter.

2.6.2.6. ULTRAVIOLET RADIATION:

In the food industry, UV light is commonly used to disinfect
packaging surfaces. Sumner et al. (1995) reported that UV radiation (2000 uW
sec cm'2) effectively destroyed S.Typhimurium on agar plates and poultry skin;-
but the bacterial reduction on poultry skin was less effective. Yndestad et al.
(1972) also found that UV radiation (10,000 uW sec cm‘z) decreased the
superficial microflora on poultry carcasses but did not have any prolonged effect

on shelf-life.

2.6.2.7. ELECTRICITY:

Effects of electrical stimulation in reducing the number of S.Typhimurium
attached to chicken legs were studied by Slavik et al. (1991). Their results
indicated that electrical stimulation killed bacteria in solution and reduced the
number of salmonellae attached to chicken legs when the legs were attached to
anodes. However, slight damage to the meat was reported when the chicken

legs were connected to the electrodes.

35

2.6.2.8. IRRADIATION:

This method has been approved by more than 40 countries and also by
the FDA in the United States to reduce the incidence of harmful pathogens on
meat and poultry products. The three types of irradiation currently permitted to
be used on raw meat and poultry include gamma irradiation, electron beams, and
X-ray (CFR, Title 21, vol 3, part 179). Gamma irradiation at a dose of 3.0 KGy
inactivated 99.9% of common foodbome pathogens (Frenzen et al., 2001).
However in the United States, irradiation of poultry meat was accepted by only
49.8% of the population. If not applied carefully at the correct temperature and
dose, irradiation can lead to undesirable textures, off-flavors and inadequate
inactivation of pathogens (Mucklow and Cross, 2002). This method is currently.

being test marketed with ground beef products.

2.6.2.9. OTHER POST-HARVEST CONTROL MEASURES:

The antibacterial effect of the Iactoperoxidase (LP) system on
growth and survival of S.Typhimurium on poultry was studied by Wolfson et al.,
(1994). They found that an LP system treatment consisting of Iactoperoxidase
(mg/ml); potassium thiocyanate (5.9 mM) and hydrogen peroxide (2.5 mM) in
water reduced levels of Salmonella on inoculated chicken legs. They reported a
reduction of 80.6% when treated for 15 min at 60°C and 13.6% when treated for

30 min at 25°C.

36

EFFECTS OF MONENSIN AND SODIUM CHLORATE ON SALMONELLA
INFANTIS COLONIZATION IN BROILERS
BY
Geetha S.Kumar‘, Elliot Ryserz, Richard M.Fulton3, Sam K.Varghese', and

Steve Bursian1

Department of Animal Science1
Department Of Food Science & Human Nutrition2
College of Agriculture & Natural Resources
Pathobiology & Diagnostic Investigation3
College of Veterinary Medicine

Michigan State University

37

CHAPTER 3
EFFECTS OF MONENSIN AND SODIUM CHLORATE ON SALMONELLA

INFANTIS COLONIZATION IN BROILERS

3.1. ABSTRACT:

Intestinal colonization and fecal shedding of Salmonella in broilers has
been an on-going food safety concern. Any reduction of Salmonella colonization
in the gastrointestinal tract of broilers will ultimately lead to a decrease in carcass
contamination during processing and thus yield a safer product. Two studies
were conducted to determine the effects of monensin and sodium chlorate on
Salmonella lnfantis colonization in broilers. In the first study, 210 Salmonella--
free birds were fed either an antimicrobial-free control diet or a treatment diet
containing monensin. All chicks were orally infected on day 3 with 1 X 108 CFU
of S. lnfantis. When caecal colonization by S. lnfantis was evaluated on days 7,
10, 14, 21, 28, 35, and 42, monensin failed to reduce S. lnfantis colonization. In
the second study, 150 Salmonella-free broilers were divided into 4 groups (3
treatment groups and a control group) and similarly infected on day 3. The three
treatment groups received a commercial sodium chlorate solution ad libitum, on
day 10, 20, or 38 for four consecutive days followed by drinking water whereas
the control group received only drinking water. Caecal colonization and liver
invasion by S. lnfantis in all groups were assessed on days 7, 10, 14, 17, 20, 24,
27, 30, 38, and 42 with fecal swab samples also analyzed on days 11, 12, 13, 15,
16, 18, 19, 21, 22, 23, 25, 26, 28, 29, 39, 40, and 41. Birds receiving sodium

38

chlorate on days 10 and 20 had significantly lower caecal populations of S.
lnfantis four days after treatment (P<0.02 and P<0.0001, respectively) compared
to the untreated control group with these lower populations persisting up to seven
days after treatment. Fecal shedding of S. lnfantis also decreased >1 and >2
logs three days after sodium chlorate treatment was begun on days 10 and 20,
respectively, with no effect observed in birds treated on day 38. While. no
difference in liver invasion by S. lnfantis was seen, lower caecal populations as
well as reduced fecal shedding of S. lnfantis indicates that sodium chlorate may
be potentially useful for decreasing the incidence of Salmonella in broilers

entering poultry processing facilities.

3.2. INTRODUCTION:

Each year the United States experiences an estimated 1.4 million cases of
foodbome salmonellosis, making Salmonella a leading cause of the 76 million
cases of foodbome illnesses reported annually (Mead et al., 1999). According to
the Center for Science in the Public Interest (CSPI, 2001), about 29% of these
outbreaks are caused by poultry products, which makes poultry the most
important vehicle for foodbome salmonellosis.

Salmonella lnfantis is among the 10 most commonly reported strains of
Salmonella isolated in the United States (CDC, 2000). In 1999 the State of
Michigan experienced an increase in S. lnfantis infections with this serovar
recovered from both patients and poultry farm environments (Wilkins et al., 2002)
with subsequent investigations linking this outbreak to the handling of chicks and

ducklings.

39

Broilers are widely recognized reservoirs for Salmonella infections in
humans. The primary source of Salmonella in the poultry processing
environment is the gastrointestinal tract of broilers where the organism
proliferates (Hinton et al., 1990) with cross-contamination of the carcass after
evisceration leading to further contamination of the processing environment.
Efforts that minimize cross-contamination of poultry carcasses with intestinal
contents will improve the microbiological quality of carcasses (Bailey, 1993).
Therefore, control measures aimed at reducing Salmonella in the gastrointestinal
tract will likely lead to a reduction in poultry-associated salmonellosis.

Monensin, an ionophore antibiotic, is commonly used as a feed additive in
the poultry industry for the control of coccidiosis. However, the impact of
monensin use alone on shedding Salmonella has not been reported. A more
recent approach for controlling Salmonella in domestic livestock is feeding of
sodium chlorate which has been shown to minimize Salmonella colonization in
pigs (Anderson et al., 2001). Hence, the purpose of this study was to investigate
the effects of monensin and sodium chlorate on S. lnfantis colonization and

shedding in broilers.

3.3. MATERIALS AND METHODS
3.3.1. EFFECT OF MONENSIN ON 8. INFANTIS IN BROILERS

A total of 70 newly-hatched Salmonella-free broiler chicks were
randomly allotted to one of two groups of 35 chicks. One group received feed

containing monensin (treatment group) whereas the other group received

40

antimicrobial-free feed. Caecal samples were collected from five randomly
selected birds on days 7, 10, 14, 21, 28, 35, and 42 and examined for numbers

of S.lnfantis with the experiment replicated three times.

3.3.2. CONTROL OF S. INFANTIS USING SODIUM CHLORATE

Fifty Salmonella-free broiler chicks were randomly allotted to one of three
treatment groups (12 birds each) or a control group (20 birds). Treatment groups
received sodium chlorate solution ad libitum for four days in place of drinking
water while the control group received only drinking water. The three treatment
groups were given sodium chlorate solution beginning on day 10, 20 and 38.
Two birds from each group were sacrificed at random after which caecal and liver”
samples were quantitatively examined for S.lnfantis on days 7, 10, 14, 17, 20,
24, 27, 30, 38, and 42. In addition, cloacal swab samples were randomly taken
from two birds in each group on days 11, 12, 13, 15, 16, 18, 19, 21, 22, 23, 25,
26, 28, 29, 39, 40 and 41, and examined for numbers of S.lnfantis. This

experiment was also replicated three times.

3.3.3. SALMONELLA INFANTIS CULTURE

Salmonella lnfantis strain 99 EN 151, originally isolated in 1999 from an
infected poultry flock in Michigan, was obtained from the Michigan Department of
Community Health (MDCH, Lansing, MI). The isolate was maintained on a
trypticase soy agar slant at 4°C and transferred four times in trypticase soy broth

(35°C/24h) (Becton Dickinson, Cockeysville, MD) before use. After the last

41

incubation, populations were determined by serial dilution in buffered peptone
water 0.1% (wlv) followed by spiral plating (AutoplateQ 4000; Spiral Biotech Inc;
Bethesda; MD) on Xylose Lysine Desoxycholate (XLD) agar (Difco Laboratories,
Detroit, MI). On the day of oral challenge, the overnight culture was centrifuged
(Sorvall®, Newtown, CT) at 10,000 x g l 10 minutes / 4°C and resuspended in
buffered peptone water (Difco) to a final concentration of 1 x 10° CFU/0.25ml

with the viable cell count confirmed by spiral plating on XLD agar.

3.3.4. CHICK HATCHING AND HOUSING

Salmonella-free broiler eggs were obtained from Aviagen (Huntsville, AL)
and refrigerated at 4°C. The following day, eggs were set for incubation at the
Michigan State University (MSU) Poultry Research & Teaching Center in an
incubator (Petersime model # 5) at 375°C dry bulb and 289°C wet bulb for 17
days. On day 18, all eggs were candied and the fertile eggs were transferred to
a hatching cabinet (Petersime model) at 375°C dry bulb and 322°C wet bulb.
The newly hatched chicks were randomly assigned to one of two or one of four
groups to receive monensin or sodium chlorate, respectively, and housed in
brooder cages in the University Lab Animal Resources containment facility.
Before infecting the chicks with S. lnfantis on day 3, caecal samples from nine
broiler chicks per group were analyzed to confirm that the chicks were free of
Salmonella. After three weeks, the birds were transferred to cages for the

remainder of the study. Room temperature ranged from 18.3 to 239°C. All

42

animal related aspects of these experiments were approved by the Michigan

State University All University Committee on Animal Use and Care.

3.3.5. TREATMENT WITH MONENSIN AND SAMPLING

Day-old Salmonella-free broiler chicks were randomly allotted to one of
two groups, each containing 35 birds. The treatment group was fed a diet
containing monensin (Coban-20®; Elanco, Indiana) which was added at the rate
of 100 glton of feed (100 mglKg) whereas the control group received feed without
monensin. The diet, which met National Research Council requirements for
poultry (NRC, 1994), was prepared at the MSU feed mill. On day 3, all chicks
were infected with S.lnfantis (10° CFU/bird) by administering 0.25ml of the
buffered peptone suspension by oral gavage. All birds were given feed and
water ad libitum throughout the experiment. On day 7, five chicks per replicate
(15 chicks from each group) were randomly selected from each group and
humanely euthanized. The caeca were aseptically removed, placed in individual
Whirl-Pak° bags (Nasco, Fort Atkinson, WI) and transported to the laboratory on
ice within 1 h of collection. The same procedure was repeated on days 10, 14,

21, 28, 35, and 42, with all samples analyzed for numbers of S.lnfantis.

3.3.6. TREATMENT WITH SODIUM CHLORATE AND SAMPLING
Fifty day-old Salmonella-free broiler chicks were randomly allotted to one
of four groups consisting of three treatment groups (day 10 treatment group, day

20 treatment group and day 38 treatment group) and a control group. On day 3,

43

all chicks were infected with S.lnfantis (10° CFU/bird) by administering 0.25ml of
the buffered peptone suspension by oral gavage. The control group received
only drinking water, whereas birds in the treatment groups were given a
commercial sodium chlorate solution containing 0.16% (wlv) sodium chlorate;
0.18% (wlv) sodium lactate and 0.021% (wlv) sodium nitrate (EKA Chemicals;
Marletta; GA) ad libitum, on days 10, 20, and 38 in place of drinking water. The
treated birds were maintained on the sodium chlorate solution for four
consecutive days and then switched back to drinking water. All birds were given
an antimicrobial-free feed ad libitum for the duration of study. Three days before
treatment on day 10, caecal and liver samples were aseptically collected from
two birds in each replicate (six birds from each group), transported on ice to the
laboratory within 1 h of collection and examined for S.lnfantis. Similar samples
were also similarly collected after treatment on days 14, 17, 20, 24, 27, 30, 38
and 42 and examined for S.lnfantis. On days 11, 12, 13, 15, 16, 18, 19, 21, 22,
23, 25, 26, 28, 29, 40, and 41, cloacal swab samples were collected at random
from birds to determine the fecal shedding pattern of S.lnfantis with the sodium
chlorate treatment. On day 42, the body weights of birds from all the four groups

were recorded before sacrifice.

3.3.7. MICROBIOLOGICAL ANALYSIS
Caecal and liver samples collected in individual Whirl-Pak° bags were
weighed, diluted 1:10 in lactose broth (Difco), minced with a pair of flame-

sterilized scissors and homogenized in a Stomacher (Seward Stomacher° 400;

44

London, England) for 2 minutes. A 1-ml aliquot from each bag was serially diluted
in 0.1% peptone water and spiral-plated on duplicate plates of Hektoen Enteric
(HE) agar (Difco) and XLD agar (Difco) with the lactose broth enrichment
incubated at 35°C for 24 j: 2 h. After 24 i 2 h of incubation at 35°C, colonies
resembling Salmonella (black, round and glistening on XLD and HE) were
counted as S. lnfantis. In cases where salmonellae were not observed by direct
plating, 1ml aliquots of the 24 h-old lactose broth enrichment were transferred to
10 ml of selenite cysteine broth (Difco) and 10 ml of tetrathionate broth (Difco)
and incubated for 24 i 2 h at 35°C. After incubation, the contents of each tube
were streaked to duplicate plates of HE and XLD agar. These plates were again
examined for the presence of typical colonies of Salmonella following 24 i 2 h of
incubation at 35°C. All cloacal swab samples, which contained 0.2 g of fecal
material, were diluted in 10 ml of lactose broth and analyzed as described for
caecal and liver samples. Selected isolates were streaked to trypticase soy agar
containing 0.6 % yeast extract (Difco) for purification and then confirmed as S.

lnfantis by serotyping at the MDCH.

3.3.8. STATISTICAL ANALYSIS

A repeated measures two-way Analysis of Variance was performed on the
data using the Statistical Analysis System (Proc ANOVA, SAS© Version 8, SAS
Institute, Inc., Cary, NC). A type III F test was used to assess overall effects of
treatments. The least square means were compared using Fisher’s LSD (Least

Significant Differences) and the significance of mean differences was judged

45

using the t-test. Since the lowest detectable limit by direct plating for cloacal
swab samples was 2.7 log CFU/g, all plates which were positive after enrichment
were assigned a maximum possible value of 2.7 log CFU/g for statistical

purposes.

3.4. RESULTS

3.4.1. TREATMENT WITH MONENSIN

Following oral infection, caecal samples collected from both groups of
birds on day 7 contained 7.0 log CFU/g with these counts decreasing from day 7
to day 42 (Table 3.1). In both groups, counts on day 10 were lower (p< 0.5) '
than those on day 7 with 6.5 log CFU/g for the control group and 6.4 log CFU/g
for monensin-treated group (Figure 3.1). This decrease in the caecal count was
only temporary with S. lnfantis populations remaining relatively constant
throughout the remainder of the study. For the control group, caecal S. lnfantis
counts on day 42 were 6.2 log CFU/g which were only about 0.3 log lower than
those observed at day 10. The monesin-treated group yielded a caecal count of
5.8 log CFU/g on day 42 which was 0.6 log lower compared to day 10. Overall,
no significant difference in the numbers of S. lnfantis retrieved from the caeca of

monensin-treated birds was observed when compared to the untreated controls.

46

Table 3.1 Mean Caecal populations of S. lnfantis (CFU/g) in birds raised on

a diet with and without monensin

 

 

Age of Monensin 1 Without monensin 1
birds

7 7.0;r0.17"2 7.01017“

10 6.41017 b 6.51:0.17 b

14 63:01?" 652017 b

21 6.1:l:0.17° 6510.17"

28 6.3017 b 65:0.17 b

35 6220.17 b 6.3:t0.17 b
42 5.23017 b 6.21:0.29 b

 

1) Data expressed as mean 1: standard deviation n= 15.

2) Data followed by different superscripts are significantly different with p< 0.5.

47

mV NV

 

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.9550 1.01

 

 

 

0.3a.

mm mm ..N

3

 

3 +3

5.2. 0.0.5.5 ho 5.85530 .0003 0.23:. . m ....n 0.50.“.

 

:.0:0:oE «.55.? 0:0

o
_.

N
m
v
m.
o
N
m

6 1 mo 601 alluvial 's

48

3.4.2. TREATMENT WITH SODIUM CHLORATE:

3.4.2.1. CAECAL COLONIZATION

When 10- and 20-day old birds were given sodium chlorate in
drinking water, caecal colonization by S. lnfantis decreased significantly (P<0.05)
4 and 7 days after treatment (Table 3.2). In birds given sodium chlorate on day
10 through 14, S. lnfantis populations were 1.2 and 1.0 logs lower on days 14
and 17, respectively, compared to the untreated controls (Figure 3.2). For the
group treated with sodium chlorate on day 20, this difference was even greater
with numbers of S.lnfantis in the caeca being 2.9 and 2.8 logs lower on days 24
and 27, respectively, compared to the control group. In contrast, no significant
difference in caecal colonization was seen in birds when sodium chlorate

treatment was given for 4 days starting on day 38.

3.4.2.2. ORGAN INVASION

No significant differences in S.lnfantis isolation rates from liver
samples were observed between the three treatment groups and the control
group (Table 3.3). Since S. lnfantis was not detected by direct plating, (< 2.0 log
CFU/g), all liver samples were subsequently enriched. When the sodium
chlorate treatment was initiated on day 10, 33% of the liver samples were free of
S.lnfantis 4 and 7 days post-treatment. The same results were observed four
days after treatment when birds began receiving sodium chlorate on day 20 with

66% of the samples negative 7 days after treatment. For birds receiving sodium

49

chlorate on day 38, all liver samples were positive on day 42, as were the

controls.

Table 3.2 Mean S. lnfantis caecal colonization (log CFU/g) using sodium

 

 

 

chlorate.
Age of Sodium chlorate treatment 1
Control 1

birds Day 10 2 Day 20 2 Day 38 2
7 71:03 “3 63:03 a 62:03 a 66:03 a
10 65:03 a 65:08 3 NA‘ NA
14 57:031 45:03 h NA NA
17 54:03 a 44:04 b NA NA
20 64:03 a 52:03 a 61:03 3 NA
24 69:03 3 NA 60:06 ° NA
27 57:03 3 NA 29:04 ° NA
30 62:03 3 NA 48:04 8 NA
38 66:03 9 NA NA 58:03 a
42 63:04 ' 61:08 a 6:04 a 5:04 a

 

1) Data expressed as mean :I: standard deviation (n=6).

2) Day sodium chlorate treatment was started.

3) Data followed by different superscripts are significantly different with p< 0.5.

4) Samples not analyzed at these time points as birds were not sacrificed.

50

gun

mVOVNMVm wmwNmNNNmrmmeoF N v

- n n p - n — p n - n n -

 

oimulul
63:8 -9 -

||.

 

 

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322:0
Eaton mEm: :o_umN_:o_ou .moomo mac—3:. .m .N.no._:m_u_

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51

Table 3.3 Invasion of S.lnfantis into the liver using sodium chlorate.

 

 

 

Age of Sodium chlorate treatment started
Control

birds Day 10 1 Day 20 7 Day 38 1
7 6 l 62 2 I6 6 I6 6 I6
10 6/6 6/6 NA NA
14 5/6 4J6 NA NA
17 6/6 4/6 NA NA
20 6/6 6/6 6/6 NA
24 5 / 6 NA3 4 I 6 NA
27 6/6 NA 2/6 NA
30 6/6 NA 6/6 NA
38 6/6 NA NA 6/6
42 6/6 6/6 6/6 6/6

 

1) Date sodium chlorate treatment was started.
2) Number of positive samples I number of samples.

3) No samples analyzed as birds were not sacrificed at these time points.

52

3.4.2.3. FECAL SHEDDING:

Fecal shedding of S. lnfantis was highly irregular. A significant
difference (P< 0.0008) between the four groups was observed with lower counts
in cloacal swabs after treatment with sodium chlorate on days 10 and 20 (Table
3.4). A reduction of > 1.4 and > 0.6 log CFU/g was observed after treatments on
days 10 and 20 respectively (Figure 3.3). Although Salmonella counts
decreased below the level of direct detection (2.0 log CFU I g) on day 13, the
levels started increasing on day 15. When treatment was given on day 20, the
levels decreased below detectable limit on day 23, as in the previous case with
fecal shedding only increasing on day 28. When 8. lnfantis was not detected by
direct plating, the enriched samples were invariably positive indicating that low'

levels of S. lnfantis were being shed at the above time points.

3.4.2.4. CONFIRMATION OF ISOLATES:

Twenty isolates from caecal, liver or cloacal swab samples in the
sodium chlorate study were serotyped at MDCH. Twelve of the 20 isolates were
confirmed as S. lnfantis with the remaining 8 isolates also identical to S. lnfantis
(6,7:-:1,5 or 6,7:r—) except for a phase change in one of the H-antigens (Table

3.5).

53

Table 3.4 Mean Fecal shedding of S. lnfantis (log CFU/g) using sodium

 

 

 

chlorate.
Age of Sodium chlorate treatment 7
Control 1
birds Day 10 2 Day 20 2 Day 38 2
11 4.3:038a3 3.2:038b
12 3.1:038 a 4.4:038 “
13 4.3:038 ‘ < 2.7 b‘
15 4.0:038 a 2.8:038 ”
16 3.7:038 ‘ 4.0:038 a
18 4.0:038 ‘3 3.2:038 "
19 3.0:0.38 ‘ 3.8:0.38 ‘
21 3.2:038 ‘ 3.3:038 '
22 4.0:038 a 3.2:038 "
23 4.0:038 ‘ < 2.7 b‘
25 4.0:038 " < 2.7 b‘
26 4.1:038 ‘ < 2.7 b‘
28 3.2:038 ‘ 2.8:038 “
29 3.6:038 a < 2.7 b‘
39 3.3:038 a 3.2:038 a
40 3.0:038 ’ 2.9:038 a
41 3.8:038 ’ 2.9:038 a

 

1) Data expressed as mean 1: standard deviation (n=6).

2) Day sodium chlorate treatment started.

3) Data followed by different superscripts are significantly different with p< 0.5.

4) S. lnfantis only detected post-enrichment and these were given a value of log

2.7 for statistical purposes.

54

gun.
mv ow mm vm 5 mm mm mm 9 2. me or

 

 

 

......<.<<.<. . ...-w
-N
Wm ........... WafWTXV/Kwawflw
F

32020 E:=oom
mEm: 9:285 .89— mzcfls .w

Bingo 50| snuelw 's

55

Table 3.5. Confirmation results on S. lnfantis selected isolates

 

 

MDCH Lab # MSU Lab # Serotype
1 D-15-F(10) lnfantis
2 D-30—Liv (C) lnfantis
3 D-17-F(10) lnfantis
4 D-28-F(20) lnfantis
5 D-42-Ce(20) lnfantis
6 D-42-E-g&p(31-33) lnfantis
7 D-14(10) lnfantis
8 D-42-Ce(38) 6,7:-:1 ,5
9 D-41-F(38) 6,7:-:1,5
10 D-40-F(38) 6,7:-:1,5
1 1 D-25-Ce(C) 6,7:-:1,5
12 D-30-Ce(20) lnfantis
1 3 D-29—F(C) lnfantis
14 D-21-F(20) 6,7:r:-
1 5 D-17-F(C) lnfantis
16 D-24-Liv(C) lnfantis
1 7 D-20—Liv(C) 6,7:r:-
18 D-19—F(10) 6,7:-:1,5
19 D-13-F(C) 6,7:r:-
20 D-24-E-Ce(20) lnfantis

 

56

3.4.2.5.

BODY WEIGHTS:

A significant difference (P < 0.0001) in bird body weight was observed

between the groups (Table 3.5). Birds treated with sodium chlorate on day 10

and 20 as a group had greater weight gain when the study concluded on day 42.

Weight was gained during the period after treatment when the birds had

decreased caecal populations of Salmonella. However, weights of birds treated

on day 38 were similar to the controls. No significant differences between bird

body weights in groups were observed.

Table 3.5. Body weights of broilers using sodium chlorate as recorded on

 

 

 

 

day 42.
Birds Weight in pounds
Control Sodium chlorate started on
Day 10 Day 20 Day 38
1 0.25a 0.875 b 0.5 b 0.25a
2 0.25a 0.8125 b 0.875 b 0.375 a
3 0.375 a 1.0 b 0.875 b 025‘
4 0253 1.0 b 0.875 b 0.253
5 0.4375a 0.8125 b 1.0625 b 0.3125 3
6 025‘ 1.0625 b 0.8125 b 025°

 

Data followed by different superscripts are significantly different.

57

3.5. DISCUSSION:

No difference in caecal colonization of S. lnfantis was observed when
broilers were given feed containing monensin or an antimicrobial-free feed, with
Salmonella populations only decreasing about 1 log during the lifetime of the
birds. Hence, the ionophore antibiotic monensin had no effect on S.lnfantis
colonization of the caeca in broilers.

Relatively few reports concerning the effect of monensin on Salmonella
have appeared in the literature since monensin is primarily used as a coccidiostat
in the poultry industry. However, when broilers were fed avoparcin (10 ppm) and
monensin (90 ppm), Holmberg et al., (1984) did report higher caecal levels of
S.lnfantis in comparison to feeding only avoparcin during a 2-week challenge
period. A significant increase (p<0.5) in numbers of Salmonella in caeca 7, 10
and 14 days after coccidia infection was observed by Arakawa et al. (1981),
indicating that treatment with an anticocidiostat would decrease salmonellae.
Work by Morishima et al. (1984) also suggests that Salmonella counts can
increase when chickens are simultaneously infected with coccidia.

Many studies have dealt with the effect of monesin on coccidia (Chapman
and Saleh, 1999; Hooge et al., 1999). When fed at a rate of 59.5 ppm, monensin
reportedly reduced mortality and improved feed conversion, leading to increased
weight gain in turkey poults (Chapman and Saleh, 1999). Monensin in
combination with sodium bicarbonate was also found to significantly reduce
mortality due to coccidia in broilers (Hooge et al., 1999). In our study which did

not assess the impact of coccidia, failure of monensin to decrease colonization

58

by Salmonella might be due to the continuous exposure of our broilers to high
levels of Salmonella in the environment.

Treatment with sodium chlorate reduced caecal colonization of Salmonella
in broilers by up to 2 logs, with birds receian sodium chlorate on days 10 and
20 having significantly lower levels of S.lnfantis in the caeca, when compared to
the untreated control birds. These findings confirm the bactericidal effectiveness
of sodium chlorate on Salmonella in birds as previously reported by Anderson et
al. (2000) in in vitm studies with rumen contents. Salmonella possess respiratory
nitrate reductase activity, which is responsible for the anaerobic reduction of
nitrate to nitrite. This same enzyme will also catalyze the intracellular reduction
of chlorate to cytotoxic chlorite with this end product being lethal to Salmonella
and other bacteria such as Escherichia coli O157:H7 that possess nitrate
reductase activity. An added advantage of sodium chlorate is that the normal
gastrointestinal flora is not affected (Anderson et al., 2000).

Despite these reports, we found sodium chlorate to be ineffective when
given to 38-day old birds that were sacrificed 4 days later. The lack of any
reduction of S. lnfantis in the caeca of 38- to 42-day old birds may be due to the
establishment and spread of S. lnfantis from the gastrointestinal tract to the
internal organs since these birds were showing overt signs of salmonellosis as
evidenced by reduced feed intake, droopiness, greenish diarrhea and a high
mortality rate. Also the fact that these birds were raised in an environment with

high levels of Salmonella may have decreased the effectiveness of the treatment

59

with reinfection of the gastrointestinal tract occurring in those birds which had
decreased levels of Salmonella after treatment on days 10 and 20.

S. lnfantis reductions of 1 and 2 logs in the caeca were observed in birds
given sodium chlorate beginning on days 10 and 20, respectively. These
findings agree those of Anderson et al., (2001) who treated pigs with sodium
chlorate. Caecal levels of Salmonella were found to decrease four days after
treatment in both of the above groups, with this decrease evident up to seven
days post-treatment in birds treated on day 20. However, in our study
populations of S. lnfantis began increasing 7 days post-treatment due to
continuous environmental exposure associated with fecal shedding of the
organism.

In contrast to our favorable results for cecal colonization, sodium chlorate
failed to prevent liver invasion by S. lnfantis. Although all liver samples were
negative by direct plating,‘ most liver samples were positive on enrichment.
These findings are similar to those observed by Brown et al., (1976) who also
reported frequent recovery of S. lnfantis from the liver as well as the lungs and
spleen of cockerals. A lower concentration of the organism in liver might result
from the fact that the caecum is the main colonization site for Salmonella (Hinton
et al., 1990). Although frequently positive for S. lnfantis, cloacal swab samples
showed wide variability within treatment groups. lnterrnittent shedding of S.
lnfantis was also observed by Brown et al., (1976) in a study wherein birds were

orally inoculated with the organism into the middle third of the esophagus.

60

In our work, feeding of sodium chlorate significantly decreased fecal
shedding of S. lnfantis with counts decreasing > 0.6 to > 1.4 logs I 9 after
treatment on days 10 and 20. Hence, four-day long sodium chlorate treatment
reduced both caecal colonization and fecal shedding. When the birds were
treated with sodium chlorate on day 20, S. lnfantis colonization decreased in
young broilers (24 days of age) with sodium chlorate remaining effective for up to
7 days after treatment. Thus, these findings suggest that sodium chlorate may
be helpful for reducing Salmonella colonization in incoming birds and thereby
decreasing carcass contamination during slaughter and further processing. In
conjunction with the USDA-FSIS mandated HACCP programs, sodium chlorate
treatment of broilers may help processing plants meet and/or exceed the.

Salmonella performance standards established by the USDA baseline study.

61

CHAPTER 4
CONCLUSIONS AND FUTURE RESEARCH

Salmonella colonization in the gastrointestinal tract of broilers leads to
cross-contamination of poultry carcasses, thus increasing the number of
foodbome salmonellosis outbreaks. According to the Pathogen Reduction I
Hazard Analysis and Critical Control Point final rule issued by FSIS-USDA, all
poultry processing plants are required to meet the Salmonella performance
standards which have been fixed at 20%. Control of Salmonella in integrated
poultry operations is often very difficult as there are numerous sources of
Salmonella in the poultry industry such as the feed, water, flies and other insects, '
farm environment and processing environment. Thus, the poultry industry is
always seeking easier, more effective and more economical ways to control
Salmonella.

The primary source of Salmonella contamination in poultry processing
plants is the gastrointestinal tract of broilers. In the present study, we evaluated
two different treatments for reducing I eliminating Salmonella colonization in the
intestinal tracts of broilers. The first treatment, in which broilers received feed
supplemented with monensin, did not provide a method to control Salmonella in
the gastrointestinal tract. These findings were not entirely unexpected since
monensin is used primarily as a coccidiostat in the poultry industry. In contrast,
treatment of broilers with sodium chlorate showed some promise for decreasing

the incidence of S.lnfantis in broilers. Caecal colonization of Salmonella

62

decreased when broilers received sodium chlorate in their drinking water over a
period of four days.

Our present study only evaluated the effects of sodium chlorate and
monensin on S. lnfantis in broilers. The effectiveness of sodium chlorate in
poultry needs to be further assessed against a wider variety of Salmonella
species in poultry. Future studies should examine the effect of sodium chlorate
on other strains of Salmonella particularly Salmonella Typhimurium DT 104 and
other multi-antibiotic strains. Efficacy of sodium chlorate as a terminal treatment
in broilers, the exact dose of sodium chlorate required to eliminate Salmonella
from the gastrointestinal tract of birds and field trials using sodium chlorate as a
preventive measure are also worth investigating. Key insights into the reduction
of poultry-home diseases could be gained by examining the effectiveness of
sodium chlorate against other poultry-bome pathogens such as Escherichia coli.
Other areas of research could also include the effect of long term treatment with
sodium chlorate as well as the effect of sodium chlorate on the end product in
terms of safety. The outcome of further investigations will help in coming to a

definite conclusion as to the effectiveness of the current method.

63

APPENDIX

School Project:

Broiler chicks raised by FFA (Future Farmers of America) students in different
schools in Michigan State were processed at the Fowlerville processing unit on
the 1’t and 2"d of November, 2001. Samples were collected from 25 of 39
schools participating. Caecal samples were taken from five birds at random from
each school. Sample collection was done as aseptically as possible in the
processing unit. Caecal samples were collected in individual stomacher bags,
transported from the processing unit to the laboratory in ice boxes and analyzed
immediately. Each sample was tested for the presence of Listeria and
Salmonella using the REVEAL microbial screening test for Salmonella and.
Listeria (Neogen Corporation, Lansing, MI). Of 75 birds tested, 57 and 56 were
positive for Salmonella and Listeria respectively. The positive REVEAL
enrichment cultures for Salmonella were plated on Hektoen enteric agar, Bismuth
sulfite agar and Xylose lysine desoxycholate agar for verification. After plating,
only 49 cultures were found positive for Salmonella. Similarly, Listeria-positive
REVEAL enrichment cultures were plated on Oxford agar and Trypticase soy
yeast extract agar (Difco) for verification with only 48 samples positive for
Listeria. Picking and inoculating two colonies per plate into Triple sugar iron and
Lysine iron agar (Difco) slants was used to presumptively confirm Salmonella
isolates. These isolates which were then grown in Tryticase soy broth, were

frozen down to —80°C.

64

Table: List of schools positive for Salmonella and Listeria

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number Name of school No. of samples positive No. of samples
for Salmonella positive for Listeria
1 North Adams-Jerome - 5
2 Dansville 5 5
3 Olivet 4 5
4 Akron-Fairgrove 4 3
5 Springport 5 -
6 Durand 3 1
7 Alpena 1 2
8 Milan 3 1
9 Dundee 1 4
10 Ubly 2 3
11 Vicksburg - -
12 Bronson - 2
13 Beal City 2 5
14 Roosevelt 2 -
15 USA 1 -
16 Byron 3 -
17 River Valley - -
18 Alma 2 3
19 Waldron 1 1

 

 

 

 

65

 

 

 

 

 

 

 

 

20 Branch ACC
21 lonia

22 Laingsburg
23 Port Hope
24 Chesaning
25 Corunna

 

 

 

66

 

 

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