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This is to certify that the

dissertation entitled

EPIDEMIOLOGY AND ANTIMICROBIAL RESISTANCE OF

"CAMPYLOBACTER SPP." FROM FOOD ANIMALS AND

HUMANS IN NORTHERN THAILAND
presented by

PAWIN PADUNGTOD

has been accepted towards fulfillment
of the requirements for

DOCTORAL LARGE ANIMAL CLINICAL

SCIENCES (EPIDEMIOLOGY)

degree in

 

 

W

U - Major professor

 

PROF JOHN B KANEENE

Date figl/ 7 / 0 Z

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

UBRARY
Michigan State
University

 

 

 

PLACE IN RETURN Box to remove this checkout from your record.
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6/01 C‘JCIRC/Dateouepes-p. 15

 

EPIDEMIOLOGY AND ANTIMICROBIAL RESISTANCE OF CAMPYLOBAC T ER
SPP. IN FOOD ANIMALS AND HUMANS IN NORTHERN THAILAND

By

Pawin Padungtod

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Large Animal Clinical Sciences
(Epidemiology)

2002

ABSTRACT
EPIDEMIOLOGY AND ANTIMICROBIAL RESISTANCE OF
CAMPYLOBA C TER SPP. IN FOOD ANIMALS AND HUMANS IN NORTHERN
THAILAND
BY

Pawin Padungtod

Campylobacter spp. have been recognized as major foodbome pathogens in
developed and developing countries. Recently, Campylobacter spp. with resistance to
antimicrobial agents have been identified in various parts of the world, including
Thailand. It is also widely speculated that the use of antimicrobial agents in food animals
may be contributing to the antimicrobial resistance problem in humans. Because there
was limited information on Campylobacter spp. in food animals in Thailand, and to
determine whether food animals were an important source of Campylobacter spp. with
resistance to antimicrobial agents, a study was designed to address six objectives: 1)
validate the use of a fluorogenic PCR assay to identify C. jejuni fi'om field samples; 2)
determine the frequencies and the antimicrobial susceptibility level of Campylobacter
spp. isolated from food animals and farm workers at farms, slaughterhouses, and markets;
3) compare the frequencies of Campylobacter spp. with resistance to antimicrobial agents
in food animals, food products and farm workers; 4) determine what risk factors
associated with the observed frequencies of antimicrobial resistance; 5) determine
whether antimicrobial use in feed and treatment on pig and chicken farms is associated
with the frequency of antimicrobial resistance in Campylobacter; and 6) determine the
association between mutation in the gyrA gene of C. jejuni and level of resistance to

ciprofloxacin.

 

A combination of cross-sectional and prospective study designs were used.
Samples were collected from pigs and chickens at the farm, slaughterhouse, and market
in 2000 and 2001. Farm and slaughterhouse worker stool samples were also collected.
Isolation of Campylobacter spp. was done using enrichment and selective media, and
suspect colonies were confirmed using oxidase test, catalase test and gram staining. In
vitro susceptibility testing was done using the microbroth dilution technique, for
ciprofloxacin, erythromycin, gentamycin, azithromycin, clindamycin, chloramphenicol,
nalidixic acid, and tetracycline. Results of susceptibility testing were reported in
minimum inhibitory concentrations (MICs), and isolates were classified as resistant based
on breakpoints from the National Committee for Clinical Laboratory Standards.
Fluorogenic PCR was used to identify C. jejuni and the Thr-86-to-Ile mutation in the
gyrA gene of C. jejuni.

The prevalence of Campylobacter spp. in pigs was found to be 73.6% at the farm,
45.6% at the slaughterhouse, and 24.6% at the market. In chickens, the prevalence was
73.4% at the farm, 50.6% at the slaughterhouse, and 47.2% at the market. In humans, the
prevalences of Campylobacter spp. were12.5% and 0% in pig farm workers and chicken
farm workers, respectively. Resistance was found to all antimicrobial agents tested in
chickens and pigs at farms, slaughterhouses and markets. The most prevalent forms of
resistance seen were to ciprofioxacin, nalidixic acid, and tetracycline. The prevalence of
resistance was higher for those antimicrobial agents to which animals were exposed.
There was very high agreement between ciprofloxacin resistance and the presence of the

Thr-86-to-Ile mutation in gyrA gene.

ACKNOWLEDGMENTS

I would like to thank the following institutions for their generosity; Institute of
International Health, MSU, National Food Safety and Toxicology Center, MSU,
Population Medicine Center, MSU, and Chiang Mai University, Chiang Mai, Thailand
for financial support, Faculty of Veterinary Medicine, Chiang Mai University for
technical assistance.

I would like to thank all my committee members, graduate students, technicians
and secretaries in all laboratories I worked in including AHDL bacteriology lab,
Dr.Holland’s E.coli lab, Dr.Linz’s lab, Dr.Mansfield’s lab, Faculty of Medicine-Chiang
Mai University bacteriology lab, and Faculty of veterinary medicine-Chiang Mai
university central diagnostic lab, and all the students who helped me with specimen
collection and laboratory work.

Special thanks to Prof. John Kaneene who has been a great mentor, and one of
the persons I like working with most. He constantly shows me the power of optimism and
the power of seeing thing in epidemiological way. Special thanks also to graduate
students and secretaries in the Population Medicine Center. Ifcarrying out a research
study is like going to war, Prof. Kaneene would be a great general, and the Population
Medicine Center would be a great company that I feel most comfortable going to war
with.

Finally, I would like to thank the 3 most important women in my life. My mother,
who built in me the love to read and to believe in education. My sister, who is always my

gold standard of doing things. And my wife, who makes it easy for me to be happy.

iv

TABLE OF CONTENTS

LIST OF TABLES x
LIST OF FIGURES xii
INTRODUCTION
RATIONALE .............................................................................. l
PROBLEM STATEMENT .............................................................. 3
OBJECTIVES ............................................................................. 3
HYPOTHESES ............................................................................ 4
OVERVIEW ............................................................................... 5
CHAPTER 1

 

CAMPYLOBACT ER SPP. IN HUMANS CHICKENS PIGS
AND THEIR ANTIMICROBIAL RESISTANCE : A REVIEW

INTRODUCTION ........................................................................ 6
PREVALENCE AND INCIDENCE OF CAMPYLOBA C T ER SPP.
Campylobacter spp. in humans ............................................ 7
Campylobacter spp. in food Animals ..................................... 8
EXPOSURE AND TRANSMISSION OF CAMPYLOBACT ER SPP.
In humans .................................................................... 12
In food Animals ............................................................ 13

CLINICAL DISEASE EXPRESSION OF CAMPYLOBA C T ER SPP.
INFECTION IN HUMANS AND ANHVIALS

In human ..................................................................... 16

In food Animals ............................................................. 17
ANTIMICROBIAL RESISTANCE

Mechanism of resistance to antimicrobial agents

in Campylobacter spp ....................................................... 18

Antimicrobial susceptibility testing ....................................... 18

Antimicrobial resistance in human isolates .............................. 19

Antimicrobial resistance in isolates from food animals ................ 21

Link between resistance in food animals isolates to

resistance in human isolates ............................................... 21
REFERENCES ........................................................................... 26

 

CHAPTER 2
REVIEW : MOLECULAR TECHNIQUES IN EPIDEMIOLOGICAL
STUDIES OF CAMPYLOBA CTER SPP.
INTRODUCTION ....................................................................... 38
AVAILABLE MOLECULAR TECHNIQUES FOR
EPIDEMIOLOGICAL STUDIES OF CAMPYLOBA CT ER SPP.
Molecular Techniques for Detection and Identification of

Campylobacter spp .......................................................... 40
Molecular techniques for Determining Relationship among
Campylobacter spp .......................................................... 43
Molecular Techniques for Determining Marker of Virulence

and resistance in Campylobacter spp ..................................... 46

APPLICATION OF MOLECULAR TECHNIQUES IN
EPIDEMIOLOGICAL STUDIES OF CAMPYLOBA C T ER SPP.

Outbreak Investigation ..................................................... 47

Genetic Evidence of Relationships among Isolates .................... 48

Detection and Identification of Bacteria, Virulence Factors,

and markers for antimicrobial resistance ................................ 48
REFERENCES ........................................................................... 52

CHAPTER 3

IDENTIFICATION OF CAMPYLOBA C T ER JEJUNI ISOLATES
FROM CLOACAL AND CARCASS SWABS OF CHICKENS IN
THAILAND BY USE OF A 5’ NUCLEASE FLUOROGENIC PCR

ASSAY

ABSTRACT ............................................................................... 60

INTRODUCTION ........................................................................ 61

MATERIAL AND METHODS
Sample Collection and Primary Isolation ................................ 63
Fluorogenic PCR Assay .................................................... 64
Conventional test kits ...................................................... 65
DNA Extraction for PCR-RF LP .......................................... 66
PCR-RFLP of the 23S rRNA .............................................. 66
Statistical Analysis .......................................................... 67

RESULTS ................................................................................. 67

DISCUSSION AND CONCLUSION .................................................. 69

REFERENCES ............................................................................ 76

vi

CHAPTER 4
PREVALENCE OF CAMPYLOBA C T ER SPP. IN PIGS, FARM
WORKERS AND PORK PRODUCTION SYSTEMS IN NORTHERN

THAILAND, 2000 — 2001
ABSTRACT .............................................................................. 79
INTRODUCTION ....................................................................... 8O
MATERIAL AND METHODS
Study Design and Population ............................................. 81
Description of the Pork Production System ............................. 82
Sample Size .................................................................. 83
Specimen and Data Collection ............................................ 83
Isolation and Identification of Campylobacter spp ..................... 84
Statistical Analysis ......................................................... 84
RESULTS
Frequencies of Campylobacter isolation ................................ 86
Risk Factors ................................................................. 86
DISCUSSION AND CONCLUSION ................................................. 87
REFERENCES ........................................................................... 95
CHAPTER 5

PREVALENCE OF CAMPYLOBA C T ER SPP. IN BROILER CHICKENS,
FARM WORKERS, AND THE CHICKEN PRODUCTION SYSTEM IN
NORTHERN THAILAND, 2000 — 2001

ABSTRACT .............................................................................. 99
INTRODUCTION ....................................................................... 100
MATERIAL AND METHODS
Study Design and Population .............................................. 101
Description of Poultry Production System .............................. 101
Sample Size .................................................................. 102
Specimen and Data Collection ............................................ 102
Isolation and Identification of Campylobacter spp ..................... 103
Statistical Analysis ......................................................... 104
RESULTS
Frequency of Campylobacter Isolation .................................. 105
Risk Factors ................................................................. 106
DISCUSSION AND CONCLUSION ................................................. 106
REFERENCES ........................................................................... 114

vii

CHAPTER 6
ANTIMICROBIAL RESISTANCE IN CAMPYLOBA CT ER SPP.
ISOLATED FROM FOOD ANIMALS AND HUMANS IN NORTHERN

THAILAND, 2000 — 2001
ABSTRACT ............................................................................... 116
INTRODUCTION ....................................................................... 117
MATERLAL AND METHODS
Study Design and Sample Size ............................................ 119
Study Population and Specimen Collection ............................. 119
Data collection .............................................................. 121
Isolation and Identification of Campylobacter spp ..................... 121
In Vitro Susceptibility Testing ............................................ 122
Statistical Analysis ......................................................... 123
RESULTS ................................................................................. 124
DISCUSSION AND CONCLUSION ................................................. 126
REFERENCES ........................................................................... 140
CHAPTER 7

DETERMINATION OF FLUOROQUINOLONES RESISTANCE IN
C.JEJUNI USING FLUOROGENIC PCR

ABSTRACT .............................................................................. 144

INTRODUCTION ....................................................................... 145
MATERIAL AND METHODS

Source of Campylobacter isolates ........................................ 147

Isolation and identification of Campylobacter spp ..................... 148

In Vitro Susceptibility Testing ............................................ 148

F luorogenic PCR assay .................................................... 149

Statistical Analysis .......................................................... 150

RESULTS ................................................................................. 150

DISCUSSION AND CONCLUSION ................................................. 151

REFERENCES ........................................................................... 157

CONCLUSION ................................................................................ 160

viii

 

APPENDICES
Appendix 1

Data collection form for pig farms ........................................ 166

Appendix 2
Sample collection form for pigs at farms ................................ 167

Appendix 3
Sample collection form for workers at pig farms ....................... 168

Appendix 4
Data collection form for chicken farms .................................. 169

Appendix 5
Sample collection form for chickens at farms ........................... 170

Appendix 6
Sample collection form for workers at chicken farms ................. 171
REFERENCES ................................................................................ 172

ix

 

Table 1-1

Table 1-2

Table 2-1

Table 2-2

Table 3-1

Table 3—2

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 5-1

Table 5-2

Table 5-3

Table 6-1

LIST OF TABLES

Prevalence of antimicrobial resistance in Campylobacter spp.
from humans ................................................................. 24

Prevalence of antimicrobial resistance in Campylobacter spp.
from food animals, foods, and water ..................................... 25

PCR protocol for detecting and identifying Campylobacter spp ..... 50
Molecular techniques for genotyping Campylobacter spp... . . . . . . . . .. 51

Proportion of samples yielding Campylobacter from farms
and slaughterhouses ........................................................ 74

Number of Cjejuni samples identified by flyorogenic PCR
assay and conventional test kit (n=59) ................................... 75

Prevalence of Campylobacter in pigs and farm workers in
Northern Thailand, 2000-2001 ............................................ 91

Incidence of Campylobacter colonization/contamination in pigs
in northern Thailand, 2000-2001 ......................................... 92

Full multivariable logistic regression model, with random
effects, for an individual pig being colonized with Campylobacter.. 93

Effect of source farm on the on the odds of finding
Campylobacter in pigs at farm and slaughterhouse .................... 94

Prevalence of Campylobacter spp. in chickens in northern
Thailand, 2000-2001 ........................................................ 111

Prevalence of Cjejuni in chickens in northern Thailand,
2000-2001 .................................................................... 112

Results of a multivariable logistic regression model, with random
effects, for a single chicken being infected with Campylobacter

at the farm .................................................................... 113

Number and type of samples collected from pig and chicken

farms in northern Thailand, 2000-2001 .................................. 132
x

 

Table 6-2

Table 6-3

Table 6-4

Table 7-1

Table 7-2

Minimum Inhibitory Concentration (MIC) dilution ranges and
breakpoint values for determination of antimicrobial resistance

for Campylobacter, based on the National Committee on Clinical
Laboratory Standards (N CCLS) recommendations .................... 133

Prevalence (%) of Campylobacter spp. with resistance to
antimicrobial agents, fi'om chickens, pigs, and farms and

slaughterhouse workers in northern Thailand, 2000-2001 ............ 134
Odds ratio for resistance in Campylobacter isolated fiom pigs

compared to chickens ....................................................... 135
MIC levels and mutation in gyrA gene in Cjejuni ..................... 153
Proportion of Cjejuni with mutation and resistance ................... 154

xi

Figure 3-1
Figure 3-2

Figure 6-1

Figure 6-2

Figure 6-3

Figure 6-4

Figure 7-1

Figure 7-2

LIST OF FIGURES

PCR-RFLP Tsp 5091 digestion patterns ................................. 72
PCR-RFLP Alu I digestion patterns ...................................... 73

Proportion (%) of Campylobacter spp. with resistance to
antimicrobial agents from pigs ............................................ 136

Relationship between resistance level and antimicrobial use
on farms ...................................................................... 137

Number of antimicrobial agents Campylobacter spp. were
resistance to .................................................................. 138

Minimum inhibitory concentration (MIC) of Campylobacter
Isolated from pigs and chickens .......................................... 139

Number of Cjejuni isolates with the Thr-86-to-Ile mutation
by MIC for ciprofloxacin (total 84 isolates, 60 with mutation) ....... 155

Number of Cy'ejuni isolates with the Thr-86-to-Ile mutation
by MIC for nalidixic acid (total 84 isolates, 60 with mutation) ....... 156

xii

INTRODUCTION
RATIONALE

Foodbome diseases have recently emerged as major public health issues around
the world. The consequences of infection by foodbome bacteria may vary depending on
the specific agent and characteristic of the patient. The most important consequence of
foodbome bacteria affecting millions of people each year is gastroenteritis or diarrhea.
Diarrhea is one of the top causes of mortality in children in developing world,
particularly in children less than one year old.

Until recently, Salmonella and Shigella were the most important foodbome
pathogens, in terms of virulence and the number of people affected. Laboratory methods
and surveillance systems for those two pathogens were established in developed and
developing countries. However, since the early 19903, Campylobacter spp., especially C.
jejuni and C. coli, have emerged as major foodbome pathogens, with higher incidences
than Salmonella and Shigella, and foods of animal origin have been implicated as a major
source of Campylobacter spp. infection in humans. Recently, there have been major
improvements in detection and identification techniques for Campylobacter spp. , and
surveillance systems for Campylobacter spp. have been put in place in many developed
nations. However, there is very limited information regarding the epidemiology of
Campylobacter spp. in developing nations.

The scope of food borne bacteria nowadays can be best viewed as an international
issue, considering the number of travelers and trades of food commodities around the
world. Bacteria originating in one part of the world may easily end up causing disease in

place far away from its’ origin. Clarifying the epidemiology of Campylobacter spp. in

 

 

 

developing countries will contribute to the global picture of food borne disease resulting
from Campylobacter spp. infection. This clarification will benefit both producers and
consumers of foods of animal origin around the world.

Another major public health issue at the turn of the century is the emergence of
food borne bacteria with resistance to antimicrobial agents. The fact that bacteria can
develop resistance to antimicrobial agents was recognized a long time ago. However,
most of those cases were limited to bacterial isolates found in hospitals or health settings,
which were constantly exposed to antimicrobial agents. The impact of those nosocomial
infections with resistance bacteria was limited to those requiring hospitalization. Now
that food borne bacteria have also been found with resistance to antimicrobial agents, the
impact may be far greater than those nosocomial pathogens.

It is widely speculated that antimicrobial used in food animal production
contribute significantly to the development of food borne bacteria with resistance to
antimicrobial agents. One of the reasons was that amount of antimicrobial agents used as
growth promoters in food animal production far exceed those used in human medicine. In
developed countries, antimicrobial usage in humans and animals are strictly regulated,
whereas in developing countries, antimicrobial usage is not strictly regulated. Therefore,
the prevalence of resistance bacteria can be expected to be higher in developing
countries. With the loose control of antimicrobial usage in human and animals and the
large size of its food animal industry, Thailand offers a unique opportunity to study this

problem.

There is a critical need for epidemiological information on foodbome bacteria
with resistance to antimicrobial agents from developing countries. Apart from public
health concerns, the increasing trade in foods of animal origin around the world makes
the safety of such foods an important issue for food-producing countries such as
Thailand. The information will be important for regulatory agencies overseeing the use of
antimicrobial agents in food animals and humans, and agencies overseeing the trade of

foods fiom animal origin.

PROBLEM STATEMENT

Despite the public health and economic significance of Campylobacter spp. in
food animals in Thailand, there is only a limited base information available.
Campylobacter spp. with resistance to antimicrobial agents have been reported in both
developed and developing countries, and the prevalence of potentially antimicrobial-
resistant Campylobacter spp. in food animals indicates that foods of animal origin may be
a source of resistant bacteria to humans. Consequently, there is an urgent need to fill this

gap in knowledge.

OBJECTIVES

In order to determine whether food animals are an important source of
antimicrobial-resistant bacteria to humans, a thorough, stepwise research approach is
needed. This thesis focuses on the epidemiology and antimicrobial resistance of
Campylobacter spp. in food animals and humans in Thailand. The main objectives of the

study were to:

1. Validate the use of a fluorogenic PCR assay to identify C. jejuni from field samples.

2. Determine the frequencies and the antimicrobial susceptibility level of
Campylobacter spp. isolated from food animals and workers at pig and chicken
farms, slaughterhouses, and markets.

3. Compare the frequencies of Campylobacter spp. with resistance to antimicrobial
agents in food animals, food products and farm workers.

4. Determine the risk factors associated with the observed frequencies of Campylobacter
spp. and frequencies of Campylobacter spp. with resistance to antimicrobial agents
isolated from various sources.

5. Determine whether antimicrobial use in feed and treatment on pig and chicken farms
is associated with the frequency of antimicrobial resistance in Campylobacter.

6. Determine the association between mutation in gyrA gene of Campylobacterjejuni
and ciprofloxacin resistance.

HY POTHESES

The following hypotheses were tested:

1.

A fluorogenic PCR assay can be used to identify C. jejuni from field samples,
providing results comparable to conventional phenotypic tests.

Campylobacter spp. are prevalent in pig and chicken production systems in northern
Thailand.

Major factors influencing the observed prevalence of Campylobacter spp. in pig and

chicken production systems can be determined.

 

 

4. Campylobacter spp. with resistance to antimicrobial agents are prevalent throughout
the pig and chicken production systems and farm and slaughterhouse workers in
northern Thailand.

5. Antimicrobial use in pig and chicken production systems is associated with the
frequency of antimicrobial resistance in Campylobacter isolated from these animals

and farm workers.

 

6. The Thr-86-to-Ile mutation in the gyrA gene of C. jejuni is associated with resistance

to ciprofloxacin. 1"

OVERVIEW

Chapter 1 is a literature review of the epidemiology and antimicrobial resistance

 

of Campylobacter spp. in humans and food animals, focusing on comparisons between
developed and developing countries. Chapter 2 is a literature review of molecular
techniques used in epidemiological studies of Campylobacter spp., focusing on the
available molecular techniques and application of those techniques.

Chapter 3 addresses Hypothesis 1, by reporting a laboratory test of a fluorogenic
PCR assay in comparison with conventional phenotypic tests. Chapters 4 and 5 address
Hypotheses 2 and 3, and describe results of an epidemiological study of Campylobacter
spp. in pig, chicken and farm workers. Chapter 6 addresses Hypotheses 4 and 5, which
involve an epidemiological study of antimicrobial resistance in Campylobacter spp. from
pigs, chickens, and farm workers. Chapter 7 addresses Hypothesis 6, reporting a
laboratory study of the mechanism of fluoroquinolone resistance in C. jejuni. The last

section of this thesis is a summary of the results of these results.

 

CHAPTER 1
CAMPYLOBA CTER SPP., AND THEIR ANTIMICROBIAL RESISTANCE, IN
HUMANS, CHICKENS, AND PIGS : A REVIEW
INTRODUCTION

Campylobacter spp. are gram-negative, non-spore forming curved or spiral
bacilli which grow under microaerophilic conditions. The first Campylobacter may have
been isolated in 1913, but were classified as Vibrio spp. until the genus Campylobacter
was established in 1963. Currently, the Campylobacteriaceae include the genera
Campylobacter and Arcobacter (Vandamme, 2000), with the related genera Helicobacter
and Wolinella in the novel family Helicobacteriaceae (V andamme, 2000). There are 14
species of Campylobacter (Vandamme, 2000), of these C. jejuni is frequently associated
with human gastroenteritis (Friedman, et al., 2000), and C. fetus subsp. fetus and
venerealis are important animal pathogens (Garcia, etal., 1983).

The purpose of this literature review was to compare Campylobacter spp. in
food animals and humans in developed and developing countries. In particular,
comparisons were made on: 1) prevalence/incidence of carnpylobacteriosis, 2) major
means of exposure and transmission, 3) clinical disease expression, and 4) problems of
antimicrobial resistance observed in these organisms, including factors associated with
the occurrence and development of resistance. It is believed that a comparative analysis
of the literatures will provide some insight into identifying gaps in our knowledge
regarding the epidemiology of Campylobacter infections and associated disease, different

ways to control the problem, and suggest critical areas for future research.

 

PREVALENCE AND INCIDENCE OF CAMPYLOBACTER SPP.

Campylobacter in Humans. Of all Campylobacter spp., C. jejuni is the
species most frequently isolated in cases of human infection (Tay, et al., 1995). In
developing countries, most reported Campylobacter infections are in children. Peaks in
Campylobacter infection rates have been reported in children less than one year of age
(Friedman, et al., 2000), and children less than 5 years old in Southeast Asia
(Oberhelrnan and Taylor, 2000). Previously reported prevalences of Campylobacter spp.
in children in Southeast Asia range from 2.9% to 15% (Phetsouvanh, et al., 1999;
Rasrinual, et al., 1988; Taylor, et al., 1993; Varavithya, et al., 1990). In children in
Thailand, Campylobacter spp. and Salmonella are common causes of diarrhea (Rasrinual,
et al., 1988; Varavithya, et al., 1990), and co-infection. with Campylobacter and E. coli,
Salmonella or Shigella is common (Poocharoen and Bruin, 1986). This exposure to
Campylobacter spp. early in life, and reports of levels of Campylobacter-specific
antibodies increasing with age (Taylor, et al., 1993), may result in less severe clinical
symptoms in adults, which makes detection and reporting of cases rare. Since
Campylobacter is commonly isolated with other enteric pathogenic bacteria in
developing countries, reports of Campylobacter cases may be largely underestimated.
There have been no reports of seasonal patterns in the occurrence of human
Campylobacter infection in developing countries

In developed countries, Campylobacter spp. have been identified as etiologic
agents in outbreaks and sporadic cases of gastroenteritis (Lehner, et al., 2000). The
reported incidence of Campylobacter infection in US. was 20.1 cases per100,000 in 2000

(Acheson, 2001), and from 60 to 90 cases per 100,000 in northern Europe (Friedman, et

 

al., 2000). When taking under-reporting into consideration, the true incidence rate is
estimated to be 1,000 - 2,300 cases per 100,000 in Europe (Friedman, et al., 2000).
Campylobacter affects all age groups in developed countries, with one peak in children
less than 4 years old, and in young adults from age 15 to 44 (Friedman, et al., 2000). The
incidence of Campylobacter in developed countries also showed seasonal patterns, with
peaks during the warmer months of the year (Friedman, et al., 2000), which may be a
result of increasing survival of Campylobacter in the environment in warm weather.
The frequency and pattern of occurrence of Campylobacter spp. differ
between developed and developing countries, especially in the number of cases reported
in adults and the presence of any seasonal patterns in occurrence. It should be noted that
there is limited information on the incidence of Campylobacter infections from
developing countries. Most developed countries in Europe and North America have
surveillance programs for Campylobacter and other foodbome pathogens, such as
FoodNet in the US. (Acheson, 2001). Establishment of similar surveillance programs

would be beneficial in developing countries.

Campylobacter in Food Animals. Many different animal species maintain
Campylobacter spp. with no clinical signs (Steinhauserova, et al., 2001). The most
important species of Campylobacter in veterinary medicine are C. fetus subsp. fetus and
venerealis (Garcia, et al., 1983). Of Campylobacter spp. that are pathogenic in food
animals, C. fetus can cause reproductive disorders in cattle and sheep (Quinn, et al.,
1994), and C. hyointestinalis and C. mucosalis have been associated with enteritis in pigs

and cattle (Garcia, et al., 1983).

There is limited information on Campylobacter spp. in food animals or foods
of animal origin from developing countries. In Thailand, Campylobacter spp. was
isolated from 12% of various food samples including pork, chicken and vegetables in
Bangkok (Rasrinual, et al., 1988), 40% of poultry ceca in India (Das, et al., 1996), and
68-100% of poultry samples from retail markets in Taiwan (Shih, 2000). The low
prevalence of Campylobacter reported in Thailand was probably due to the fact that
various types of meats and vegetable were included in the study, while the other two
studies examined only poultry products. Campylobacter was isolated more in samples
from fresh markets than in supermarkets in Taipei, Taiwan (Shih, 2000). In Kenya,
Campylobacter was found on 77% of poultry products at the market (Osano and Arirni,
1999), while the prevalence of Campylobacter at the poultry farm was found to be 64-
100% (Kazwala, et al., 1990; Simango and Rukure, 1991). By species, C. jejuni was
isolated more frequently than C. coli in live chickens in Afiica (Simango and Rukure,
1991)

There have been many studies on Campylobacter spp. in food animals, and
foods of animal origin, in developed countries. In the past, research in cattle was limited,
but has been increasing as outbreaks of human carnpylobacteriosis have been traced to
foods of cattle origin (Kalman, et al., 2000). The prevalence of Campylobacter was
found to be 15% in beef calves (Busato, et al., 1998), 37.7% in dairy herds (Wesley, et
al., 2000), and 89.4% on beef cattle at slaughter (Stanley, et al., 1998). The prevalence in
beef cattle at slaughter peaked in the summer (Stanley, et al., 1998), which coincides with
the seasonal peak of human Campylobacter infections (Friedman, et al., 2000).

Extensive research has been conducted on Campylobacter in pigs. Pigs carry

higher proportions of C. coli than C. jejuni, whether they have enteritis or not (Harvey, et
al., 1999; Steinhauserova, etal., 2001). In the US, Campylobacter spp. was isolated
from 76% of gilts, 100% of pregnant sows, 57.8% of newborn piglets, and 100% of
weaning pigs (Young, et al., 2000). Fifty percent of piglets were infected with the same
serotype as their sows by seven days of age in the Netherlands (Weijtens, et al., 1997).
The average number of Campylobacter colonizing the gut decreased toward the end of
the rearing period (Weijtens, et al., 1999). A study in Belgium reported the prevalence of
Campylobacter spp. on pig carcasses at slaughterhouses to be 2% (Korsak, et al., 1998),
and Campylobacter was found in 1.3% of samples from pork from a retail market in the
US. (Duffy, et al., 2001).

There is a large body of research on Campylobacter in poultry. The reported
prevalence of Campylobacter in broiler flocks ranged between 35-57% in Europe
(Atanassova and Ring, 1999) (Pearson, et al., 1996; Refregier-Petton, et al., 2001; Van de
Giessen, et al., 1996). The prevalence of Campylobacter colonization was higher in
organic farms (100%) when compared to extensive indoor system (49.2%) or
conventional farms (36.7%) (Heuer, et al., 2001). A study at Swedish slaughterhouses
showed a 27% flock prevalence, which increased with bird age and flock size (Bemdtson,
et al., 1996). The prevalence of infection in chickens has been shown to increase linearly
with the age of the birds (Genigeorgis, et al., 1986). At broiler farms, day-old chickens
can be colonized with Campylobacter (Stern, et al., 2000), with rates of 100%
colonization by 3-4 weeks (J acob-Reitsma, et al., 1995). Up to 40% of the flocks in the
UK. were found to be colonized with Campylobacter spp. by four weeks, and up to 90%

by seven weeks (Evans and Sayers, 2000). After transportation, counts of

10

 

Campylobacter in chickens increased (Stem, et al., 1995; Whyte, et al., 2001), although
there was no significant increase in prevalence (Whyte, et al., 2001). The reported
prevalence of Campylobacter spp. in retail chickens varied from 28.5-100% (Table 1-1).
Lower levels of contamination were found in chicken pieces without skin (Berrang, et al.,
2001; Uyttendaele, et al., 1999), but removal of skin did not change the prevalence of
Campylobacter spp. found on chicken carcasses (Berrang, et al., 2001). Frozen chickens
had lower bacteria counts than fresh chickens (Dufrenne, et al., 2001; Stem, et al., 1984).
The highest levels of recovery usually occurred during the warmer months (Jun - Oct)
(Jacob-Reitsma, et al., 1994; Stem, et al., 1985; Wedderkopp, et al., 2000; Willis and
Murray, 1996), and more C. jejuni (43-86%) than C. coli (1 1-39%) were recovered
(Atanassova and Ring, 1999; Jacob-Reitsma, et al., 1994; Shih, 2000; Wedderkopp, et al.,
2000). The seasonality of Campylobacter found in poultry products at the market
coincide with the peak of incidence of Campylobacter in humans, which demonstrates
the importance of chicken as a source of Campylobacter infection for humans in
developed countries.

There do not appear to be significantly different colonization rates of
Campylobacter in food animals between developed and developing countries. However,
since the housing and management of food animals differs between developing and
developed countries, the rate of colonization in developing countries, which are mostly
located near the equator, should be higher than those in developed countries. This

speculation should be confirmed by field observation.

ll

EXPOSURE AND TRANSMISSION OF CAMPYLOBACTER

In Humans. In developed countries, Campylobacter spp. are associated with
sporadic cases and outbreaks of infection. Outbreaks of Campylobacter are usually
associated with raw milk (Altekruse, et al., 1998; Kalman, et al., 2000), whereas sporadic
illnesses are often associated with consumption of chickens (Deming, et al., 1987; Effler,
et al., 2001; Kapperud, et al., 1992; Schorr, et al., 1994; Studahl and Andersson, 2000).
Other reported risk factors for Campylobacter spp. infection included handling of
chickens or preparing chickens (Hopkins and Scott, 1983), contact with cats (Deming, et
al., 1987), consuming antibiotics before illness (Effler, et al., 2001), eating pork (Studahl
and Andersson, 2000), barbequing (Kapperud, et al., 1992; Studahl and Andersson,
2000), living or working on farms (Studahl and Andersson, 2000), working in
slaughterhouses (Cawthraw, et al., 2000), exposure to animals with diarrhea (Saeed, et
al., 1993), and travel abroad (Schorr, et al., 1994). Traveling to developing countries was
associated with Campylobacter with resistance to antimicrobial drugs (Gaunt and
Piddock, 1996). Foreigners residing in countries where Campylobacter is prevalent also
have high risks of infection (Gaudio, et al., 1996). In addition to traditional outbreak
investigational techniques, various molecular identification techniques are now available
for epidemiological typing of Campylobacter spp.(Wassenaar and Newell, 2000). These
techniques have been used to trace outbreaks back to broiler or dairy farm and food
handlers (Kalman, et al., 2000; Olsen, et al., 2001; Pearson, etal., 2000). A recent
molecular study also suggested a link between the Campylobacter spp. found in farm

environments and those causing diseases in local communities (Fitzgerald, et al., 2001).

12

In developing countries, chicken products were found to be an important
source of Campylobacter in humans in both Asia (Rasrinual, et al., 1988) and Africa
(Simango and Rukure, 1991), and chickens were also found to be a potential source of
Campylobacter in farm workers (Simango and Rukure, 1991). Although higher
prevalences of Campylobacter on poultry products was reported in developing countries,
the local customs of eating well-cooked meat and poultry may reduce the risk of
infection, when compared to developed countries, where the consumption of

undercooked meat is more common.

In Food Animals. There have been several studies on the epidemiology of
Campylobacter in poultry in developed countries. Sources of infection were more likely
to be horizontal contamination from the environment (Jacob-Reitsma, 1997; Van de
Giessen, et al., 1992) or water system (Kazwala, et al., 1990; Pearson, etal., 1993), rather
than from direct flock-to-flock transmission (Pearson, et al., 1996). Although prevalence
of Campylobacter spp. is high (67%) in breeder flocks, serotyping of the organism did
not support the hypothesis of vertical transmission (Chuma, et al., 1997; Jacob-Reitsma,
1995; Petersen, et al., 2001). Additional risk factors for Campylobacter colonization
include housing with no air circulation, more than two workers working in the same
poultry house, more than three poultry houses, acidified drinking water, the presence of
insects or litter in worker changing rooms (Refiegier-Petton, et al., 2001), batch depletion
of the flock (not all in/all out) (Hald, et al., 2001), and the presence of other animals (pig,
cattle, sheep) on the farm (Van de Giessen, et al., 1996). Wild birds have been found

with Campylobacter spp., with prevalences up to 50% in birds near chicken houses

13

(Craven, et al., 2000) and 40% in dead wild birds found in broiler houses (Jones, et al.,
1991), and has been found in wild birds not associated with poultry facilities (Chuma, et

al., 2000).

A longitudinal study that followed the same flock of chickens fiom farm to
processing plant and market found the highest prevalence of Campylobacter at the
processing plant (32.5%), especially on carcasses after chilling (52%), suggesting that
contamination of carcasses most likely occurred at the processing plant(J ones, et al.,
1991). Counts of Campylobacter were highest in caeca and colon of birds at slaughter
(Berrang and Dickens, 2000), and the number of Campylobacter declined after scalding,
but increased during picking and evisceration (Berrang and Dickens, 2000; Whyte, et al.,
2001), which suggested that the caecum and colon are the most probable sources of
contamination in the slaughterhouses, which has been confirmed by molecular typing
(Newell, et al., 2001). After processing contaminated flocks, Campylobacter spp. can be
found on all slaughter equipment (Bemdtson, et al., 1996; Newell, et al., 2001), making
cross-contamination between different flocks at slaughter possible (N ewell, et al., 2001;
Rivoal, etal., 1999). Other risk factors for the isolation of Campylobacter at
slaughterhouses include short empty period between flocks, the lack of strict baniers
such as changing boots between houses, wet litter or bedding, the presence of other
poultry on the farm, staff handling other poultry on the farm, dividing the flock before
slaughter, slaughterhouse staff loading birds from several different farms, and the

presence of mice (Bemdtson, et al., 1996).

14

In developed countries, the permanent colonization of Campylobacter spp. in
pigs is probably related to constant exposure to other colonized pigs, since experiments
have shown that pigs reared off-sow had less prevalence of Campylobacter spp. (Harvey,
et al., 2000).

Very little research has been done on the epidemiology of Campylobacter in
food animals in developing countries. In one study, Campylobacter were isolated
frequently from surface water and foot baths, which may be a potential source of
Campylobacter on the farm (Kazwala, et al., 1990). Because of the differences in the
rearing systems and the slaughtering processes, risk factors for Campylobacter
contamination in food animals in developing countries may be significantly different
from those in developed countries. Some factors on the farm, such as biosecurity
measures and the presence of wild birds, may be similar between developed and
developing countries. In many developing countries, the majority of the slaughterhouses
that process meat for local consumption commonly have lower standards of hygiene,
which may also result in higher prevalence of Campylobacter on their products.
However, meat in local markets in developing countries usually does not remain in the
market more than one day after slaughter, so bacteria do not have the opportunities to
multiply as much as in developed countries, especially for slow-growing organism like
Campylobacter. Epidemiological studies of Campylobacter in food animals under
different rearing systems in developing countries should be conducted to clarify the risk
factors associated with the presence of Campylobacter in food animal and food animal

products.

15

CLINICAL EXPRESSION OF CAMPYLOBACTER INFECTION IN HUMANS
AND ANIMALS

In Humans. Campylobacterjejuni and Campylobacter coli have been
recognized as important etiologic agents of gastrointestinal infection since the 1970s
(Nachamkin, et al., 2000). In developed countries, typical clinical feature of
Campylobacter spp. infection include acute, self-limiting gastroenteritis, characterized by
diarrhea, fever and abdominal cramps, with a 24 to 72 hour incubation period (Allos,
2001). Diarrhea is initially watery, which may last more than two weeks in travellers’s
diarrhea (Gallardo, et al., 1998), or may become bloody as a result of diffirse
inflammatory colitis and enteritis (Blaser, 1997). An important post-infection sequela of
Campylobacterjejuni infection is Guillain-Barré syndrome (GBS), an acute
demyelinating disease of the peripheral nervous system that results in a flaccid paralysis
(Acheson, 2001; Altekruse, et al., 1998).

In developing countries, the clinical picture of Campylobacter infection is
characterized by a milder form of gastroenteritis (Oberhehnan and Taylor, 2000), and
symptomatic infection is uncommon in adults (Blaser, 1997). Severe diarrhea requiring
hospitalization is usually the result of co-infection with other virulent bacteria or viruses
(V aravithya, et al., 1990). The severity of clinical symptoms and fecal excretion in
children was inversely related to age (Taylor, et al., 1993). Although the severity of
Campylobacter infection in adults was different between developed and developing
countries, the clinical symptoms of infection in adults resulting from infection in
developing countries was similar to those in developed countries (Oberhelrnan and

Taylor, 2000).

16

In Food Animals. In cattle, C. fetus subsp. venerealis is the most frequently
encountered Campylobacter in bovine abortion or infertility, while C. fetus subsp. fetus
causes sporadic abortion in cattle and enzootic abortion in sheep (Garcia, et al., 1983).
Bovine venereal carnpylobacteriosis is a chronic infection of the female genital tract,
characterized by mild endometritis and transient infertility (Garcia, et al., 1983).
Infection in bulls is accompanied by neither histological changes nor modifications in the
characteristic of semen (Garcia, et al., 1983). C. fetus subsp. fetus infection in ewes

resulting in abortion commonly in the last six week of gestation (Garcia, et al., 1983).

The role of C. jejuni as a primary pathogen in farm animals is uncertain. C. jejuni can be
found in feces of diarrheic and healthy calves (Radostits, et al., 2000) and piglets
(Steinhauserova, et al., 2001), but both C. jejuni and C. coli can cause a mild self-limiting
enteritis and bactererrria when inoculated orally to newborn calves (Radostits, et al.,
2000). In cows, there has been a single report of mastitis caused by C. jejuni (Morgan, et

aL,1985)

ANTIMICROBIAL RESISTANCE IN CAMPYLOBACTER

Antimicrobial resistance has become a major public health concern in both
developed and developing countries in recent years (Isenbarger, et al., 2002; Witte,
1998). Resistance to antimicrobial agents in respiratory and enteric bacteria poses the
risk of treatment failure and increased cost of treatment (V asallo, et al., 1998).

Campylobacter with resistance to antimicrobial agents have been reported in both

17

developed and developing countries (Table l-l), and the situation seems to deteriorate
more rapidly in developing countries, where there is widespread and uncontrolled use of
antibiotics (Hart and Kariuki, 1998). F luoroquinolones and macrolides are the
antimicrobial agents of choice for empirical treatment of gastroenteritis, and therefore
resistance to these classes of antimicrobial agents are of paramount concern in

Campylobacter (Engberg, et al., 2001).

Mechanisms of resistance to antimicrobial agents in Campylobacter spp. Resistance to
aminoglycosides is normally mediated by enzymes that modify the drugs (Aarestrup and
Engberg, 2001). Mechanism of resistance to chloramphenicol depends on the ability to
produce enzymes that block binding of the drug to the ribosome, which is the target site
of action (Aarestrup and Engberg, 2001). In Cjejuni and C. coli, resistance to tetracycline
was found to be located on a transmissible plasmid encoding ribosomal protection gene
(Aarestrup and Engberg, 2001). Macrolide resistance in Cjejuni is chromosomally
mediated through mutation of the 23S rRNA gene (Engberg, et al., 2001). Mechanism of
fluoroquinolone resistance in C.jejuni was also found to be chromosomally mediated
through mutation of the gyrA gene (Wang, et al., 1993) and parC gene (Gibreel, et al.,
1998). Resistance to azithromycin was found to be correlated with resistance to
quinolones (Isenbarger, et al., 2002). Resistance to more than one group of antimicrobial

agents in Cy'ejuni may be the result of efflux mechanism (Charvalos, et al., 1995).

Antimicrobial susceptibility testing. There are several methods currently

being used for antimicrobial susceptibility testing of Campylobacter. Agar dilution,

18

commonly used in Europe, gives qualitative results but requires a high level of
standardization for comparison (Caprioli, et al., 2000). Broth dilution provides
quantitative data and is highly reproducible, but is more expensive than agar dilution
(Caprioli, et al., 2000). The E-test (AB Biodisk, Culver city,CA) was found to give
results comparable to the agar dilution test (Huang, et al., 1992), but is also expensive.
Various genetic methods for assessing antimicrobial resistance are also available
(Cockerill III, 1999). Comparisons of the results of antimicrobial susceptibility testing
should be made with care, since different methods from the same laboratory or similar
methods in different laboratories may yield different results (Caprioli, et al., 2000). For
surveillance, quantitative data may be more desirable, since it can detect trends that
indicate reduced susceptibility, and interventions can be implemented before high levels

of resistance have developed (Walker and Thomsberry, 1998).

Antimicrobial resistance in human isolates. Increasing prevalence of
quinolone-resistant Campylobacter spp. has been observed in the Netherlands since the
early 1990’s (Endtz, et al., 1990; Talsma, et al., 1999), where resistance to
fluoroquinolones increased from 11% in 1994 to 29% in 1997 (Talsma, et al., 1999).
Quinolone-resistant Campylobacter have recently been reported in many other countries,
including Canada (Gaudreau and Gilbert, 1998) and Spain (Prats, et al., 2000). In the
US, Campylobacter with resistance to nalidixic acid increased fi'om 1.3% in 1992 to
10.2% in 1998 (Smith, et al., 1999).

Resistance to erytlrromycin appeared in the Netherlands in the 1990s (Talsma,

et al., 1999). Resistance to macrolides has been reported to be as high as 90% in Spain

19

(Mirelis, et al., 1999), but trends over time for erythromycin resistance show stable and
low rates in Japan, Canada and Finland (Engberg, et al., 2001). Resistance to macrolides
was found to be more prevalent in C. coli than C. jejuni (Mirelis, et al., 1999; Saenz, et
al., 2000).

There are several risk factors associated with the isolation of antimicrobial-
resistant Campylobacter. Isolation of Campylobacter with resistance to quinolones was
found to be associated with foreign travel and use of quinolone prior to isolation (Smith,
et al., 1999). Exposure to fluoroquinolones in both humans and animals was found to
induce resistance in Campylobacter (McDermott, et al., 2002; Segreti, et al., 1992;
Wretlind, et al., 1992). The frequency of isolation also showed seasonal patterns with
lower levels of resistant isolates in the summer months, when the overall incidence of
Campylobacter isolation was higher (Talsma, et al., 1999). Consumption of
contaminated foods is also a source of resistant bacteria: it has been estimated that, in the
worse case scenario where prevalence of resistance Campylobacter is as high as 84%, 3-4
people per year in US will die as a result of infection with fluoroquinolone resistant
Campylobacter spp. from beef consumption (Anderson, et al., 2001).

Antimicrobial resistance in human Campylobacter isolates has been found in
developing countries. In Kenya, 51% of diarrhea patient had isolates of bacteria that
were not susceptible to antimicrobial treatment, and 24% of Campylobacter was
resistance to nalidixic acid (Shapiro, et al., 2001). In Thailand, resistance to azithromycin
remained low (6%) (Isenbarger, et al., 2002) and therefore may be used empirically to
treat gastroenteritis (Kuschner, et al., 1995). Similar to developed countries, resistance to

macrolides was found to be more prevalent in C. coli than C. jejuni in Taiwan (Li, et al.,

20

1998), with resistance to erythromycin in 50% of C. coli isolated from human patients
(Li, et al., 1998). In Thailand where fluoroquinolones are widely used in broiler farms to
control respiratory disease, the prevalence of quinolone resistance Campylobacter spp.
was found to increase from 0% in 1987 to 84% in 1995 (Hoge et al., 1998).
Antimicrobial resistance in isolates from food animals. The resistance to
fluoroquinolones in food animal was observed in the Netherland since early 1990’s
(Endtz, et al., 1990). Since then, there have been several reports of antimicrobial
resistance in Campylobacter spp. isolated from food animals in both developed and
developing countries (Table 1-2). High level of resistance to fluoroquinolones and
tetracycline were reported in the Netherland (J acob-Reitsma, et al., 1994), Spain (Saenz,
et al., 2000), Ireland (Lucey, et al., 2000) and Taiwan (Li, et al., 1998). Resistance to
tetracycline was much higher in Taiwan compared to other countries in Europe (Table 1-
2). Resistance to gentamycin was relatively low in Spain (Saenz, etal., 2000), Ireland
(Lucey, et al., 2000) and Taiwan (Li, et al., 1998). Only resistance to chloramphenicol
was found in Campylobacter spp. isolated from chickens in Switzerland (Frei, et al.,

2001).

Link between resistance in food animal isolates to resistance in human
isolates. There has been speculation that the use of antimicrobial agents, such as
quinolones, in food animals resulted in increasing the prevalence of quinolone resistance
in Campylobacter spp. from humans (Endtz, et al., 1991; Gaunt and Piddock, 1996;
Piddock, 1996) followed the approval of fluoroquinolone use in food animals. There

seems to be temporal relationship between use of fluoroquinolones in animals and the

21

finding of resistant Campylobacter in humans (Engberg, et al., 2001). In Spain, the
proportion of human isolates of Campylobacter resistant to quinolones was found to be
72%, while 99% of Campylobacter isolated from broilers and pigs were resistant to
quinolones (Saenz, et al., 2000). In Taiwan, 52% of C. jejuni isolated from human
patients were resistance to ciprofloxacin, while 92% of isolates from chickens were
resistant to the same agent (Li, et al., 1998). Since C. coli was the the most common
species of Campylobacter isolated from pigs (Steinhauserova, et al., 2001), resistance to
macrolides in human isolates may reflect the use of macrolides in pork production in
Taiwan. In Denmark, it was shown that proportion of Enterocooci with resistance to
avoparcin, tylosin, erythromycin, Virginiamycin and avilarnycin reduced after those
agents were banned from the market (Aarestrup, et al., 2001). This observation indicated
significant association between antimicrobial use as growth promoter in food animal and
prevalence of resistance bacteria. Similar phenomenon may be observed in

Campylobacter spp., which should be confirmed.

In summary, resistance was observed at high levels in food animals in both
developed and developing countries. Studies suggested an association between
antimicrobial use in food animals and the development of resistance in human isolates in
developed countries. Traveling to developing countries has also been related to isolation
of resistant Campylobacter. However, there have been no reports of risk factors for the
development of antimicrobial resistance in developing countries. In most developing
countries, while antimicrobial use is usually less regulated than developed countries,

economic necessity may limit the use of newer, more expensive antimicrobial agents. An

22

epidemiological study of risk factors associated with the development of antimicrobial
resistance in developing countries will be valuable, in order to prolong the usefulness of

the available antimicrobial agents for treatment of important human pathogens.

23

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37

CHAPTER 2
MOLECULAR TECHNIQUES IN EPIDEMIOLOGICAL STUDY OF
CAMPYLOBA CTER SPP. : A REVIEW

INTRODUCTION

Campylobacter spp. Have been recently recognized as an emerging food borne
bacteria in many developed countries (Acheson, 2001; Pearson, et al., 2000;
Phetsouvanh, et al., 1999; Tay, et al., 1995). C. jejuni and C. coli have been recognized as
important etiologic agent of gastrointestinal infection since 1970 (Nachamkin, et al.,
2000). Typical clinical features of Campylobacter spp. infection include acute, self-
limiting gastroenteritis characterized by diarrhea, fever and abdominal cramps (Allos,
200]). The incubation period usually ranges between 24-72 hours. Diarrhea is initially
watery then becomes bloody, which is a result of diffuse inflammatory colitis and
enteritis (Blaser, 1997). In developing countries, symptomatic infection is uncommon in
adult (Blaser, 1997). C. jejuni is also considered a cause of travelers’ diarrhea resulting in
watery diarrhea which last more than 14 days (Gallardo, et al., 1998). An important post-
infection sequel of C. jejuni infection is Guillain-Barre syndrome (GBS), an acute
demyelinating disease of the peripheral nervous system resulting in flaccid paralysis
(Acheson, 2001; Altekruse, etal., 1998).

Campylobacter spp. are associated with both sporadic and outbreak infection. In
the US, the incidence of Campylobacter spp. infection was found to be 20.1 cases per
100,000 population in 2000 (Acheson, 2001). Outbreaks of Campylobacter spp. are

usually associated with raw milk (Altekruse, et al., 1998; Kalman, et al., 2000), whereas

38

 

sporadic illnesses are often associated with consumption of chickens (Deming, et al.,
1987; Effler, et al., 2001).

In Southeast Asia, Campylobacter spp. infection was found predominantly in
children less than 5 years old. Previously reportedprevalences of Campylobacter spp. in
this area range from 2.9% - 15% (Phetsouvanh, et al., 1999; Rasrinual, et al., 1988;
Varavithya, et al., 1990) in children, and up to 10% in adults (Gaudio, et al., 1996). In
diarrheic children in Thailand, Campylobacter spp. was isolated as frequently as entero-
toxogonic E.coli and Salmonella (Rasrinual, et al., 1988). There was no obvious seasonal
pattern, and childrens with Campylobacter spp. are often co-infected with E.coli,
Salmonella or Shigella (Poocharoen and Bruin, 1986).

Because of the growing concern over the public health significance of
Campylobacter spp., various molecular techniques have been developed and applied in
epidemiological studies of Campylobacter spp. A review of genotyping techniques for
Campylobacter spp. was recently published (Wassenaar and Newell, 2000). However, a
review of the application of molecular techniques in epidemiological studies is not
available. The objectives of this review, therefore, were to 1.) summarize available
molecular techniques applicable to Campylobacter spp. and 2.) review the applications of
molecular techniques in epidemiological studies of Campylobacter spp. A comparison of
available techniques and suggestions for the application of molecular techniques in

epidemiological studies of Campylobacter spp. will be discussed.

39

AVAILABLE MOLECULAR TECHNIQUES FOR EPIDEMIOLOGICAL
STUDIES OF CAMPYLOBA CT ER SPP.

Molecular techniques for detection and identification of Campylobacter spp.
Campylobacter spp. are slow-growing, fastidious organisms, which prefer
microaerophilic conditions (5% Oz, 10% C02, 85% N2) (Nachamkin, et al., 2000). Most
Campylobacter grow well at 37°C, but a group of thermophilic Campylobacter, such as
C. jejuni and C. coli, also grow well at 42°C. Isolation and identification of
Campylobacter usually begins by culture in selective enrichment media, which includes
antibiotics to limit the grth of competitive organisms, and ingredients to neutralize the
toxic effects of oxygen and light (Corry, et al., 1995). There is no single selective media
able to recover all types of Campylobacter spp. (Moore and Murphy, 2000). Because of
the strict conditions required for bacterial growth, the length of time required for culture
(6 days) (Nachamkin, et al., 2000), and the varying phenotypes of isolates within a single
species (Rautelin, et al., 1999), other methods of detecting and identifying
Campylobacter are attractive alternatives. Molecular assays for Campylobacter and other
bacteria (Hoorfar, et al., 2000; Shanna, et al., 1999) offer the means to detect the
presence of bacteria without culture.

The polymerase chain reaction (PCR) is one of the most widely used molecular
techniques in detection and identification of bacteria (Saiki, et al., 1985), and various
protocols for detecting and identifying Campylobacter spp. have been developed (Table
2-1). PCR employs Taq polymerase to synthesize double stranded DNA specific to the
primers used in the reaction. PCR can be carried out in a thermocycler in which the DNA

product doubles every cycle, creating a specific product from a small amount of starting

40

template. Steps in doing PCR involve DNA extraction, amplification and visualization of
PCR product.

DNA extraction There are different protocols for DNA extraction such as
phenol-chloroform extraction (Ausubel, et al., 1997), and guanidine-thiocyanate
extraction (Boom, et al., 1990). Basically, DNA extraction exploit the ability of DNA to
bind to organic salts. This complex can be separated from other cell debris after lysis.
Then DNA can be purified using an organic solvent.

The sample matrix and DNA extraction process may substantially affect PCR
efficiency (Englen and Kelly, 2000; Ludwig and Schleifer, 2000), resulting in a loss of
sensitivity when applied to certain types of samples (Chuma, et al., 1997). Different
protocols for DNA extraction have successfirlly extracted Campylobacter DNA from
fecal specimens (Lawson, etal., 1998; Rasmussen, et al., 1996; Vanniasinkam, et al.,
1999) and food samples (O'Sullivan, et al., 2000). A recently developed immuno-
magnetic separation procedure has been used to separate Campylobacter cells fiom foods
(Y u, et al., 2001), and has yielded satisfactory results (Waller and Ogata, 2000).
Mechanical disruption of cells in the presence of DNAzol has proven useful when
extracting DNA from heat-resistant Campylobacter in environmental samples (Englen
and Kelly, 2000).

AmplificaLtion Several different primers have been used to achieve identification
of Campylobacter to the species level (Lawson, et al., 1998). The most commonly used
gene for PCR amplification is the 16S rRNA gene. Several other genes have proven
useful, such as flaA (Rasmussen, eta1., 1996),flaB (Rasmussen, et al., 1996), ceuE

(Houng, et al., 2001), 23S rRNA (Eyers, et al., 1993) and cadF (Konkel, et al., 1999).

41

Visualization of PCR products PCR coupled with ELISA utilizing specific
probes (Metherell, et al., 1999) or using restriction fi'agment length polymorphism
(RFLP) profiling has been used to identify species of Campylobacter after amplification
(Fermer and Engvall, 1999; Hurtado and Owen, 1997; Korolik, et al., 2001; Marshall, et
al., 1999). PCR coupled with other product visualizing techniques, such as colorimetric
assay (Casademont, et al., 2000; O'Sullivan, et al., 2000), has been developed, allowing
automated and rapid reading of results. Some colorimetric systems can yield quantitative
results (Waller and Ogata, 2000). A fluorogenic PCR has been developed for identifying
C. jejuni (Wilson et al., 2000), and has been shown to be more specific than standard
phenotypic tests using a conventional test kit (Padungtod, et al., 2002).

Advantages of PCR The advantages of using the PCR technique include
increased specificity (Lilja and Hanninen, 2001), sensitivity (V anniasinkam, et al., 1999)
and reduction in time required (Denis, et al., 1999). PCR was shown to yield 97.8%
specificity when detecting C. jejuni from stool samples, when compared to culture
technique (W aegel and Nachamkin, 1996). The detectability level (concentration of
bacteria ; CFU/ml) of PCR was lower than plate culture, however, the total number of
cells require for detection was much less (Lawson, et al., 1998). PCR was shown to yield
comparable result to selective agar for detection of thermophilic Campylobacter spp. in
food samples (Thunberg, et al., 2000). F luorogenic PCR cuts down diagnostic time to
one hour after DNA extraction (Logan, et al., 2001), compared to the six days required by
standard culture methods and three days needed for PCR afier enrichment (Denis, et al.,

1999; Lilja and Hanninen, 2001). Bypassing the enrichment step by using

42

irnmunomagnetic separation of cells resulted in shortening the total turnaround time to

eight hours (Waller and Ogata, 2000).

Molecular techniques for determining relationship among Campylobacter
spp. The main objective for genotyping Campylobacter spp. in epidemiological studies is
to provide evidence that strains related by epidemiological factors (time, place, host) are
also genetically related (Tenover, et al., 1995). It was shown over a decade ago that
genotyping techniques offer greater discriminatory power than biotyping or phage typing
of Campylobacter (Patton, et al., 1991). An extensive review of genotyping techniques
for Campylobacter (Wassenaar and Newell, 2000) included fla-typing (PCR-Randon
Amplified Fragment-Length Polymorphism; PCR-RFLP) , pulsed-field gel
electrophoresis (PF GE), ribotyping, and random amplified polymorphic DNA (RAPD) as
major molecular techniques employed for genotyping of Campylobacter (Table 2-2).
Other genotyping techniques for Campylobacter spp. include Amplified fragment length
polymorphism (AFLP), multiplex PCR-RF LP, and nucleotide sequencing (Wassenaar
and Newell, 2000).

AF LP is based on complete digestion of whole chromosomal DNA followed by
selective amplification of the digested product with specific restriction site recognized by
the specific amplification primers (Wassenaar and Newell, 2000). AF LP has been used
successfully to identify species, subspecies, and genetic relationships of Campylobacter
spp. (Duirn, et al., 2000; On and Harrington, 2001; Wagenaar, et al., 2001). AFLP is
more specific than phenotyping or ribotyping in ability to identify C. fetus to species

level (Wagenaar, etal., 2001).

43

PCR-RFLP has been used in epidemiological studies of samples from humans
(Waegel and Nachamkin, 1996), animals (Chuma, et al., 1997; Petersen and On, 2000),
and is sometimes called macrorestriction profiling (Petersen and On, 2000) or flagellin
typing when the fla gene is used (Nachamkin, et al., 1993). Because the fla gene of
Campylobacter contains both conserved and variable sequences, primer can be designed
to amplify the conserved target areas. Then restriction enzymes can be used to digest the
amplified product which will show different banding patterns among different genotype
due to variability at the locus (Nachamkin, et al., 1993). Because flagella typing is based
on only one genetic locus, it may be able to discriminate at the species level (De Boer, et
al., 2000). RFLP was found to correlate well with results from heat-labile serotyping,
and offers higher discriminatory power (Nachamkin, et al., 1993).

RAPD is most useful when no previous information on the target bacteria is
available. This procedure has been shown to be useful for discriminating virulent strains
of Campylobacter (Carvalho, et al., 2001), typing rare Campylobacter (Misawa, et al.,
2000) or characterizing serologically non-typable Campylobacter (Mazurier, et al., 1992).
RAPD use arbitrary primers to amplify chromosomal DNA under low stringency
conditions. Generally, 10-mer primers are used under condition that allow mismatch
amplification, which occur randomly throughout the chromosome (W assenaar and
Newell, 2000).

PFGE is one of the most popular techniques and is most widely used in
epidemiological studies (Fitzgerald, et al., 2001; Fujita, etal., 1995; Hanninen, et al.,
1998) and outbreak investigations (Fitzgerald, et al., 2001; Olsen, et al., 2001). In PFGE,

restriction enzyme(s) are used to digest chromosomal DNA in situ (immobilize in agarose

prior to cell lysis). After digestion, electrophoresis of the product is achieved under
conditions in which the electric field is changed in a pulsed manner (Wassenaar and
Newell, 2000). The choice of enzymes using in PFGE varies significantly, but the most
discriminatory enzymes found for typing Campylobacter so far are SmaI combined with
SalI (Fitzgerald, et al., 2001; Steele, et al., 1998), HaeIII, HindIII, PruII, and PstI (F ayos,
et al., 1992; Owen, et al., 1993). PFGE was shown to be reproducible in different
laboratories (Ribot, et al., 2001), but PFGE profiles may change following several
passages of the bacteria on culture media (On, 1998).

Ribotyping is another form of genotyping based on agarose gel electrophoresis of
digested genomic DNA, followed by Southern blot hybridization with rRNA gene
specific probe (W assenaar and Newell, 2000). Ribotyping has been shown to be more
discriminatory than serotyping in some instances (Smith, et al., 1998), but there was no
direct association between ribotypes and serotypes of Campylobacter (Fitzgerald, et al.,
1996). Species discrimination among Campylobacter using automated ribotyping was
shown to be unreliable (De Boer, et al., 2000), and the discriminatory power of
ribotyping to type C. jejuni is limited because C. jejuni only have 3 copies of rRNA genes
(W assenaar and Newell, 2000).

When comparing these techniques, AF LP has the highest discriminatory power
when typing C. jejuni and C. coli (De Boer, et al., 2000; Lindstedt, et al., 2000). PFGE
(using SalI, SmaI, or both) was more discriminatory than ribotyping, flagellin typing (De
Boer, et al., 2000), fatty acid profile typing, serotyping, and biotyping (Steele, et al.,

1998). Overall, the ranking of the discriminatory power of these tests, fiom highest to

45

lowest, is AFLP, PFGE, PCR-RFLP, and ribotyping (De Boer, et al., 2000; Lindstedt, et
al., 2000).

One problem affecting the reproducibility of any genotyping method is the
genetic instability of the target DNA, which may occur through recombination,
transformation or mutation. Techniques utilizing whole genomic DNA that generate
several small bands, such as RAPD, PF GE and AFLP, should be the least sensitive to
genomic instability (Wassenaar and Newell, 2000). Additionally, PCR-RFLP should be
relatively reproducible, if the RFLP target genes are stable (Wassenaar and Newell,

2000).

Molecular techniques for identifying markers of virulence and resistance in
Campylobacter spp. Molecular techniques can be used to identify new virulence factors
(Carvalho, et al., 2001), and known genetic markers for virulence in Campylobacter
(Wilson, et al., 2000). Carvalho et a]. reported finding of invasive-associated marker in
C. jejuni and C. coli resulting from examination of RAPD profiles (Carvalho, et al.,
2001).

There is a large body of work on markers for resistance in Campylobacter. A
marker has been identified for the efflux mechanism of C. jejuni, with is associated with
resistance to more than one antimicrobial agent (multi-resistance) (Charvalos, et al.,
1995). It has been shown that specific mutations in g1yA or parC gene of C. jejuni confer
resistance to quinolones (Gibreel, et al., 1998; Wang, et al., 1993). Although various
mutations have been reported, the Thr-86-to-Ile (C-T transition) was most frequently

associated with high levels of resistance to fluoroquinolones (Ruiz, et al., 1998; Wilson et

46

al., 2000). Several molecular techniques have been developed to detect this mutation in
the gyrA gene in C. jejuni, including fluorogenic PCR (Wilson, et al., 2000), mismatch
PCR amplification (Glaab and Skopek, 1999), and nonradioisotopic single strand
conformation polymorphism (Charvalos, et al., 1996). These techniques may be of value
in screening C. jejuni for antimicrobial resistance without conducting in vitro

susceptibility testing.

APPLICATIONS OF MOLECULAR TECHNIQUES IN EPIDEMIOLOGICAL
STUDIES OF CAMPYLOBA CTER SPP.

Outbreak investigation. Molecular techniques can provide laboratory evidence
that an outbreak resulted from a common source (Hanninen, et al., 1998; Harrington, et
al., 1999), or even pin point the source of the outbreak if isolates from suspected sources
are available (Olsen, et al., 2001). Although biotyping and serotyping have proven useful
in outbreak investigations (Pearson, etal., 2000), numerical analysis resulting from
molecular techniques such as PFGE or AF LP offers higher discriminatory power, and the
resulting data can be used to compare isolates from wider geographical areas or from
different hosts at different times to yield more information.

Outbreak investigations commonly involve verification of the case and
determination of the risk factors associated with increased probability of becoming a
case. Establishing a genetic relationship between bacteria isolated fi'om the case and
suspected source provides very specific evidence of causation. However, since outbreak

investigation is commonly conducted retrospectively, as in a case-control study, the

47

temporal sequence of events should be ascertained before definitive conclusions can be

drawn.

Genetic evidence of relationships among isolates. Molecular techniques can be
used to compared isolates from various sources to determine whether there is any
relationship among them (Chuma, et al., 1997; Duim, et al., 2000; Fitzgerald, et al.,
2001). Clarifying relationships among epidemiologically-related isolates may lead to
novel hypotheses about the development and transmission of Campylobacter infection,
which may increase our understanding of how Campylobacter disseminates among
various species of animals (Misawa, et al., 2000) and environments (Petersen, et al.,
2001), and the transmission dynamics of Campylobacter in specific hosts (Harrington, et
al., 1999) or across hosts (Duim, et al., 2000).

When conducting epidemiological studies, it is desirable to use highly
discriminating assays with the ability to generate data which can be shared among
various laboratories. Molecular techniques offer both of these qualities, once
improvement and standardization of procedures are established. Disease monitoring and
surveillance, or other epidemiological studies of Campylobacter, using molecular
techniques to provide genetic information will yield more precise information and may

lead to more interesting hypotheses on the epidemiology of Campylobacter.

Detection and identification of the bacteria, virulence factors, and markers

for antimicrobial resistance. Molecular techniques can be used to detect and identify

Campylobacter with virulence factor(s) as mentioned earlier. The use of genetic markers

48

 

is of great value in epidemiological or clinical studies involving difficult to measure
outcomes such as low levels of bacteria contamination in a food matrix, and can be useful
in monitoring or surveillance of Campylobacter with specific qualities, such as resistance
to antimicrobial agents. Techniques like fluorogenic PCR may offer specific and rapid
assays for monitoring C. jejuni with virulence or resistance markers in food products.
Improving bacteria separation technique from various matrices, such as irnmunomagnetic
separation will also allow shorter time require for bacteria contamination diagnostic. This

would allow food producers to better monitor their products for contamination.

In conclusion, molecular techniques offer valuable assistance to the
epidemiological study of epidemiology of Campylobacter. The selection of specific
techniques to employ may depend on the specific needs and resources of different
laboratories. Combinations of different protocols can increase the ability to genetically
discriminate Campylobacter. However, a combination of protocols may result in longer
processing time. Standardization of protocols among various laboratories will allow
comparison of result, especially those obtained from numerical analysis of genetic
materials, such as PFGE and AF LP. PF GE and AF LP are the most promising techniques

to provide useful information for the epidemiological study of Campylobacter.

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2-1. PCR protocols for the detection and identification of Campylobacter spp.
Target gene Discriminate Processing of References
species PCR products*
16S rRNA, hip, Yes Agarose gel (Lawson, et al., 1998),
asp electrophoresis and (Linton, et al., 1997)
southern blot
16S rRNA No Agarose gel (V anniasinkam, et al.,
electrophoresis 1999)
16S rRNA Yes I Melting peak analysis (Logan, etal., 2001)
of biprobes in real time
PCR
16S rRNA No 2 RFLP (Marshall, et al., 1999)
16S rRNA Yes ELISA (Metherell, et al., 1999)
16S rRNA Yes Agarose gel (Denis, et al., 1999)
electrophoresis
16S rRNA Yes Avidin capture assay (Waller and Ogata,
2000)
16S -— 23S rRNA Yes Southern blot and (O'Sullivan, et al.,
spacer region colorimetric reverse 2000)
hybridization
16S — 23S rRNA Yes I PAGE (Christensen, et al.,
spacer region 1999)
23S rRNA Yes RFLP (Eyers, et al., 1993),
(F ermer and Engvall,
1999), (Steinhauserova,
et al., 2001),
23S rRNA Yes RFLP [Korolik, 2001 #573;
(Korolik, et al., 1995)
23S rRNA Yes RFLP (Hurtado and Owen,
1993
flaA,flaB No Internal probe (Rasmussen, et al.,
hybridization 1996)
ceuE Yes Agarose gel (Houng, et al., 2001)
electrophoresis
gyrA Yes Fluorogenic PCR (Padungtod, et al.,
2002)
IG02 fragment Yes Non-radioactive (Casademont, et al.,
sandwich hybridization 2000)

l

 

 

 

 

 

(except C. jejuni from C. coli)
2 (can separate C. jejuni from C. coli with additional primer)
* RFLP — randomly amplified fragmented length polymorphism, PAGE —

Polyacrylarnide agarose gel electrophoresis, ELISA — enzyme linked immunosorbent
assay

50

 

 

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59

CHAPTER 3
IDENTIFICATION OF CAMPYLOBACTER JEJUNI ISOLATES FROM

CLOACAL AND CARCASS SWABS OF CHICKENS IN THAILAND BY USE OF
A 5’ NUCLEASE FLUOROGENIC PCR ASSAY

ABSTRACT

A rapid PCR-based 5’ nuclease fluorogenic PCR assay for identifying
Campylobacterjejuni was applied to Campylobacter isolates from chicken cloacal and
carcass swabs collected from chicken farms and a slaughterhouse in Thailand. The
primers and probe were based on the sequence of the gyrA gene in C. jejuni. C. jejuni
isolates were identified using fluorogenic PCR assay of bacterial cells directly from
Campylobacter selective agar medium. This assay allowed identification of C. jejuni
within 1 day after colonies appeared on selective media. The fluorogenic PCR assay
yielded comparable results to the conventional phenotypic test kit (kappa = 0.76), but
required less time. When species identification by the two methods was in conflict,
results were confirmed by PCR-RFLP of 23S rRNA genes. In these instances, the
fluorogenic PCR assay identified more isolates of C. jejuni correctly than the
conventional phenotypic test kit (6 of 7 unidentifiable by conventional test kit). The
fluorogenic PCR assay offers a rapid and specific method that outperforms the
conventional phenotypic test kit in identification of C. jejuni fiom environmental

samples.

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INTRODUCTION

Campylobacter has been implicated as a major cause of food-bome disease in
many countries, including Thailand (Mead, et al., 1999; Varavithya, et al., 1990). In
particular, Campylobacterjejuni has been cited as the most frequent cause of food-bome
disease (Acheson, 2001), and has been associated with Guillain-Barré syndrome (GBS), a
neurodegenerative disease in humans (Allos, 2001). Campylobacter is frequently
isolated from poultry meat products (Atanassova and Ring, 1999; Ono and Yamarnoto,
1999; Uyttendaele, et al., 1999), which have been fi'equently identified as sources of
sporadic cases of carnpylobacteriosis (Pearson, et al., 2000). Contamination of poultry
products can be traced back to farms (Berrang, et al., 1999) where chickens are
commonly found with Campylobacter (Evans and Sayers, 2000). In addition to its
presence in meat products, Campylobacter has been shown to rapidly develop resistance
to clinically-relevant antimicrobial agents such as fluoroquinolones (J acob-Reitsma, et
al., 1994) Since 1970, there has been substantial improvement in isolation and
identification techniques for Campylobacter. Common techniques employed to isolate
Campylobacter include culture on selective growth media and filtration methods
(Nachamkin, et al., 2000). There are numerous culture media available for the isolation
of Campylobacter (Corry, et al., 1995), but the antibiotics included as selective agents to
control competing flora may also inhibit some strains of Campylobacter (Moore and
Murphy, 2000). Identification methods for C. jejuni include assays such as the
hippuricase test, serological tests, lectin agglutination, and cellular fatty acid profiles,
each of which has its own advantages and disadvantages (On, 1996). However, these

procedures are time-consuming due to the slow grth rate of Campylobacter in culture.

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Polymerase chain reaction (PCR) techniques for detection and identification of
Campylobacter have been developed and successfully applied to samples from foods
(Thunberg, et al., 2000), fecal samples (Linton, etal., 1997), and poultry products
(Waller and Ogata, 2000). PCR-based assays offer a rapid alternative for identifying
Campylobacter without sacrificing specificity (On, 1996).

A 5’ nuclease fluorogenic PCR was recently developed (Holland, et al.,
1991)which can be used to rapidly identify specific target genes or discriminate between
different alleles of the same target gene (Lee, et al., 1993). Fluorogenic PCR assays use a
non-extendable oligonucleotide hybridization probe that contains a fluorescent reporter
dye and a quencher dye. During PCR cycling, the probe first specifically hybridizes to
the corresponding template, but is digested by the exonuclease activity of Taq DNA
polymerase as it moves along the template strand. This cleavage results in an increase of
fluorescent emission reporter dye, which can be measured by fluorescence spectrometry.
The use of an internal probe carrying a signal-generating system in combination with
target-specific primers increases the specificity of the PCR reaction. The level of
fluorescence measured at the end of the PCR cycling provides qualitative information on
the presence or absence of nucleic acid target (Livak, et al., 1995). A fluorogenic PCR
assay using primers and probes specific for the gyrA gene was recently developed in our
laboratory. This assay successfully identified and enumerated a variety of laboratory and
clinical C. jejuni isolates (Wilson, et al., 2000). The present study was conducted to
evaluate the performance of this fluorogenic PCR assay for identification of C. jejuni in

samples derived from poultry farms and slaughterhouses and to compare its performance

62

with a widely-accepted conventional phenotypic test kit for species identification of

Campylobacter spp..

MATERIALS AND METHODS

Sample collection and primary isolation. This study was conducted as part of a
three year epidemiological study designed to determine the prevalence and antimicrobial
susceptibility profiles of E. coli, Salmonella, and Campylobacter in food animals and
meat products in Thailand. Samples were collected during the rainy season fi'om May to
July, 2000. One hundred and fifty-five 5-6 week old chickens were randomly selected
for sample collection from 3 convenience-sampled chicken farms in Thailand. Farms
were selected according to their willingness to participate, and possessed chickens at
market age (6-7 weeks) at the time of sample collection. Each farm had from 5000 to
8000 chickens, and were within 20 kilometers from the laboratory facilities at Chiang
Mai University. One hundred and three chickens from a slaughterhouse were
systematically selected for sample collection after defeathering by selecting two birds
from every batch of 10 chickens processed on a single day.

Cloacal swab samples were collected by swabbing inside the cloacal area of each
chicken with a sterile cotton swab, which was then placed in Stuart’s transport medium
(RCM supply, Bangkok, Thailand). At the slaughterhouse, samples were collected from
the uneviscerated chicken carcasses after killing and defeathering, before putting in the
chilling tank. Cloacal swabs were collected along with swab of the area surrounding the
cloaca and under the wing of each bird at the slaughterhouse, using sterile cotton swabs

that were placed in Stuart’s transport medium. Samples were kept on ice during

63

transportation and refiigerated until processing, within 12 hours after collection. Swabs
were streaked directly onto Karrnali agar plates (KSA ; Oxoid, Basingstoke, UK), and
incubated in plastic bags at 42°C for up to 48 hours under microaerobic conditions (5%
02, 10% C02), using the Anaerocult C system (Merck ; Whitehouse Station, New
Jersey). Suspected Campylobacter colonies were confirmed by oxidase test, catalase test,
and gram stain. Gram-negative spiral rods that were oxidase and catalase positive were
identified as Campylobacter, frozen and stored in 30% glycerol with Mueller-Hinton
broth at -70EC and shipped to Michigan State University on dry ice. Upon arrival,
Campylobacter isolates were subcultured onto Brucella agar medium (Becton Dickinson ;
Franklin Lake, New Jersey) supplemented with 5% defibrinated sheep blood (BASB ;
Cleveland Scientific, Bath, Ohio), and on Karmali agar medium. Agar plates were
incubated at 37°C under 5% CO2 for 36 to 48 hours. A single colony was selected from
each plate and subcultured on BASB and KSA agar plates for species identification.
Fluorogenic PCR assay. After bacteria were grown on BASB or KSA at 37°C ,
5% CO2, for 36 to 48 hours, cells were harvested and suspended in Mueller-Hinton broth
for chromosomal DNA extraction(Wilson, et al., 2000). Primers J L238 and JL239 and
the fluorogenic probe TAQl , were used. The fluorogenic PCR assay PCR reaction mix
contained the following: 1X Taqman buffer (PE Applied Biosystem, Branchburg, NJ),
0.2 mM (each) dNTP (0.4 mM dUTP), 0.5 pmol of each primer/mL, 200 nM fluorogenic
probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/mL, 0.01 U of Amperase
UNG (Perkin-Elmer)/mL, 4.5 mM MgCl2, 0.05% gelatin, 0.01% Tween20. A sterile
toothpick was used to transfer bacteria from a single colony from BASB or KSA agar

medium into the fluorogenic PCR reaction mix. Prior to initial PCR denaturation, all

64

Fluorogenic PCR reaction mixtures were incubated at 50°C for 2 min in the presence of
Amperase UNG to prevent PCR product carryover. Using 50 mL PCR reactions, initial
denaturation was conducted at 95EC for 10 min ; the annealing and polymerization steps
were combined at 60°C for 1 min and followed by denaturation at 95EC for 30 s. This
process was cycled 40 times. Fluorescence emissions were monitored with an ABI Prism
7700 sequence detection system (Perkin-Elmer). The assay was performed on each
sample twice. If the Fluorogenic PCR assay results fiom identical samples were not
consistent, or if they were in conflict with conventional test kit results, PCR-RFLP of the
23S rRNA gene was used to confirm the species identification as described below. C.
jejuni 43429 and C. coli 1777208 were used as positive and negative controls
respectively.

Conventional test kit. The API CAMPY test strips (Biomerieux, Marcy l'Etoile,
France) were utilized as recommended by the manufacturer. Bacterial cells from BASB
agar plates were used to inoculated both portions of the strip. The first half of the test
strip, which was incubated at 37°C under aerobic conditions for 24 hours, included tests
for urease, reduction of nitrate, esterase, hippurase, gamma glutamyl transferase,
reduction of chloride to triphenyl tetrazolium, pyrrolidonyl arylarnidase, L-arginine
arylamidase, L-aspartatearylarnidase, and alkaline phosphatase. The second half of the
strip, including tests for production of H28; glucose, succinate, acetate, propionate,

malate, and citrate assimilation; and susceptibility to nalidixic acid, cefazoline and
erythromycin was incubated at 37°C under 5% CO2 for up to 48 hours. The results of

each test were read as specified by the manufacturer, and the final identification was

65

achieved by referring to the analytical profile index (20890) provided by the
manufacturer. C. jejuni 43429 was used as positive control.

DNA extraction for PCR-RFLP. Bacterial cells scraped fiom BASB agar plates
inoculated 24 hours earlier were pelleted, and DNA was extracted by standard methods
(Ausubel, et al., 1997). Briefly, cells were resuspended in TE buffer (IOmM Tris-HCl, 1
mM EDTA[pH8.0]) and lysed with 0.5% sodium dodecyl sulphate in the presence of 100
mg of proteinase IOmL. Cellular debris was removed by complexing with
hexadecyltrimethyl ammonium bromide followed by phenol-chloroform extraction and
Rnase I digestion. DNA was precipitated with isopropanol, redissolved in TE, and its
concentration determined using a DU530 spectrophotometer (Beckrnan Instruments ;
Schaumburg, IL).

PCR-RFLP of the 23S rRNA. PCR-RFLP of the 23S rRNA was used to
confirm the identity of Cjejuni isolates. The protocol described by F errner and Engvall
(F errner and Engvall, 1999) was used. The primer pair consisting of THERMl (5' TAT
TCC AAT ACC AAC ATT AGT 3') and THERM4 (5' CTT CGC TAA TGC TAA CCC
3') was used to amplify 491 bp of a highly-polymorphic region of the 23S rRNA gene;
RFLP banding patterns of this region were previously shown to be specific for all species
of thermophilic Campylobacter (F errner and Engvall, 1999). The PCR reaction mix
contained 0.25 mM of each primer/mL, 1X PCR buffer, 1.5 mM MgCl2/mL, 0.1 mM
each dNT P, 0.05 u/mL Amplitaq (PE Applied Biosystem ; Branchburg, NJ), and 1.6
ng/mL DNA template. The PTC-lOO thermocycler (MJ Research ; Watertown, MA) was

used as follows. The cycling protocol was preceded by a 12 min incubation at 94°C.

Initial denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C

66

for 1 min. The 50mL PCR sample was cycled 45 times, followed by a final extension at
72°C for 5 min. PCR products were visualized by electrophoresis on a 2% agarose gel.
For species identification, PCR products were digested with AluI and T sp5091 in
separate reactions, according to the sequence detection system manufacturer‘s
recommendations (F ermer and Engvall, 1999). DNA of known Campylobacter species
was used as controls, including C. jejuni 43429, C. call 1777208, C. upsaliensis 43954,
C. lari 3121, C. fetus 27374 ; E. coli 25922 was used as a non-Campylobacter DNA
control. The resulting band patterns were visualized by electrophoresis on 2% agarose
gels, ethidiurn bromide staining, and UV illumination.

Statistical analysis. Statistical analysis was conducted using SAS v. 8 (SAS
Institute, Cary, NC). The kappa statistic was calculated to determine the agreement
between results obtained via fluorogenic PCR assay and conventional test kit. Kappa
statistic determine the agreement between two tests without making an assumption that

one is more efficient than another (Rosner, 1995).

RESULTS

From a total of 361 samples collected fiom chickens from 3 farms and one
slaughterhouse, 143 (40%) yielded presumptive Campylobacter after primary isolation
and biochemical analyses (Table 3-1). The overall prevalence of Campylobacter from
chicken cloacal swab samples on the 3 farms was found to be 62%, while the
Campylobacter prevalence of the same sample type at the slaughterhouse was 41%. But

the Campylobacter prevalence on the finished carcasses was only 3.9%.

67

Of the 143 isolates, 86 isolates were transported back to the US in dry ice. They
were subsequently kept at —80°C in the lab at Michigan State university. Of those 86
isolates transported and frozen, 79 were recovered (92%) from frozen stocks and were
analyzed further by the fluorogenic PCR assay. However, due to limited resources, only
59 were subjected to analysis by conventional phenotypic test kit for species
identification (Table 3-2). The remaining isolates were not successfully recovered from
frozen stocks. In the 59 isolate subset, good agreement was observed between results
obtained from the fluorogenic PCR assay and conventional test kit for identification of C.
jejuni (kappa = 0.76). Of seven isolates with discordant test results, one was identified
as C. jejuni by conventional test kit, but was negative by the fluorogenic PCR assay.
The remaining six isolates were identified as C. jejuni by the fluorogenic PCR assay, but
were unidentifiable by conventional test kit. Of these six isolates, two discordant isolates
were not successfully recovered from frozen stocks, while the remaining four isolates
were analyzed further by PCR-RFLP of the 23S rRNA gene (sample identification
numbers 408, 411, 488, and 559). The results of PCR-RFLP supported the fluorogenic
PCR assay identification of these isolates as C. jejuni (Figure 3-1 and Figure 3-2).
Additionally, there were seven isolates that gave inconsistent fluorogenic PCR assay
results in two separate runs of the test (one positive and one negative result). Of these,
five isolates were recovered and subjected to PCR-RFLP (sample identification numbers
408, 481, 502, 526, and 556). PCR-RFLP supported identification of all five of these
isolates as C. jejuni. Fluorogenic PCR assay results using cells grown on BASB or KSA
and tested in parallel also showed significant agreement (kappa = 0.69), suggesting that

direct analysis of cells from either medium is effective.

68

DISCUSSION AND CONCLUSION

The proportion of cloacal swab samples yielding Campylobacter isolates from the
slaughterhouse (41%) in this study was higher than levels previously observed in
America (20%; Jones, et al., 1991) in a similar sample types. However, the prevalence of
Campylobacter on chicken farms was comparable to what was previously found in
China(6l%) (Wu, et al., 2000) , but lower than in Britain (90%;Evans and Sayers, 2000).
The lower proportion of isolates found in chicken carcasses was possibly due to the
incubation temperature. The selective plates were only incubated at 42EC, eliminating
the possibility of detecting non-therrnophilic Campylobacter, which may have
contributed to the low levels detected.

In another study, the conventional test kit was shown to yield good agreement (up
to 94% agreement at species level) with a conventional biochemical test panel of 11 tests
in identifying species of Campylobacter (Shih, 2000). However, the time required to
obtain these results was 24 hours after the test strip was inoculated, which followed a 24-
48 hours initial culture on a blood agar plate to generate an inoculurn for the test. The
major advantage of the fluorogenic PCR assay was the reduction of time and labor for
obtaining results. Colonies from selective media, such as KSA, yielded comparable
results with colonies from enrichment media, such as BASB, providing some flexibility
in choice of growth media. Additionally, the fluorogenic PCR assay could be performed
as soon as colonies appeared on selective media. This assay eliminated the need to
extract DNA or visualize results by gel electrophoresis, which reduced the amount of

time and labor considerably. Finally, the fluorogenic PCR assay showed a higher success

69

rate in detecting C. jejuni than the conventional phenotypic test kit under our sampling
protocol. We are currently developing primers specific to other Campylobacter species,
such as C. coli (now available), that can be used in place of the primer for C. jejuni for
rapid and specific identification of these other species (unpublished data).

Recently, there have been several studies focused on shortening the detection time
for Campylobacter in food and fecal specimens. These techniques include PCR of the
168 rRNA (Thunberg, et al., 2000), 238 rRNA (Fermer and Engvall, 1999), and Fla
(Rumussen, et al., 1996) genes, multiplex PCR of 16S rRNA, mapA, and ceuE genes,
and enzyme immunoassay (EIA; (Hoorfar, et al., 1999). However, each of these
techniques, with the exception of EIA required isolation on selective medium, DNA
extraction, and visualization of PCR products by gel electrophoresis with or without
Southern blotting. The EIA method, although very rapid, does not allow firrther
susceptibility testing or storage of isolates, since no colonies are obtained. When
selective medium is used, it is not possible to precisely estimate the number of
Campylobacter in the sample, because the selective agent may also inhibit the growth of
strains of interest (Moore and Murphy, 2000).

Our fluorogenic PCR assay also yielded reproducible and accurate results when
applied directly to identification of C. jejuni from selective medium. Although we did
not comfinn all the Cjejuni positive isolates by our assay with the PCR-RFLP because of
limited resources, PCR-RFLP of the discrepant isolates supported the identification by
our assay. If the rest of the fluorogenic PCR assay positive isolates were confirmed, it
should strengthen the specificity of our assay. Our PCR-RFLP restriction pattern using

AluI yielded different patterns for Cjejuni from those reported by Fermer and Engvall

7O

(F ermer and Engvall, 1999). This may be the result of different genotypes or the
incomplete digestion of the PCR product due to high starting number of templates. We
are working on methods to allow us to simultaneously identify and quantify the number
of cells in environmental samples without the need to isolate colonies on selective
medium. One method under study, immunomagnetic separation (Lea, et al., 1985), has
been used successfully in combination with other techniques to detect Campylobacter
(Lamoureux, et al., 1997) and other food-bome bacteria from various sources (Cudjoe
and Krona, 1997; Skjerve, et al., 1990). Irnmunomagnetic separation was shown to
improve the detectability level, particularly in environmental sarnples(Elder, et al., 2000).

In conclusion, the F luorogenic PCR assay assay is a sensitive and accurate
method for identifying C. jejuni directly from a colony on selective medium without the
need to extract DNA or perform gel electrophoresis. It can be applied for detection of
other species of Campylobacter, and is a promising technique, when used in combination
with cell separation techniques, to provide a rapid assay for the identification and

enumeration of Campylobacter in food and environmental samples.

71

Figure 3-1 PCR-RFLP T sp 5091 digestion patterns.

Lane: 123456789101112131415

 

_ n — _~...
-—~I‘~———O—-—-

Lanes: 1) 100 bp DNA ladder; 2) E. coli 25922; 3) C. jejuni 43429; 4) C. coli

1777208; 5) C. upsaliensis 43954; 6) C. lari 3121; 7) C. fetus 27374; 8) 408; 9) 411;

10) 488; 11)481; 12) 526; 13) 556; 14) 559; 15) 502; 16) no template control

72

Figure 3-2 PCR-RFLP Alu I digestion patterns.

Lan6212345678910111213l415

 

Lanes: 1) 100 bp DNA ladder; 2) E. coli 25922; 3) C. jejuni 43429; 4) C. coli
1777208; 5) C. upsaliensis 43954; 6) C. lari 3121; 7) C. fetus 27374; 8) 408; 9) 411;

10) 488; 11)481; 12) 526; 13) 556; 14) 559; 15) 502; 16) no template control

73

Table 3-1 Proportion of samples yielding Campylobacter from farms and slaughterhouse

 

Source

chickens (percent)

Number of Sample yielding Campylobacter / Number of

 

Chicken farms

 

Farm A 38/55 (69)

Farm B 17/50 (34)

Farm C 42/50 (84)

Total from Chicken farms 97/155 (63)
Slaughterhouses

Cloacal swab 42/103 (41)

Under wing swab 4/103 (4)
Total fi'om slaughterhouse 46/206 (22)

 

Grand total

143/361 (40)

 

74

Table 3-2 Number of C. jejuni samples identified by fluorogenic PCR assay and
conventional test kit (n=5 9).

 

 

Fluorogenic PCR Percent Conventional test Percent
assay kit
Cjejum' 33* 56 28* 48
Not Cjejuni 26 44 31 52

 

* kappa = 0.76 (95% CI: 0.60 - 0.93) , 1 indicate perfect agreement, 0 indicate no
agreement.

75

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Jacob-Reitsma, W. F., C. A. Kan, and N. M. Bolder. 1994. The induction of quinolone
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Lamoureux, M., A. Mackay, S. Messier, I. Fliss, B. W. Blais, R. A. Holley, and R. E.
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78

CHAPTER 4
PREVALENCE OF CAMPYLOBA CTER SPP. IN PIGS, FARM WORKERS AND
PORK PRODUCTION SYSTEMS IN NORTHERN THAILAND, 2000-2001.
ABSTRACT

A combination of cross-sectional and prospective studies were conducted in pig
farms, slaughterhouses and fresh markets in northern Thailand, from 2000-2001 to: 1)
compare the frequency of Campylobacter spp. on the farm, at the slaughterhouses, and at
the market, and 2) identify factors associated with the observed frequency of
colonization/contamination.

Fecal samples were collected from 76 pigs at the farm, and the same pigs were
followed to the slaughterhouses and markets, where additional samples were collected.
Isolation and identification of Campylobacter was done using enrichment broth, selective
agar, biochemical tests and 5'-nuclease fluorogenic PCR.

The incidence of colonization/contamination was highest at the farms (61.1%),
followed by the slaughterhouses (41.7%) and markets (33.3%). Lymph node and fecal
samples yielded more Campylobacter than carcass swabs from the same pig at the
slaughterhouses (p<0.01). The prevalence of Campylobacter in healthy farm workers
was 12.6%. Sampling location and source farms were significantly associated with the

observed prevalence.

79

INTRODUCTION

Campylobacter spp. have been recognized as major food-bome pathogens
throughout the world (Acheson, 2001; Pearson, et al., 2000; Phetsouvanh, et al., 1999;
Tay, et al., 1995). The majority of human cases of carnpylobacteriosis are caused by C.
jejuni and C. coli (N achamkin, et al., 2000). Infection with C. jejuni has been reported to
result in severe gastroenteritis, which may be fatal in immunocompromised patients
(Allos, 2001). One important sequelae of C. jejuni infection is Guillain-BarrS Syndrome
(GBS), an acute demyelinating disease of the peripheral nervous system resulting in
flaccid paralysis (Acheson, 2001; Altekruse, et al., 1998). Rates of Campylobacter
infections was 20.1 cases per 100,000 people in the US. (Acheson, 2001), 60 — 90 cases
per 100,000 people in Europe (Friedman, et al., 2000), and, in Southeast Asia,
Campylobacter infections were found predominantly in children less than 5 years old,
with prevalences ranging fiom 2.9% - 15% (Phetsouvanh, et al., 1999; Rasrinual, et al.,
1988; Taylor, et al., 1993; Varavithya, et al., 1990). Children with Campylobacter spp.
are often co-infected with E. coli, Salmonella or Shigella (Poocharoen and Bruin, 1986).

Foods of animal origin are commonly implicated as sources of Campylobacter
infection in humans, both in sporadic cases and disease outbreaks (Acheson, 2001). In
developed countries, milk is a common source of Campylobacter in disease outbreaks
(Evans, et al., 1996; Lehner, et al., 2000), while chicken is a common source of infections
for sporadic cases (Effler, et al., 2001; Studahl and Andersson, 2000). Reports of
outbreaks of foodbome illness due to Campylobacter are rare in developing countries,

even though there are reports of Campylobacter isolated fiom foods of animal origin

(Rasrinual, et al., 1988).

80

Campylobacter species most commonly associated with human disease, C. jejuni
and C. coli, have been isolated frequently from domestic animals (Garcia, et al., 1983).
In food animal species, pigs commonly carry more C. coli than C. jejuni (Steinhauserova,
et al., 2001). The Campylobacter spp. considered to be major veterinary pathogens
include C. fetus, not commonly found in pigs, and C. mucosalis and C. hyointestinalis,
which have been associated with diseases in pigs (Quinn, et al., 1994).

Because of the increasing public health importance of Campylobacter spp. in
terms of disease and associated antimicrobial resistance, it is important to study the
epidemiology of this infection in both animals and humans. There have been very few
studies examining the presence of Campylobacter through the food production system
from farm to market, in both the food animals and the workers in direct contact with
these animals. The basic hypothesis underlying this research is that Campylobacter spp.
are prevalent in pigs throughout the production system in Thailand, and major factors
influencing the observed prevalence can be determined. The objectives of this study,
therefore, were to: 1) determine the frequency of Campylobacter spp. in pigs and workers
at farms, slaughterhouses, and markets, 2) determine the risk factors associated with the

observed frequencies.

MATERIALS AND METHODS
Study design and population. This study is a part of a larger epidemiological
study of Campylobacter in food production systems in northern Thailand. The study was

conducted in two phases: the first phase consisted of a cross-sectional study of local pig

81

producers from May to July of 2000, and the second phase was a prospective study of
pigs from local producers from May to July of 2001.

Pig farms under consideration for participation in the study were finishing pig
operations, raising pigs from ages 30 to 110 days, that were subcontractors for a company
that maintains a large, lOOO-sow operation in Chiang Mai province in northern Thailand.
In both phases, pig farms were selected to participate in this study based on their
willingness to participate. Additional criteria for inclusion were: 1) the farms had to have
pigs at the age of approximately 95-100 days (1-2 weeks prior to slaughter) at the first
sampling time; and 2) be located within 80 kilometers radius fi'om the laboratory.

Description of the pork production system. Piglets, feed, medication, and
veterinary services were provided by the company to each of the contract farms. Once the
pigs reached market weight, the company transported the pigs to one of the two
municipal slaughterhouses in the Chiang Mai area. All slaughterhouses are government
operated and the government provides the facilities and personnel for inspection of
animals and carcasses. In general, the company transported the pigs to holding pens at
the company headquarters one day before slaughter, and the meat vendors purchased live
pigs at the holding pens. The company then delivered the pigs to the specific
slaughterhouse selected by the meat vendor, who hired personnel to slaughter and process
the carcasses. Once processed, the vendors picked up the carcasses from the
slaughterhouse to sell the next morning at the flesh markets.

In the second study phase (year 2001), an arrangement was made with the
company and the meat vendors so that the pigs sampled at the farm would be delivered to

a specific slaughterhouse and to a specific vendor at the market. This arrangement

82

allowed us to follow the same pigs from the farm, slaughterhouse, and eventually to the
market.

Sample size. Because there were no reports of the prevalence of Campylobacter
at the farm level in Thailand, the reported prevalence of 12% in food (9) was used to
estimate the required sample size. In order to estimate the frequency of Campylobacter in
pigs within 11% of the true prevalence, and with Type I error of 0.10, a sample size of 22
pigs per farm was derived using a previously published formula (18). To account for
attrition, 25 pigs were considered to be an adequate sample size fiom each farm.

Specimen and data collection. From 10 available pens in pig house at each farm,
five pens were systematically selected for the study (every other pen). Each pen had 20-
25 pigs, from which five pigs were randomly selected for sample collection.
Approximately 10 grams of fecal material were evacuated from the rectum of each pig
and stored in plastic cups on ice. Swabs of pen floors and feed trays (pooled sample of
two swabs fiom each pen) were collected using sterile gauze soaked with 10 ml of sterile
skim milk. All farm workers were provided with plastic cups containing Cary-Blair
medium and asked to submit 10 grams of stool in the cups provided. All samples were
kept on ice during transportation and processed within six hours after collection. In the
second phase, An ear tag was put on each pig sampled to facilitate tracking of the pig
identity fi'om farm through the slaughterhouse and the market.

At slaughter, mesenteric lymph nodes were collected after evisceration.
Approximately 20 grams of fecal material were collected from the intestine of each pig.
Carcass swabs were collected by wiping an area approximately 40 cm2 from around the

thigh area and inside the rib cage with sterile gauze pads, which were put in plastic bags

83

with 10 ml sterile skim milk for transport. In the second phase of the study,
approximately 100 grams of meat from the neck area attached to the head (with the ear
tag) were purchased at the fresh market. Samples were stored on ice during transportation
to the laboratory and processed within 12 hours after collection.

Isolation and identification of Campylobacter spp. . Fecal samples were directly
inoculated, using sterile swabs, on Karmali agar with antimicrobial supplements
(including cefoperazone, vancomycin, and amphotericin B), and incubated at 42°C
under10% CO2 for up to 5 days. All swabs were put in 90 ml of Bolton broth with
antimicrobial supplements (cefoperazone, trimethoprim, vancomycin, and amphotericin
B), and incubated for 48 hours at 42°C under10% C02, and a swab of the supernatant
from each sample was inoculated on Preston agar with antimicrobial supplements
(polyrnixin B, rifampicin, trimethoprim, and cycloheximide). Approximately 10 grams of
the inside of lymph node were collected using sterile equipment, minced, and then
processed as described above for swabs.

The agar plates were examined everyday for positive colonies. Suspected
Campylobacter colonies were confirmed by oxidase test (Dryslide, BBL), catalase test
(3% H202), and gram stain. Gram-negative spiral rods that were oxidase and catalase
positive were identified as Campylobacter, frozen and stored in 30% glycerol with
Mueller-Hinton broth at ~70°C for firture analysis. Campylobacter isolates were subjected
to speciation using a 5'-nuclease fluorogenic PCR (Padungtod, et al., 2002).

Statistical analysis. A pig was classified as positive if it had at least one sample
with positive Campylobacter spp. identification. Incidence was calculated as the number

of new positive samples at each sampling point, divided by number of samples with

84

negative isolation results at previous sampling point. Relative risk was calculated as a
ratio of incidence of Campylobacter between two sampling locations. Prevalence was
calculated as the number of positive samples divided by total number of samples tested.
A Chi-square test of independence was used to determine the significance of any
association between sampling location and the proportion of positive pigs in 2000.

A multivariable logistic regression model with random effects was used to model
the odds of finding Campylobacter in pigs at different location in 2001. Pig identification
was included in the model as random effect term.

A similar technique was used to model the odds of finding Campylobacter in pigs
fiom different farms controling for year of sample collection both at farms and
slaughterhouse. Batch of pigs was included in the model as random effect term.

Model parameters were estimated using Generalized Estimating Equation (GEE;
Zeger and Liang, 1986). GEE can be applied to repeated measures data with missing
value, and consistent estimates can be obtain under mild assumption of correlation among
observation (Stokes et al.,2000). All analysis was done using SAS V8.01 (SAS institute

Inc., Cary, NC).

RESULTS

A total of five farms, three slaughterhouses, and one fresh market participated in
the study. In 2000, three pig farms and Lampoon municipal slaughterhouse participated
in the study. In 2001, three pig farms, and the Sansai and Chiang Mai municipal
slaughterhouses participated in the study. Two of the three farms participating in 2001

were also participants in 2000. Samples were also collected from Muang Mai market in

85

Chiang Mai city. A total of 402 samples from pigs, 32 farm worker samples, 92
environmental samples, and 67 pork samples were collected.

Frequencies of Campylobacter isolation. Campylobacter spp. were isolated at
higher rates from the pigs at farms than those at slaughterhouses (Table 4-1). The
prevalence of Camplyobacter was highest in pigs and lowest in environmental samples.
Farm environmental samples yielded higher rate of Campylobacter isolation than samples
fi'om slaughterhouse environment. The average sample prevalence of C. jejuni for both
years of study was 1.1%. At slaughterhouses in 2001, significantly more Campylobacter
were isolated from lymph node samples (68.6%) than carcass swab samples (36.5%; chi-
square p < 0.01). In 2000, significantly more Campylobacter were isolated fiom fecal
samples (21.2%) than carcass swab samples (6.0%; chi-square p < 0.01). In 2000, other
species of Camplyobacter identified included C. coli (72.2% of isolates), C. cryaerophila
(1.3%), C. fetus (1 .3%), C. sputorum (1.3%), and C. upsaliensis (1.3%).

The incidence of Campylobacter colonization/ contamination was highest at the
farms, followed by the slaughterhouse and market respectively (Table 4-2). However, the
higher risk of contamination reflected by the relative risk was not statistically significant.

Risk factors. The effect of sampling location on the odds of finding
Campylobacter in pigs was summarized in Table 4-3. The odds of finding
Campylobacter in pigs at the farm was significantly higher than slaughterhouse in both
years of the study.

The effect of overall management of the farm, which was represented by source
farm of the pigs, on the odds of finding Campylobacter both at the farm and the

slaughterhouse was shown in Table 4-4. Pigs fiom farm C had significantly higher odds

86

of being found with Campylobacter than other farms. Both at the farm and the

slaughterhouse.

DISCUSSION AND CONCLUSION

By prospectively follow the same pigs from farm to slaughterhouse and market,
we were able to demonstrate the presence of Campylobacter through the pig production
system. This study design allowed us to determine the risk of acquiring Campylobacter
in pigs at farm, slaughterhouse, and market. Other study design such as case-control or
cross-sectional study would not be able to clarify the time sequence of events as shown in
our study. Our study clearly showed that 61.1% of negative pigs became positive for
Campylobacter isolation during one week period at the farm, 41.7% of negative pigs at
the farms became positive at the slaughterhouse, and 33.3% of negative pigs at the
slaughterhouse became positive at the market.

The prevalence of Campylobacter spp. observed on the farm was, surprisingly,
comparable to what was previously reported in the US. (86-99%; Harvey, et al., 1999),
and the Netherlands (85%; Weijtens, et al., 1997). The high incidence of colonization
observed at the farm may largely be attributed to the open housing in which birds can
freely fly through. Wild birds have been implicated as possible source of Campylobacter
contamination in chicken farms (Jones, et al., 1991). The high prevalence observed may
also be the result of colonization before the pigs were transported to the fattening farms.
Young (Young, et al., 2000) reported 57.8% colonization in newborn piglets and 100%
colonization in weaning pigs in the US. Because of the time limitation, we did not

sample the pigs before they were initially transported to the farms, which would have

87

provided information on this possible source of infection. Molecular identification
techniques, such as PFGE or PCR-RFLP, could be employed to clarify the relationship
among isolates from the same location (Wassenaar and Newell, 2000).

The prevalence of Campylobacter in pig house environment samples was lower
than from samples collected directly fiom the pigs. This difference may be due to the
sample collection method (Hoar, et al., 1999) or the fact that Campylobacter do not
survive well in aerobic conditions in the environment. Employing a manure drag through
the house may have improved sensitivity of detection in environmental sample (White, et
al., 1997).

Our observations confirmed that C. coli seems to be the predominant species of
Campylobacter in pigs in Thailand. Similar observations have been made for pigs in
America (Young, et al., 2000) and Europe (Steinhauserova, et al., 2001).

There were significant differences in the prevalence of Campylobacter observed
in samples from carcass at the slaughterhouse between 2000 and 2001 (chi-square p <
0.01). This may be due to differences in the sample collection and isolation protocols
between 2000 and 2001. Larger pieces of gauze were used in 2001, which might have
increased the number of bacteria picked up when compared to the cotton swabs used in
2000 (Korsak, et al., 1998). Also, the enrichment step of all swab samples was only
implemented in 2001, which might have increased the sensitivity of the isolation protocol
in the second year (Corry, et al., 1995).

Lymph node and fecal samples yielded more Campylobacter than carcass swabs.
Unfortunately, lymph node and fecal samples were not collected from the same animal in

2000, therefore comparison between those two type of samples may not be valid.

88

Although lymph node samples may be more sensitive for detecting Campylobacter at the
slaughterhouse, the finding may only reflect the level of colonization at the farm, and not
the contamination at the slaughterhouse, which carcass swabs may better represent. Fecal
samples may reflect colonization at the farm, and may indicate the risk of contamination

posed by the carcass at slaughter.

Approximately half of the pigs with no Campylobacter at the farms were
contaminated at the slaughterhouse. This observation may be the result of increased
shedding of the organism following transportation (Whyte, et al., 2001) or contamination
from water used in the slaughterhouse (Hanninen, et al., 1998).

The prevalence of Campylobacter spp. fi‘om meat samples at the market was
slightly higher in our study (24.6%) than previously reported in the US. (1 .3%; Duffy, et
al., 2001) and Thailand (12%; Rasrinual, et al., 1988). The reported prevalence of
Campylobacter in foods from Thailand included other types of meat besides pork
(Rasrinual, et al., 1988), which may make comparison of findings inappropriate. The
prevalence at the market was comparable to prevalence of Salmonella in pork reported in
Thailand (24%; Boonmar, et al., 1997).

Four out of thirty-two stool specimens from healthy farm workers yielded
C.jejuni. This may be the result of asymptomatic Campylobacter infection, which is
common in adults in developing countries (Allos, 2001). It was also suggested that the
isolation of Campylobacter fiom healthy persons in developing countries may be the
result of constant reinfection (Oberhelrnan and Taylor, 2000). This may be the case for
farm and slaughterhouse workers, who are frequently in contact with animals with

diarrhea (Saeed, et al., 1993).

89

Sampling location and source farm of the pigs were shown to be associated with
the prevalence of Campylobacter. Although Campylobacter were found at lower level at
the market, it may present greater risk to consumer, compared to workers at the farms and
slaughterhouse. Prevalence of Campylobacter in pig from farm C was much higher than
other farms both at the farm and at the slaughterhouse. This observation suggested
increase probability of finding Campylobacter, in pigs at the slaughterhouse, if the pigs
were raised in farm with high prevalence of Campylobacter. The fact that workers in
farm C also work in the chicken house might have contributed to the higher prevalence
observed at farm C, compared to other farms.

In conclusion, our study demonstrated a high prevalence of Campylobacter spp.
throughout the pig production system in northern Thailand. This poses a direct risk of
infection to consumers, particularly children in Thailand. These high prevalence levels,
coupled with the documented ability of Campylobacter to develop and/or acquire
resistance to antimicrobial agents, pose a potentially serious problem of antimicrobial
resistance in a widespread human pathogen in Thailand. Long-term epidemiological
studies, employing a longitudinal study design, should be conducted to clarify the risk
factors for Camplyobacter colonization at pig farms. New studies involving more farms
should be conducted, as this will allow causal relationship to be established more

precisely, with more statistical power.

90

Table 4-1 Prevalence of Campylobacter in pigs and farm workers in northern Thailand,

 

 

 

2000-2001 .
Place Pigs Workers Environment
# % # % # %
Tested Positive Tested Positive Tested Positive
Farms 254 73.6 32 12.5 70 14.3
Slaughterhouse 1 80 45 .6 NA NA 22 4.6
Market 69 24.6 NA NA NA NA

 

NA — No available sample

91

Table 4—2 Incidence of Campylobacter colonization/contamination in pigs in northern

Thailand, 2000-2001.

 

 

 

Place # Tested % Positive Relative risk

Risk 95%C.I.
Farms 18 61.1 1.83 0.65-2.54
Slaughterhouse 12 41 .7 1 .40 0.57-3 .43
Market“ 15 33.3 - -

 

* base for comparison

92

Table 4-3 Effect of sampling location on the odds of finding Campylobacter in pig

 

 

Year Location Prevalence (%) Odds ratio 95% C1
2000 Farm 59.6 4.21 2.35 — 7.57
Slaughterhouse 26.0
2001 Farm 92.6 4.66 2.30 — 9.45
Slaughterhouse 72.4 8.28 3.73 — 18.38
Market 24.6

 

93

Table 4-4 Effect of source farm on the probability of finding Campylobacter in pigs at

farm and slaughterhouse

 

 

Farm Prevalence at Odds ratio* Prevalence at Odds ratio
farm (%) (95% CI) slaughterhouse (95% CI)
(%)
A 79'2 (0.53.5111) 70° (1.3 32in)
B 24.0 (0010;4203) No sample -
C 92" (1,425.7) 94'7 (139-6112.7)
D 88.0 - 62.5 -

 

*Control for year of sample collection

94

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Campylobacters from Malaysia. Singapore medical journal. 362282-284.

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Logtestijn, and J. H. J. Veld. 1997. The transmission of Campylobacter in piggeries; an
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Environmental survey by manual drag sampling for Salmonella enteritidis in chicken
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97

Whyte, P., J. Collins, K. McGill, C. Monahan, and H. O'Mahony. 2001. The effect of
transportation stress on excretion rates of Campylobacters in market-age broiler. Poultry
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98

CHAPTER 5
PREVALENCE OF CAMPYLOBA C T ER SPP. IN BROILER CHICKENS, FARM

WORKERS, AND THE CHICKEN PRODUCTION SYSTEM IN NORTHERN
THAILAND, 2000-2001

ABSTRACT

A combination of cross-sectional and prospective study design was used to
determine the prevalence of Campylobacter spp. in broiler chickens at farms,
slaughterhouse, market, and human workers associated with those facilities. A total of 6
broiler farms, one slaughterhouse and one meat vendor at the flesh market participated in
the study during 2000-2001. A total of 305 fecal swabs fiom cloaca from the farms, 176
fecal swabs from the cloaca and carcass swabs were collected from the slaughterhouse,
72 thigh muscles were purchased from the market, and 22 stool samples from workers at
the farms and slaughterhouse were collected. Isolation and identification of
Campylobacter spp. was done using enrichment media, selective media and biochemical
tests.

Prevalence of Campylobacter spp. in broiler chickens was found to be 73.4%,
50.6% and 47.2% at the farms, slaughterhouse and market respectively. Campylobacter
spp. were significantly more prevalent at the farms (p S 0.01) than slaughterhouse or
market. Significantly more C. jejuni were isolated fiom samples collected at the
farms(42.9%; p S 0.01) than at the slaughterhouse (19.5%) or market (11.8%). No
Campylobacter spp. were isolated from healthy workers. Older birds was less likely to be
colonized with Campylobacter spp. (p S. 0.01). Increasing flock size was significantly
associated with higher prevalence of Campylobacter in chickens, while increasing age

was significantly associated with lower prevalence.

99

INTRODUCTION

The public health consequences of Campylobacter spp. are widely recognized. In
developed countries, C. jejuni has been recognized as a major cause of foodbome
bacterial enteritis (Acheson, 2001; Pearson, et al., 2000), and can result in severe
gastroenteritis, which may be fatal in immunocompromised patients (Allos, 2001). In
contrast to the developed countries, Campylobacter spp. has been reported mainly as a
diarrheal pathogen in children in developing countries (Phetsouvanh, et al., 1999;
Rasrinual, et al., 1988; Taylor, et al., 1993; Varavithya, et al., 1990), and has been
reported as prevalent as enterotoxigenic E. coli and Salmonella in Thailand (Rasrinual, et
al., 1988). Campylobacter infections in adults in developing countries commonly do not
result in any clinical symptom (Allos, 2001).

Foods of animal origin are commonly implicated as sources of Campylobacter
spp. infection in humans (Acheson, 2001). Chicken is a common source of infection for
sporadic cases (Effler, et al., 2001; Studahl and Andersson, 2000) of carnpylobacteriosis
in developed countries. In Thailand, Campylobacter spp. was isolated from 12% of food
samples (Rasrinual, et al., 1988). Campylobacter spp. are commonly isolated from
chicken at farms, slaughterhouses and poultry products at retail markets in Germany
(Atanassova and Ring, 1999), Denmark (Wedderkopp, et al., 2000), and Japan (Ono and
Yamamoto, 1999).

Because of the increasing public health importance of Campylobacter spp. in
terms of disease and associated antimicrobial resistance, it is important to study the
epidemiology of this infection in both animals and humans. There have been very few

studies examining the presence of Campylobacter through the food production system

100

from farm to market, in both food animals and the workers in direct contact with these
animals. The basic hypothesis underlying this research is that Campylobacter spp. are
prevalent in chickens throughout the production system in Thailand, and major factors
influencing the observed prevalence can be determined. The objectives of our study,
therefore, were to: 1) determine the prevalence of Campylobacter spp. in broiler chickens
at farms, slaughterhouses, and market, and humans working on these farms and
slaughterhouses, and 2) determine the risk factors associated with the observed

prevalences.

MATERIALS AND METHODS

Study design and population. A combination of cross-sectional and prospective
studies were used. This study is part of a larger epidemiological study of Campylobacter
in food production systems in northern Thailand. The study was conducted in two phases:
the first phase consisted of a cross-sectional study fi'om May to July of 2000, and the
second phase was a prospective study from May to July of 2001. Chicken farms were
selected to participate in this study, based on their willingness to participate. Additional
criteria for inclusion were 1) the farms had chickens at the age of approximately 40 days
(1 week prior to slaughter) at the first sampling time, and 2) the farms had to be located
within 80 kilometers of the laboratory. The chicken farms in the study belonged to two
companies, three farms from each company.

Description of the poultry production system. The broiler chicken industry in
Thailand is composed of two sectors. The first sector involves large commercial farms

that produce meat primarily for export. The second sector involves small farms that

101

 

produce meat for the local consumption. Because the latter industry has a major impact
on the food safety of the local population, this study was designed to evaluate the
problem of Campylobacter in the sector that produces meat for local consrunption. Each
company supplies the farm with newborn chicks, feeds, and medications. The chickens
were raised on farms until they reached market age at about 45 to 55 days old. The
companies then sell the chickens to meat vendors, who usually operate their own
slaughterhouses. After slaughter, the meat vendors sell the chickens at the market the
next day. There were no government inspections of animals and carcasses.

In year 2001, an arrangement was made with the company and the meat vendors,
so that the chickens sampled from the same flock at the farm would be delivered to a
specific slaughterhouse and to a specific vendor at the market. This arrangement allowed
us to follow the same flock of chickens fiom the farm, to the slaughterhouse, and
eventually to the market.

Sample size. Because there were no reports of prevalence of Campylobacter spp.
in chickens at the farm level in Thailand, the reported prevalence of Campylobacter spp.
in food (12%) was used for sample size calculation (Rasrinual, et al., 1988). In order to
estimate the frequency of Campylobacter in chickens within 11% of the true prevalence,
and with Type I errors of 0.10, a sample size of 22 chickens was derived using a
previously published formula (Smith, 1995). To account for any attrition, 25 chickens
were considered to be an adequate sample size from each farm.

Specimen and data collection. At the farm, 25 chickens were randomly selected
from the chicken house. Fecal swabs were collected from the cloaca, using sterile cotton

swabs which were subsequently stored in Stuart's transport media (RCM supply,

102

Bangkok, Thailand). Samples of floors and water trays (pooled sample of five for each
house) were collected using sterile gauze pads soaked with 10 ml of sterile skim milk.
All farm workers were provided with plastic cups containing Cary-Blair medium and
asked to submit 10 grams of stool in the cups provided. Samples were kept on ice during
transportation and processed within six hours after collection. Samples were collected
from the farms twice (nine days apart) in 2001. It was not possible to sample the same
chickens twice, due to the difficulty in tracking the same chicken from farm to
slaughterhouse and market in Thailand.

At the slaughterhouse, samples were collected fiom the chickens after killing and
defeathering but before the carcasses were put into the chilling tank. Fecal swabs fi‘om
cloaca were collected using sterile cotton swabs and stored in Stuart's transport media.
Carcass swabs of the area under both wings were collected using a sterile 25 cm2 gauze
pad, which were subsequently stored in 10 ml skim milk. At the market, a thigh from
each chicken was purchased fiom the vendor. Samples were stored on ice during
transportation and processed within 12 hours after collection.

In addition to biological specimens, data concerning farm management and the
birds was collected using a pre-tested questionnaire administered to the farm owner by
one of the investigators (PP). Information on the birds included the age of birds in the
flock being sampled, and the flock size (the number of chickens in the house where
samples were collected).

Isolation and identification of Campylobacter spp. Fecal samples were directly
inoculated on Karmali agar with antimicrobial supplements (including cefoperazone,

vancomycin, and amphotericin B), and incubated at 42°C under10% CO2 for up to 5

103

days. All other samples were put in 90 ml of Bolton broth with antimicrobial supplements
(cefoperazone, trimethoprim, vancomycin, and amphotericin B), and incubated for 48
hours at 42°C under10% CO2, and a swab of the supernatant from each sample was
inoculated on Preston agar with antimicrobial supplements (polyrnixin B, rifampicin,
trimethoprim, and cycloheximide). Approximately 10 grams of meat were removed fiom
each thigh using sterile equipment, minced, and then processed as described above for the
non-fecal swab samples.

The agar plates were examined everyday for positive colonies. Suspected
Campylobacter colonies were confirmed by oxidase test (Dryslide, BBL), catalase test
(3% H202), and gram stain. Grarn-negative spiral rods that were oxidase and catalase
positive were identified as Campylobacter, fiozen and stored in 30% glycerol with
Mueller-Hinton broth at -70°C for future analysis. Campylobacter isolates were subjected
to speciation using a 5'-nuclease fluorogenic PCR (Padungtod, etal., 2002).

Statistical analysis. A positive sample was defined as one with positive
Campylobacter spp. identification. Prevalence was calculated as the number of positive
samples divided by total number of samples tested. At slaughter, where two samples
were collected from one bird, a positive bird was one where at least one of the samples
was positive for Campylobacter spp. A Chi-square test was used to determine the
significance of the association between proportion of positive birds with the location
where samples were taken (farm, slaughterhouse, or market).

A multivariable logistic regression model with random effects was used to model
the odds of finding Campylobacter in chickens at the farm. Independent variables

included: flock size (input in step of 1,000), age of the chickens (day) when samples were

104

collected, and year of sample collection. A backward elimination algorithm was used.
Variable which its removal resulted in change of odds ratio for flock size by 10% was
retained in the model. Source farm of the chicken was included in the model as random
effect term.

Model parameters were estimated using Generalized Estimating Equation (GEE;
Zeger and Liang, 1986). GEE can be applied to repeated measures data with missing
value, and consistent estimates can be obtain under mild assumption of correlation among
observation (Stokes et al.,2000). All analysis was done using SAS V8.01 (SAS institute

Inc., Cary, NC).

RESULTS

In 2000, three chicken farms, with 1000 - 5000 chickens each, from company A
participated in the study. In 2001, three chicken farms, with 3000 - 6500 chickens each,
from company B participated in the study. The same slaughterhouse participated in both
years of the study. Meat samples were collected from Nongdok market in Lampoon city,
located 30 kilometers from the laboratory. A total of 553 samples from chickens, 22
from workers, and 37 environmental samples were collected during the study.

Frequency of Campylobacter isolation. Campylobacter spp. was isolated fi'om
the chickens at farms(73.4) more than at slaughterhouses(50.6) or the market (47.2; p 5
0.01; Table 5-1). There was no significant different between the prevalence of
Campylobacter at the slaughterhouse and market (p = 0.462). No Campylobacter spp.
was isolated from healthy workers, and the prevalence of Campylobacter in

environmental samples was much lower than in chickens. At the slaughterhouse,

105

significantly more Campylobacter were isolated from cloacal samples (40.8%) than
carcass swabs (3.9%; p S 0.01) in 2000. In 2001, approximately the same proportion of
cloacal (34.7%) and carcass(37.5%) swab yielded Campylobacter (p = 0.86).
Campylobacter were isolated from carcass swabs significantly more fi‘om 2001 samples
(37.5%) than from samples taken in 2000 (3.9%; p 5 0.01).

The prevalence of C. jejuni isolation was compared with other non-jejuni isolates
(Table 5-2). The prevalence of C. jejuni was higher at the farms than at the
slaughterhouse and market (p _<_ 0.01). There was no significant difference in the
prevalence of C. jejuni between the slaughterhouse and the market.

Risk factors. The result of a multivariable logistic regression model with random
effects was shown in Table 5-3. The final model included only flock size and age of the
chickens when samples were collected. Increasing flock size was significantly associated
with higher prevalence of Campylobacter in pigs at the farm, while increasing age was

siignificantly associated with lower prevalence.

DISCUSSION AND CONCLUSION

The prevalence of Campylobacter spp. within chicken flocks reported here was
similar to reports from other developed and developing countries (Heuer, et al., 2001;
Kazwala, et al., 1990; Simango and Rukure, 1991). Only one out of 35 environmental
samples fi'om the farms yielded Campylobacter. This may reflect the inability of
Campylobacter spp. to survive in aerobic conditions, where Campylobacter may turn into

the coccoid non-culturable form (Nachamkin, 1999).

106

The prevalence of Campylobacter at the slaughterhouse was much lower than
what was previously reported in modern slaughterhouses(100%) (Bemdtson, et al., 1996).
The lower observed prevalence in our study probably reflects the differences in slaughter
and sample collection processes between modern slaughterhouses and the slaughterhouse
in this study. The slaughterhouse in this study was a small local facility that processed
only 500-800 chickens per day. Slaughtering was done manually by workers, and
carcasses were not opened or eviscerated, which would minimize fecal contamination of
the carcasses. Although fecal samples were taken from the cloaca, the fact that the
carcasses were not opened may greatly reduce the chance to find Campylobacter on those
carcasses.

The prevalence of Campylobacter spp. observed at the slaughterhouse in 2000
may not be associated with the prevalence observed at the farm since chickens sampled at
the slaughterhouse were not fi'om the same flock sampled at the farm. In 2001, although
the chickens sampled at the slaughterhouse were from the same flocks sampled at the
farm, the Campylobacter isolated at the slaughterhouse may not have been those that
colonized chickens at the farm. The fact that there was significantly less C. jejuni at the
slaughterhouse, which was the contrary to what was observed at the farms suggested
contamination at the slaughterhouse. The probable source of contamination is water in
the slaughterhouse (Bemdtson, et al., 1996). Since the carcasses were not opened, there
may only be a very limited chance that C. jejuni in the chickens would contaminated the
carcasses, compared to other modern slaughterhouses (Berrang and Dickens, 2000).

The different prevalences observed fi'om carcass swab samples in 2000 to 2001

may be a result of different sample collection and isolation protocol. A larger piece of

107

gauze was used to swab the carcasses in 2001, as opposed to the cotton swabs used in
2000. Also, the enrichment step of all swab samples was only implemented in 2001,
which might have increased the sensitivity of the isolation protocol in the second year.

The prevalence of Campylobacter spp. on meat at the market was higher than
previously reported in Thailand (Rasrinual, et al., 1988). However, the previous report
was conducted in a different city at different time, and combined various types of meat.
Since C. jejuni was not very prevalent at the slaughterhouse, it was not surprising that no
C. jejuni was found on the meat at the market, despite the fact the carcasses were opened
and eviscerated at the market before the thigh pieces were purchase from the vendors.
This may be the result of evisceration by hand, which may reduce the chance of tearing
the intestines in comparison to evisceration using machines.

This study indicated that older chickens were less likely to be colonized by
Campylobacter, which does not agree with previous studies (Kazwala, et al., 1990). Of
flocks sampled in this study, the two flocks with samples collected at age higher than 51
days yielded less Campylobacter than other flocks. The previous study which reported
the increase in prevalence of Campylobacter in chickens only followed birds up to more
than 28 days (Kazwala, et al., 1990). This difference in the maximum age of follow up
may account for this difference in findings.

Larger flock size was shown to be significantly associated with higher prevalence.
This observation may in part be due to the fact that hygiene measures in larger flocks
may be less rigorous thanwhat was practiced in smaller flocks. The farms we collected

samples from were family owned and operated only by family member. There fore,

108

limited labour for larger flocksize may play an important role in general hygienic
management of the farms.

The reason no Campylobacter spp. was found in any farm or slaughterhouse
workers was due in part to the fact that there were very few healthy workers available to
participate in the study. Five of the six farms participating in the study were operated by
family members only, with only two workers per farm. The slaughterhouse was owned
and operated by the meat vendor, and employed only four workers. Although it was
suggested that the isolation of Campylobacter from healthy persons in the developing
world was the result of constant reinfection (Oberhehnan and Taylor, 2000), we did not
observe any Campylobacter in workers constantly exposed to Campylobacter on the
poultry farms or at the slaughterhouse.

In summary, this study demonstrated high prevalences of Campylobacter through
the chicken production process in northern Thailand. The use of molecular techniques
for genotyping Campylobacter may be helpfirl in clarifying the relationships among
isolates from different sampling points (Wassenaar and Newell, 2000). This poses a
direct risk of infection to consumers, particularly children in developing countries. These
high prevalence levels, coupled with the documented ability of Campylobacter to develop
and/or acquire resistance to antimicrobial agents, pose a potentially serious problem of
antimicrobial resistance in a widespread human pathogen in Thailand. Long-term
epidemiological studies, employing a longitudinal study design, should be conducted to
clarify the risk factors for Camplyobacter colonization at pig farms. New studies
involving more farms should be conducted, as this will allow causal relationship to be

established more precisely, with more statistical power. Studies designed to conduct

109

analyses at flock level, as opposed to chicken level, will allow more farms to be included
in the studies, while maintaining the applicability of results to small poultry producers

throughout the area.

110

Table 5-1. Prevalence of Campylobacter spp. in chickens in northern Thailand,

 

 

2000-2001.
Chickens Workers Environment
# % # % # %
Place
Tested Positive Tested Positive Tested Positive
Farms 305 73.4* 18 0 35 2.9
Slaughterhouse 176 50.6 4 0 2 0
Market 72 47.2 NA NA NA NA

 

NA — No available sample

* Chi-square p .<_ 0.05

111

Table 5-2. Prevalence of C. jejuni in chickens in northern Thailand, 2000-2001.

 

 

Place # Tested % C. jejuni % others Chi-square p
Farms 205 42.9 57.1 50.01
Slaughterhouse 82 19.5 80.5 0.462
Market 34 11.8 88.2 -

 

base for comparison

112

Table 5-3. Results of a multivariable logistic regression model, with random

effects, for a single chicken being infected with Campylobacter at the farm.

 

Risk factor Range Odds Ratio 95% CI.
Flock size
1 - 8 1.3 1.1-1.6
(*1,000 chickens)
Age of bird
32 - 63 0.90 0.87-0.93
(dayS)

 

113

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Medscape Infectious diseases. 4(10):1017.

Allos, B. 2001. Campylobacterjejuni Infections: Update on emerging issues and trends.
Clinical infectious diseases. 32:1201-1206.

Atanassova, V., and C. Ring. 1999. Prevalence of Campylobacter spp. in poultry and
poultry meat in Germany. International journal of food microbiology. 51:187-190.

Bemdtson, E., M. Danialsson-Tham, and A. Engvall. 1996. Campylobacter incidence on
a chicken farm and the spread of Campylobacter during the slaughter process.
International journal of food microbiology. 32:35-47.

Berrang, M., and J. Dickens. 2000. Presence and level of Campylobacter spp. on broiler
carcasses throughout the processing plant. Journal of applied poultry research. 9:43-47.

Effler, P., M. Ieong, A. Kimura, M. Nakata, R. Burr, E. Cremer, and L. Slutsker. 2001.
Sporadic Campylobacterjejuni infections in Hawaii: Association with prior antibiotic use
and commercially prepared chicken. The journal of infectious diseases. 183:1152-1155.

Heuer, 0., K. Pedersen, J. Andersen, and M. Madsen. 2001. Prevalence and antimicrobial
susceptibility of thermophilic Campylobacter rn organic and conventional broiler flocks.
Letters 1n applied microbiology. 33: 269- 274.

Kazwala, R., J. Collins, R. Crinion, and H. O'Mahony. 1990. Factors responsible for the
introduction and spread of Campylobacterjejuni infection in commercial poultry
production. The veterinary record. 126:305-306.

Nachamkin, I. 1999. Campylobacter and Arcobacter, p. 716-726. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
microbiology, 7 ed. ASM press, Washington DC.

Oberhelman, R., and D. Taylor. 2000. Campylobacter infections in developing countries,
p. 139-153. In I. Nachamkin and M. Blaser (ed.), Campylobacter, 2 ed. ASM press,
Washington DC.

Ono, K., and K. Yamamoto. 1999. Contamination of meat with Campylobacterjejuni in
Saitarna, Japan. International journal of food microbiology. 47:211-219.

Padungtod, P., D. Wilson, J. Bell, J. Kaneene, R. Hanson, and J. E. Linz. 2002.
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Pearson, A. D., M. H. Greenwood, J. Donaldson, T. D. Healing, D. M. Jones, M.
Shahamat, R. K. A. Feltham, and R. R. Colwell. 2000. Continuous source outbreak of
Campylobacteriosis traced to chicken. Journal of food protection. 63(3):309-314.

Phetsouvanh, R., Y. Midorikawa, and S. Nakamura. 1999. The seasonal variation in the
microbial agents implicated in the etiology of diarrheal diseases among children in Lao
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health. 30(2):319-323.

Rasrinual, L., O. Suthienkul, P. Echeverria, D. Taylor, J. Seriwatana, A.
Bangtrakulnonth, and U. Lexomboon. 1988. Foods as source of enteropathogens causing
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Simango, C., and G. Rukure. 1991. Potential sources of Campylobacter species in the
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Smith, R. D. 1995. Veterinary Clinical Epidemiology, 2 ed. CRC press, Ann Arbor.

Stokes, M.E., C.S.Davis, and G.G. Koch. 2000. Categorical data analysis using the SAS
system. 2"d ed. SAS Institute, Cary, NC.

Studahl, A., and Y. Andersson. 2000. Risk factors for indigenous Campylobacter
infection: A swedish case-control study. Epidemiology and infections. 125:269-275.

Taylor, D. N., D. M. Perlrnan, P. D. Echeverria, U. Lexomboon, and M. J. Blaser. 1993.
Campylobacter immunity and quantitative excretion rates in Thai children. The journal of
infectious diseases. 168:754-758.

Varavithya, W., K. Vathanophas, L. Bodhidata, P. Punyaratabandhu, R. Sangchai, S.
Athipanyakom, C. Wasi, and P. Echeverria. 1990. Importance of Salmonellae and
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28(11):2507-2510.

Wassenaar, T., and D. Newell. 2000. Genotyping of Campylobacter spp. Applied
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Wedderkopp, A., E. Rattenborg, and M. Madsen. 2000. National surveillance of
Campylobacter in broilers at slaughter in Denmark in 1998. Avian diseases. 44:993-999.

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outcomes. Biometrics. 42: 121-130.

115

CHAPTER 6
ANTIMICROBIAL RESISTANCE IN CAMPYLOBA CTER SPP. ISOLATED
FROM FOOD ANIMALS AND HUMANS IN NORTHERN THAILAND,
2000-2001
ABSTRACT

A cross-sectional study was conducted to determine the frequency of
antimicrobial resistance in Campylobacter spp. isolated from food animals, food
products, and farm workers in northern Thailand, and the risk factors associated with the
observed frequencies of resistance. Six chicken farms, four pig farms, three
slaughterhouses, and two meat vendors participated in the study. Isolation and
identification of Campylobacter were done using enrichment media, selective media,
biochemical tests and fluorogenic PCR. In vitro susceptibility testing was done using the
microbroth dilution technique. Antimicrobial agents tested included ciprofloxacin,
erythromycin, gentamicin, azithromycin, clindarnycin, chloramphenicol, nalidixic acid
and tetracycline. Multivariable logistic regression model was used to determine the
significance of potential risk factors on the presence of resistance.

Resistance to all 8 antimicrobial agents tested was found in Campylobacter
isolated fiom pigs at farms, slaughterhouses and market, and 6 of the agents tested in
chickens. The most prevalent forms of resistance seen were to ciprofloxacin, nalidixic
acid and tetracycline. The prevalence of resistance was higher for those antimicrobial
agents to which animals were exposed. No resistance or low frequency of resistance was
observed for those antimicrobial agents not used on the farm. Campylobacter isolated
from pigs were significantly resistance to more type of antimicrobial agents, and had

significantly higher MIC for all antimicrobial agents than those isolated fi'om chickens.

116

INTRODUCTION

The public health consequences of Campylobacter spp. are widely recognized. In
developed countries, C. jejuni has been recognized as a major cause of foodbome
bacterial enteritis (Acheson, 2001; Pearson, et al., 2000), and can result in severe
gastroenteritis, which may be fatal in immunocompromised patients (Allos, 2001). In
contrast to the developed countries, Campylobacter spp. has been reported mainly as a
diarrheal pathogen in children in developing countries (Phetsouvanh, et al., 1999;
Rasrinual, et al., 1988; Taylor, et al., 1993; Varavithya, et al., 1990), and has been
reported as prevalent as enterotoxigenic E. coli and Salmonella in Thailand (Rasrinual, et
al., 1988). Campylobacter infections in adults in developing countries commonly do not
result in any clinical symptoms (Allos, 2001). Foods of animal origin are commonly
implicated as sources of Campylobacter spp. infection in humans (Acheson, 2001).
Campylobacter spp. are commonly isolated fi'om chicken at farms, slaughterhouses and
on poultry products at retail markets (Atanassova and Ring, 1999), and in Thailand,
Campylobacter was isolated from 12% of food samples (Rasrinual, et al., 1988).

Antirrricrobial resistance has become one of the major public health concerns in
both developed and developing countries in recent years (Isenbarger, et al., 2002; Witte,
1998). Resistance to antimicrobial agents in enteric bacteria poses an increased risk of
treatment failure and increased cost of treatment (V asallo, et al., 1998). The use of
antimicrobial agents has been linked to the development of resistant bacteria in humans
(Berends, et al., 2001) and livestock (Boonmar, et al., 1998; J acob-Reitsma, et al., 1994),
and levels of antimicrobial resistance appear to be increasing in developing countries

(Hoge, et al., 1998), where there is widespread and uncontrolled use of antibiotics (Hart

117

and Kariuki, 1998). In addition to bacteria showing resistance to individual antimicrobial
agents, bacteria have also exhibited resistance to more than one drug (multi-resistance)
and cross-resistance between different families of antimicrobials (Isenbarger, et al., 2002;
Prescott, 2000).

It is widely speculated that the use of antimicrobial agents in food animal species
may be contributing to the antimicrobial resistance problem in humans (Barton, 1998;
Hoge, et al., 1998; Witte, 1998). Since Campylobacter with resistance to antimicrobial
agents has been reported in both developed and developing countries (Isenbarger, et al.,
2002; Smith, et al., 1999), the prevalence of potentially antimicrobial-resistant
Campylobacter in food animals indicates that foods of animal origin may be a source of
resistant bacteria for humans (Threlfall, et al., 2000).

In order to determine whether food animals are an important source of bacteria
with resistance to antimicrobials, one of the first steps to evaluate this situation is to
compare antimicrobial resistance patterns in pathogens fi'om food animals and humans
with exposure to these animals. In this study, we hypothesized that Campylobacter spp.
with resistance to antimicrobial agents are prevalent throughout the food animal
production system, and that antimicrobial use in swine and chicken production systems is
associated with the fiequency of antimicrobial resistance. The objectives of our study,
therefore, were to: 1) determine the frequencies of Campylobacter spp. with resistance to
antimicrobial agents in food animals, food products, and farm workers in northern
Thailand, and 2) compare the level and class of antimicrobial resistance in

Campylobacter isolated from pigs and chickens.

118

MATERIALS AND METHODS

Study design and sample size. This study is a part of a larger epidemiological
study of Campylobacter in food production systems in northern Thailand. The study was
conducted in two phases: the first phase consisted of a cross-sectional study from May to
July of 2000, and the second phase was a prospective study fi'om May to July of 2001 .

Because there were no reports of prevalence of Campylobacter spp. in chickens
and pigs at the farm level in Thailand, the reported prevalence of Campylobacter spp. in
food (12%) was used for sample size calculation (Rasrinual, et al., 1988). In order to
estimate the frequency of Campylobacter in food animals with Type I and Type H errors
of 0.10, a sample size of 22 animals per farm was derived using a previously published
formula (Smith, 1995). To account for any attrition, 25 animals were considered to be an
adequate sample size from each farm.

Study population and specimen collection. Six chicken farms, four pig farms,
one chicken slaughterhouse, two pig slaughterhouses, and two meat vendors at the market
participated in the study. Specimen collection was done during May to July of 2000 and
2001. Pig farms in the study were finishing pig operations, raising pigs from ages 30 to
110 days, that were subcontractors for a company that maintains a large, 1000-sow
operation in Chiang Mai province in northern Thailand. The chicken farms in the study
belonged to two companies, and raised chickens until they reached market age at about
45 to 55 days old. F arms were selected for this study based on their willingness to
participate. Additional criteria for inclusion were: 1) the farms had to have animals of

appropriate age at the first sampling time (chickens at the age of approximately 40 days,

119

pigs at the age of approximately 95-100 days); and 2) be located within 80 kilometers
radius fiom the laboratory.

Specimens were collected at the farm approximately one week before the animals
were slaughtered, after slaughter in the slaughterhouse, and at the fresh meat markets
(Table 6-1). All samples were stored on ice during transportation to the laboratory and
processed within 12 hours after collection.

Animal samples

From available pens in pig house at each farm, five pigs were randomly selected
from five systematically sampled pens, and approximately 10 grams of fecal material
were evacuated from the rectum of each pig and stored in plastic cups on ice. At the
poultry farms, 25 chickens were randomly selected, and fecal swabs were collected fiom
the cloaca, using sterile cotton swabs which were subsequently stored in Stuart's transport
media (RCM supply, Bangkok, Thailand).

At slaughter, pig mesenteric lymph nodes were collected after evisceration,
approximately 20 grams of fecal material were collected fiorn the intestine of each pig,
and carcass swabs were collected by wiping an area approximately 40 cm2 fiom around
the thigh area and inside the rib cage with sterile gauze pads. Samples were collected
from the chickens after killing and defeathering but before the carcasses were put into the
chilling tank. Fecal swabs from cloaca were collected using sterile cotton swabs and
stored in Stuart's transport media, and carcass swabs of the area under both wings were
collected using a sterile 25 cm2 gauze pad. All carcass swabs were put in plastic bags

with 10 ml sterile skim milk for transport.

120

In the second phase of the study, approximately 100 grams of pork fiom the neck
area attached to the head (with the ear tag) and a thigh fiom each chicken were purchased
at the fresh market.

Farm and slaughterhouse worlgrs samples

Farm and slaugherhouse workers were provided with sterile plastic cups
containing Cary-Blair medium and asked to submit 10 grams of stool in the cups
provided.

Environmental samples

Swabs of pen floors and feed trays were collected using sterile gauze soaked with
10 ml of sterile skim milk.

Data collection. Data collection was accomplished by using two pre-tested
questionnaires that were administered in person by one of the investigators (PP). The first
questionnaire was used to collect data relating to the types and quantities of antimicrobial
agents used in feed and treatment of sick animals, and other farm management practices.
Other data, including the age of the animals sampled, were also collected. The second
questionnaire was used to collect data relating to the farm workers. These data included
worker’s age, gender, medical history, and whether they had consumed any antimicrobial
agents within one month prior to the day stool samples were collected.

Isolation and identification of Campylobacter spp. Isolation of Campylobacter
spp. was achieved using enrichment media (Bolton broth) and selective agar (Karmali or
Preston agar). Suspected Campylobacter colonies were confirmed by oxidase test
(Dryslide, BBL), catalase test (3% H202), and gram stain. Gram-negative spiral rods that

were oxidase and catalase positive were identified as Campylobacter, frozen and stored

121

in 30% glycerol with Mueller-Hinton broth at -70°C. The stock bacteria were transported
from Thailand to Michigan State University (MSU) where in vitro susceptibility testing
was done. Identification of C. jejuni was done using 5'-nuclease fluorogenic PCR assay
(Padungtod, et al., 2002) conducted at MSU.

In vitro susceptibility testing. In vitro susceptibility testing was done using the
microbroth dilution method, following guidelines provided by the National Committee on
Clinical Laboratory Standards (NCCLS, 1997). Bacterial isolates from frozen stock were
grown on Brucella agar supplemented with 5% defiibrinated sheep blood (BASB) for 48
hours at 37°C under microaerophilic conditions (85%N2, 5% C02, 10% 02). Individual
colonies fi'om each plate were subcultured on BASB under similar growth conditions.
Bacteria were scraped from the BASB with a sterile cotton swab and suspended in 5 ml
H2O. The turbidity was adjusted to a 0.5 McFarland standard using a standard solution,
and one ml of the bacterial suspension was then added to 9 m1 of Haemophilus testing
medium (HTM). The final concentration of the inoculum was approximately 8 x 105
CFU/ml.

Customized SensiTitre plates were purchased pre-made from TREK Diagnostic
Systems, Inc. with azithromycin, chloramphenicol, ciprofloxacin, clindarnycin,
erythromycin, gentamicin, nalidixic acid, and tetracycline. These antinricrobials were
chosen on the basis of their importance in treating human Campylobacter infections and
to provide diversity in representation of different antimicrobial classes. Antimicrobial
concentrations on the plate ranged from 0.03 to 256 ug/ml, depending on the
antimicrobial agent, with a range of seven levels of dilution for each agent. C. jejuni

ATCC33560 was used as a quality control strain since it was shown to produce

122

repeatable antimicrobial resistance profiles in rrricrobroth dilution testing. Each plate was
inoculated by adding 100 111 of the bacterial suspension to the plate using an
autoinoculator. Plates were covered with a gas-permeable seal and incubated at 42EC at
5% CO2 for 48 hours.

The minimum inhibitory concentration (MIC), the lowest concentration of an
antimicrobial agent that inhibits growth of the bacteria, was recorded manually. Wells
with turbidity or an accumulation of bacteria at the bottom were considered positive for
bacterial growth. The breakpoints used to categorize isolates as resistant or not resistant
for each antimicrobial agent for Campylobacter spp. were those recommended by the
National Antimicrobial Resistance Monitoring System (NARMS) (Table 6-2).

Statistical analysis. The prevalence of antimicrobial resistance was calculated as
the number of samples yielding Campylobacter spp with resistance to a given
antimicrobial agent divided by total number of samples tested. A chi-square test was used
to compare proportion of Campylobacter with resistance to antimicrobial agents from
different source.

Because the type and duration of antimicrobial use in pig farms was different
from those in chicken farms. All pig farms in our study shared similar protocol for using
antimicrobial agents. Similarly all chicken farms in our study also shared similar protocol
for using antimicrobial agents. The source animal of Campylobacter was used as proxy
measure of antimicrobial use when comparing antimicrobial resistance.

A Wilcoxon Rank Sum test was used to compare the number of antimicrobial

agent to which Campylobacter were resistant between Campylobacter isolated from pigs

123

and chickens. And to compare the minimum inhibitory concentration for each
antimicrobial agents between Campylobacter isolated fi'om pigs and chickens.

A multivariable logistic regression model with random effects was used to model
the odds of being resistance to each antimicrobial agent. Thus, 6 separate multivariable
models were conducted for each of the 6 antimicrobial agents tested. Independent
variables included source animal (pig, chicken), year of sample collection, location where
samples were collected, and whether or not Campylobacter isolates was Cjejuni. Source
animal was the main effect of interest. Specific farms or slaughterhouse where samples
were collected was included in the model as random effect term. All variables were
included in the model initially. The backward elimination algorithm was used. Variable
which its removal resulted in change of odds ratio for source animal by more than 10%
was retained in the model.

Model parameters were estimated using Generalized Estimating Equation (GEE;
Zeger and Liang, 1986). GEE can be applied to repeated measures data with missing
value, and consistent estimates can be obtain under mild assumption of correlation among
observation (Stokes et al.,2000). All analysis was done using SAS V8.01 (SAS institute

Inc., Cary, NC).

RESULTS

Resistance to antimicrobial agents was found in Campylobacter from farms,
slaughterhouses, and market, in both pigs and chickens (Table 6-3). In chickens,
resistance was found for six of eight antimicrobial agent tested. The most prevalent forms

of resistance seen were to ciprofloxacin, nalidixic acid and tetracycline. Over 79% of

124

Campylobacter isolated from chickens were resistant to more than one agent. In pigs,
resistance was found for all eight antimicrobial agents tested. The three most prevalent
forms of resistance were ciprofloxacin, nalidixic acid and tetracycline. Over 89% of
Campylobacter isolated from pigs were resistant to more than one antimicrobial agent.
Only two Campylobacter isolates from pig farm workers were subjected to in vitro
susceptibility testing, and both were resistant to ciprofloxacin, nalidixic acid and
tetracycline.

Comparison of proportion of Campylobacter spp with resistance to antimicrobial
agents from the same pig farms between 2000 and 2001 is shown in Figure 6-1. As seen
in Figure 6-1, the proportion of Campylobacter spp. resistant to the 8 antimicrobial
agents remained very high in the two years. Although there were minor decline in the
proportion of resistant Campylobacter spp. (Figure 6-1), these declines were not
significantly different for all antimicrobial agents except for gentamicin(p<0.01).

Antimicrobial use on the farm was recorded using questionnaires. Although
precise estimates of quantities used were not available, concentrations used in feed or
water were obtained, and provide a comparative amount of antimicrobial use. In chicken
farms, lincomycin and enrofloxacin were used in feed at 2.2 ppm and 5 ppm,
respectively. Chlortetracycline was also used in layer population in the same chicken
farms we collected samples from. However, none of the chickens we collected sample
were exposed directly to Chlortetracycline. On pig farms, amoxycillin, Chlortetracycline
and lincospectin were used in feed at 200 ppm, 2000 ppm and 1000 ppm, respectively.
These antimicrobial agents were used as growth promoters in piglets before they were

transported to the finishing farms. Gentarnicin, amoxycillin, and enrofloxacin were used

125

on pig finishing farms for therapeutic only. None of the pigs we collected sample from
were treated with these agents.

Comparisons were made between the classes of antimicrobials used on animals
and the farms and levels of antimicrobial resistance (Figure 6-2). On both pig and
chicken farms, the prevalence of resistance was higher for those antimicrobial agents
with related drug exposure to the animals. On both pig and chicken farms, the prevalence
of resistance Campylobacter was higher for those antimicrobial agents used on the farms
in feed and/or therapeutically, whether or not the animals were directly exposed to those
agents. While no resistance or extremely low frequency of resistance was observed for
those antimicrobial agent not used on the farm.

Campylobacter isolated from pigs were significantly (p<0.01) resistance to more
type of antimicrobial agents (Figure 6-3) and had significantly higher MIC for all
antimicrobial agents (Figure 6-4) than those isolated from chickens. The results of
multivariable logistic regression models with random effects (Table 6-4) also showed
increase odds of being resistance to ciprofloxacin, erythromycin, azithromycin,
clindamycin, nalidixic acid and tetracycline in Campylobacter isolated from pigs
compared to those isolated fi'om chickens. Models for clindamycin and chlorphenicol
were not possible due to lack of resistance in isolates from chickens and low frequency of

resistance in pigs.

DISCUSSION AND CONCLUSION
This study found antimicrobial resistance to a variety of agents in Campylobacter

isolates fiom food animals, farm workers, and meat samples in Thailand. Our study

126

demonstrated high prevalences of resistant isolates of Campylobacter fi'om pigs and
chickens, which are known reservoirs for Campylobacter in humans (Pearson, et al.,
2000). More importantly, this study demonstrate the association between antimicrobial
use on the farms and the prevalence of resistance Campylobacter in food animals
through the production system at farms, slaughterhouses, and markets. This association
was demonstated based on the specificity of the relationship between antirrricrobial used
on the farms and the prevalence of resistance. Our study demonstrate that resistance to
the same class of antimicrobial agents used on the farms were observed, while no
resistance or low level of resistance were observed for those antimicrobial agents not
used on the farms for both pigs and chickens.

When species of animals were used as proxy measure for antimicrobial used,
prevalence of resistance and MIC value for Campylobacter isolated from pigs which
were exposed to antimicrobial agents for longer period of time was significantly higher
than those isolated from chickens. Although we demonstrate the strength of association
between antimicrobial used on farms reflected by specie of animals and the odds of
Campylobacter being resistance to those agents, as shown by the odds ratio in Table 6-4.
However, this association reflected by comparing specie of animals should be interpreted
with caution. Since almost all Campylobacter isolated fi'om chickens were C. jejuni, the
patterns of resistance seen in chickens may only be reflective of resistance patterns
characteristic of C. jejuni. Differences in levels of resistance between species may also be
due to different exposures to antimicrobial grth promoters, and the period of time that
the animals remained on the farms. Chickens remained on the farm for less than 60 days,

while pigs were raised for more than 100 days at finishing farms. The longer time period

127

may allow the accumulation of resistance bacteria from the farm environment, and allow
longer exposure time to other animals treated with antimicrobials during the raising
period.

On: observation was consistent with the observation previously reported by other
researchers (J acob-Reitsma, et al., 1994; McDermott et al., 2002) demonstrating the
associations between exposure to antimicrobial agents and the presence of resistant
isolates in chickens and pigs. The chickens in our study were exposed to antimicrobial
grth promoters in feeds throughout the study period, and pigs were exposed to
antimicrobial growth promoters before they entered this study when they were piglets.
Patterns of resistance observed in Campylobacter isolates from pigs were probably due to
colonization of piglets by resistant bacteria before they were shipped to the finishing farm
(Young, et al., 2000). Unfortunately, our study design did not allow clarification of
temporal relationship between antimicrobial exposure and incidence of resistance
Campylobacter. A prospective study following the animals fi'om birth to slaughter will be
ideal for clarification of the temporal relationship between exposure to antimicrobial and
incidence of resistance development.

The biological plausibility that antimicrobial use in feed exerts selection pressure
for those bacteria with resistance to the exposed agents was observed not only in
Campylobacter, but also in several other bacteria (Bager et a1. 1997;Witte et al., 2000).
Antimicrobial use in the community and hospital also facilitate the resistance
development in several pathogenic bacteria (N eu, 1992). For Campylobacter, resistance
to fluoroquinolone was observed following the introduction of enrofloxacin in food

animals in the Netherland (Endtz et al. 1991) and US (Smith et al. 1999). The plausibility

128

that bacteria with resistance to antimicrobial agents developed on the farms can be
transmitted to human was demonstrated as early as 1987 (Levy, 1987). Therefore, it is
plausible that antimicrobial use in pigs and chickens farms in northern Thailand may led
to the high prevalence of resistance Campylobacter observed in our study.

The overall antimicrobial resistance profiles were generally similar among
Campylobacter isolated from farms, slaughterhouses, and markets within species. No
resistance to gentamicin and chloramphenicol was observed in chickens, regardless of
sample type or location of sampling. In pigs, resistance to gentamicin and
chloramphenicol were only observed in isolates fi'om the farm and slaughterhouse, but at
levels that were lower than for other antirrricrobial agents. By class of antimicrobial
agent, the prevalence of resistance was comparable in both pigs and chickens. The overall
prevalence of resistance in isolates from chickens was to 73% to quinolones
(ciprofloxacin) and 81% to nalidixic acid, and 98%-100% in pigs. From farm samples,
the prevalence of resistance to macrolides (erythromycin and azithromycin) and
clindamycin, a lincosarnide which commonly shows cross-resistance with macrolides
(Prescott, 2000), ranged from 9.8 to 10.4% in isolates fiom chickens and 63 to 69% in
isolates from pigs. The prevalences of resistance to these classes of agents were
comparable at slaughterhouse and market in isolates from both animals.

Multi-drug resistance was found in all types of animal samples from all sampling
locations. The overall prevalence of Campylobacter with resistance to more than one
antimicrobial agents was higher in pig samples than chicken samples. This may be due to
co-resistance among antimicrobial agents tested. In Campylobacter, resistance to

fluoroquinolones in is thought to be chromosomally mediated through the mutation in the

129

gyrA gene (Gibreel, et al., 1998; Ruiz, et al., 1998; Wang, et al., 1993), and resistance to
macrolides is due to mutation in the 23S rRNA gene (Engberg, et al., 2001; Jensen and
Aarestrup, 2001). One possible mechanism of co-resistance between quinolones and
azithromycin is the efflux pump, which may transport both agents out of bacteria cells
(Charvalos, et al., 1995). Since there are no reports of such a mechanism in
Campylobacter, the mechanism of cross-resistance between these two agents is an area
that should be investigated (Isenbarger, et al., 2002).

The Campylobacter isolated from healthy farm workers were resistant to the same
classes of antimicrobial agents commonly use in both human and animals, including
tetracycline and fluoroquinolones. The high prevalence of resistance observed in farm
workers may be due to personal use of antimicrobial agents (Berends, et al., 2001). It
should be noted, however, that interviews of farm workers showed no recollection of
using antimicrobial agents during 30 days period prior to stool collection. It has been
suggested that antimicrobial use in food animal production may result in the increasing
prevalence of bacteria with resistance to antimicrobial agents in humans (Barton, 1998;
Witte, 1998). Although there is evidence that problem of antimicrobial resistance in
humans results from personal use of antimicrobial agents (Berends, et al., 2001), the high
prevalence of resistant bacteria in food animals may pose an additional risk to the public
through consumption of foods of animal origin (Threlfall, et al., 2000). Studying patterns
of antimicrobial resistance in farm workers and their animals is an approach that can
come closer to addressing the role of antimicrobial use in food animals in the
development of antimicrobial resistance in humans. Our study collected samples from

commercial farms, slaughterhouses and markets supplying meat for local consumption.

130

Although there were a limited number of farm workers sampled, the study provides
interesting results, upon which future studies can be built.

In summary, our study demonstrated high prevalences of Campylobacter with
resistance to antimicrobial agents throughout the pig and chicken production system in
northern Thailand, and the association between antimicrobial use on the farms and
resistance to antimicrobial agents in Campylobacter. Further studies to clarify the
contribution of antimicrobial use to this problem, and assessment of risk to consumers
resulting from resistance bacteria in meat, should be conducted. Although any conclusion
regarding the quantitative contributions of antimicrobials used in farm is not possible
with this study, a surveillance system of food borne outbreaks and antimicrobial use
should be put in place in order to detect changes in antimicrobial resistance profiles in
pathogens isolated from food animals and humans, and prevent major public health

impact resulting from those resistance food borne bacteria.

131

Table 6-1 Number and type of samples collected fi'om pig and chicken farms in northern

Thailand, 2000-2001.

 

Number of Number of
Year Sampling location facilities Sample tyne samples
2000 Chicken farm 3 Cloacal swab 155
Workers’ stool 7
Chicken slaughterhouse l Cloacal swab 100
Carcass swab 100
Pig farm 3 Pig fecal 150
Workers’ stool 15
Pig slaughterhouse 1 Pig fecal 103
Carcass swab 103
2001 Chicken farm 3 Cloacal swab 150
Workers’ stool 11
Chicken slaughterhouse Cloacal swab 73
Carcass swab 73 I
Workers’ stool 4
Chicken meat vendor 1 Thigh muscle 72
Pig farm 4 Pig fecal 115
Workers’ stool 30
Pig slaughterhouse 1 Lymph node 70
Carcass swab 75
Pork vendor 1 Neck muscle 69

 

132

Table 6-2 Minimum Inhibitory Concentration (MIC) dilution ranges and breakpoint
values for determination of antimicrobial resistance for Campylobacter, based

on the National Committee on Clinical Laboratory Standards (N CCLS)

 

recommendations
Range of Concentrations Resistance Breakpoint
Antimicrobial agents Tested (pg/ml) (uiml)
Ciprofloxacin 0.03 — 64 2 4
Erythromycin 0.12 — 256 2 8
Gentamicin 0.12 — 256 2 16
Azithromycin 0.03 — 256 2 2
Chloramphenicol 1 — 64 2 32
Clindamycin 0.06 — 256 2 4
Nalidixic acid 0.12 -— 128 2 32
Tetracycline 0.25 - 256 .>_ 16

 

133

Table 6-3 Prevalence (%) of Campylobacter spp. with resistance to antimicrobial agents,
from chickens, pigs, and farm and slaughterhouse workers in northern

Thailand, 2000-2001

Chickens Pigs Worker

Farm Slaughter Market Farm Slaughter Market Farm
N 183 78 32 202 101 14 2

 

Ciprofloxacin 73.8 92.3 90.6 98.0 88.1 100.0 100.0

Erythromycin 9.8 2.6 3.1 85.6 66.3 50.0 0.0
Gentamicin 0.0 0.0 0.0 18.3 16.8 0.0 0.0
Azithromycin 10.4 2.6 3.1 85.1 63.4 50.0 0.0
Chloramphenicol 0.0 0.0 0.0 2.0 2.0 0.0 0.0
Clindamycin 9.8 2.6 3.1 85.1 69.3 57.1 0.0

Nalidixic Acid 73.2 89.7 87.5 98.0 90.1 100.0 100.0

Tetracycline 72.7 39.7 81.3 98.5 93.1 92.9 100.0

Multi-drug

79.2 97.4 96.9 95.5 89.1 100.0 100.0
Resistance

134

Table 6-4 Odds ratio for resistance in Campylobacter isolated fi'om pigs compared to

 

chickens"

Rgs‘igfzifilcfto Odds ratio for pigs" 95% CI
Ciprofloxacin 7.34 2.23 — 24.13

Nalidixic acid 7.18 2.59 - 19.88
Erythromycin 60.36 15.63 — 233.60
Azithromycin 55.94 15.04 -— 208.02
Clindamycin 65.15 17.68 - 240.07
Tetracycline 26.75 10.12 — 70.70

 

*Summary of results of 6 multivariable logistic regression models.

“Adjusted for year of sample collection, location where samples were collected, and

whether or not Campylobacter isolates was Cjejuni.

135

Figure 6-1 Proportion(%) of Campylobacter spp. with resistance to antimicrobial agents

 

       

 

 

 

 

 

 

from pigs.
r 100.0-

80.0

*5 60.0 3 =

0

§
20.0
0.0 f Tl, . . T.,.

CIP ERY GEN' AZl CHL CLI NAL TET Multi-r
2000(n=40) I 2001(n=91)

 

 

 

 

*Significantly different at pS0.05
CIP-ciprofloxacin, ERY-erythromycin, GEN-gentarnicin, All-azithromycin,
CHL-chloramphenicol, CLI-clindamycin, NAL-nalidixic acid, TET-tetracycline

Multi-r -— resistance to more than one agents

136

Figure 6-2 Relationship between resistance level and antimicrobial use on farms.

 

 

 

 

 

 

 

 

 

A. Pig farms
1 O ‘E
'3 a
§ 2
a GI!
S s
i
O % 4' 32:35? O.
TET CLI CIP GEN CHL
Antimicrobial agents

 

 

 

B. Chicken farms

 

 

 

 

 

 

1 -— "10005
3 "8° g
3 +603
0
:I 4.40 E
‘5 +20%

0 ~:&Ja ~fi+ L 0 0-

 

CIP CLI TET GEN CHL
Antimicrobial agents

 

 

 

CIP-ciprofloxacin, CLI-clindamycin, TET-tetracycline, GEN-gentamicin,

CHL-chloramphenicol

El Antimicrobial use in feed (1 = use, 0 = not use)

Antimicrobial use for treatment / in other group of animal (1 = use, 0 = not use)

Q Frequency of resistance

137

Figure 6-3 Number of antimicrobial agents Campylobacter spp. were resistant to.

 

 

 

 

 

 

 

Percent resistance

 

 

0 1 2 3 4 5 6 7 8
Number of antimicrobial agents

 

 

 

El Chickens(n=293) I Pigs(n=321)

 

 

 

 

138

Figure 6-4 Minimum inhibitory concentration of Campylobacter isolated fiom pigs and

chickens (mode)

 

 

 

Chickens (n=293)
I Pigs (n=320)

L092 MIC
.b

 

 

 

    

 

 

CIP ERY GEN AZ CHL CLI NAL TET
Antimicrobial agents

 

 

 

CIP — ciprofloxacin, ERY — erythromycin, GEN — gentamycin, AZI — azithromycin
CHL — chloramphenicol, CLI — clindamycin, NAL — nalidixic acid,

TET - tetracycline

139

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143

CHAPTER 7
USING A FLUOROGENIC PCR ASSAY FOR DETERMINATION OF
FLUOROQUINOLONE RESISTANCE
IN CAMPYLOBA CTER JEJUNI

ABSTRACT

A fluorogenic PCR assay for the gyrA gene was used to determine the frequency
of a Thr-86 mutation in Campylobacterjejuni isolates from food animals and humans in
northern Thailand, and to investigate the correlation between this mutation and bacterial
resistance to fluoroquinolones. A total of 84 isolates of C. jejuni were used: 65 from
samples from healthy chickens on farms, 16 fiom chicken samples at slaughterhouse, one
fi'om chicken meat at the market, and one isolate from a healthy farm worker. In vitro
susceptibility testing was done using the microbroth dilution technique. Minimum
inhibitory concentration (MIC) breakpoints established by the National Antimicrobial
Resistance Monitoring System were used to categorize resistance in C. jejuni to
ciprofloxacin and nalidixic acid.

Sixty of the 84 C. jejuni isolates tested carried the Thr-86 mutation in the gyrA

gene. All isolates with ciprofloxacin MIC 2 2 :g/ml carried the mutation, and all isolates

with nalidixic acid MIC 5 16 :g/ml did not carry Thr-86-to-Ile mutation. There was very

high agreement between ciprofloxacin resistance and the presence of the mutation (kappa
= 0.971 , p 5 0.01). The level of agreement between was lower for the presence of the

Thr-86-to-Ile mutation with nalidixic acid resistance (kappa = 0.859; p _<_ .01).

144

INTRODUCTION

Antimicrobial resistance has become one of the major public health concerns in
both developed and developing countries in recent years (Isenbarger, et al., 2002; Witte,
1998). The use of antimicrobial agents has been linked to the development of resistant
bacteria in humans (Berends, et al., 2001) and livestock (J acob-Reitsma, et al., 1994;
McDermott, et al., 2002), and levels of antimicrobial resistance appear to be increasing in
developing countries (Hoge, et al., 1998), where there is widespread and uncontrolled use
of antibiotics (Hart and Kariuki, 1998). Fluoroquinolones have been used successfully for
the treatment of severe gastroenteritis in human. The rapid development of
fluoroquinolone resistance, particularly in Campylobacter spp. , threaten the future
usefulness of this class of agents in the future.

Campylobacter spp. has been reported mainly as a diarrheal pathogen in children
in deve10ping countries (Phetsouvanh, et al., 1999; Rasrinual, et al., 1988), and infections
in adults commonly do not result in clinical symptoms (Allos, 2001). In Thailand,
Campylobacter has been isolated from 12% of food samples (Rasrinual, et al., 1988), and
resistance to tetracycline (Lee, et al., 1994), quinolones and macrolides (Hoge, et al.,
1998) has been documented. Campylobacter spp. are commonly isolated from chickens
at farms, slaughterhouses and on poultry products at retail markets (Atanassova and Ring,
1999), and it has been shown that Campylobacter in poultry can rapidly develop
resistance following exposure to antimicrobial agents, especially fluoroquinolones
(McDermott, et al., 2002).

F luoroquinolones are bactericidal agents acting by inhibiting the function of DNA

gyrase and DNA topoisomerase IV enzymes in bacteria, which leads to a lethal break of

145

double-strand DNA (Drlica and Zhao, 1997). Resistance to fluoroquinolones in C. jejuni
was found to be the result of mutation in gyrA or parC, which encode DNA gyrase and
topoisomerase IV, respectively (Gibreel, et al., 1998; Ruiz, et al., 1998; Wang, et al.,
1993). A single mutation in gyrA gene from Thr-86 to He (C to T transition) results in
resistance to ciprofloxacin at dilutions from 4 - 16 ug/ml (Charvalos, et al., 1996;
Zimstein, et al., 1999). Such mutations can be detected using molecular techniques such
as mismatch amplification (Charvalos, et al., 1996), nonradioactive single-stand
conformation polymorphism (Zimstein, et al., 1999), and fluorogenic polymerase chain
reaction (PCR; Wilson, et al., 2000).

Fluorogenic PCR offers several advantages over other molecular tests for
identification of mutations. The process uses a non-extendable oligonucleotide
hybridization probe that contains a fluorescent reporter dye and a quencher dye. During
PCR cycling, the probe hybridizes to the template and is digested by the exonuclease
activity of Taq DNA polymerase as it moves along the template strand. This cleavage
results in an increase of fluorescent emission of the reporter, which can be measured by
fluorescence spectrometry. Allelic discrimination testing can be done with fluorogenic
PCR, using probes and reporter dyes specific to the target allele (mutant or wild type),
and examination of the post-PCR fluorescence emission spectrum(Lee, et al., 1993). This
assay can detect a single base mismatch in probes 20 to 30 nucleotides long (Livak,
1999). The advantages of this real-time PCR allelic discrimination assay are: 1) it
employs a closed tube system, which reduces contamination of DNA; 2) the analysis is
conducted in log phase while PCR product is increasing (as opposed to end point assay in

plateau phase when PCR product concentration is stationary), allowing more than one

146

gene to be analyzed simultaneously; 3) there is no post-PCR processing step, which saves
time, labor and resources; and 4) it is compatible with automated technologies (Heid, et
al., 1996).

Isolation, identification and in vitro susceptibility test of C.jejuni is laborious and
time consuming. In order to conduct large scal epidemiological study or surveillance of
fluoroquinolone resistance C.jejuni, a diagnostic tool allowing rapid process of the
sample and compatible with with automation technologies is desirable. Because of the
advantages that fluorogenic PCR can offer to epidemiological study and surveillance of
fluoroquinolone resistance C.jejuni, it was decided to exploit the potentials of this assay
to study C. jejuni isolates obtained from a country where fluoroquinolone resistance was
prevalent. We hypothesize that the Thr-86 mutation in the gyrA gene of C. jejuni can be
observed in field isolates, and that the presence of this mutation is correlated with
resistance to ciprofloxacin and nalidixic acid. The objectives of our study were to: 1)
determine the frequency of the Thr-86 mutation in gyrA in C. jejuni isolated fiom food
animals and farm workers in northern Thailand; and 2) investigate the association
between the Thr-86 mutation in gyrA and resistance to ciprofloxacin and nalidixic acid in

C. jejuni.

MATERIAL AND METHODS

Source of Campylobacter isolates. The bacteria in this study were derived from a
cross-sectional epidemiological study of Campylobacter spp. in food animals and farm
workers in northern Thailand during the summer months of 2000 and 2001. A total of 84

isolates of C. jejuni were used in this study: 65 from samples from healthy chickens on

147

farms, 16 from chicken samples at slaughterhouse, one from chicken meat at the market,
and one isolate from a healthy farm worker.

Isolation and identification of Campylobacter spp. Isolation of Campylobacter
spp. was achieved using enrichment media (Bolton broth) and selective agar (Karmali or
Preston agar). Suspected Campylobacter colonies were confirmed by oxidase test
(Dryslide, BBL), catalase test (3% H202), and gram stain. Gram-negative spiral rods that
were oxidase and catalase positive were identified as Campylobacter, frozen and stored
in 30% glycerol with Mueller-Hinton broth at -70°C. The stock bacteria were transported
from Thailand to Michigan State University (MSU) where in vitro susceptibility testing
was conducted. Speciation of C. jejuni was done using 5'-nuclease fluorogenic PCR assay
(Padungtod, et al., 2002) conducted at MSU.

In vitro susceptibility testing . In vitro susceptibility testing was done using the
microbroth dilution method, following guidelines provided by the National Committee on
Clinical Laboratory Standards (NCCLS)(NCCLS, 1997). Bacterial isolates fi'om fi'ozen
stock were grown on Brucella agar supplemented with 5% defiibrinated sheep blood
(BASB) for 48 hours at 37°C under microaerophilic conditions (85%N2, 5% CO2, 10%
02). Individual colonies from each plate were subcultured on BASB under similar growth
conditions. Bacteria were scraped fi'om the BASB with a sterile cotton swab and
suspended in 5 ml H2O. The turbidity was adjusted to a 0.5 McFarland standard using a
standard solution, and one ml of the bacterial suspension was then added to 9 ml of
Haemophilus testing medium (HTM). The final concentration of the inoculum was

approximately 8 x 105 CFU/ml.

148

Customized SensiTitre plates were purchased pre-made fiom TREK Diagnostic
Systems, Inc., with azithromycin, chloramphenicol, ciprofloxacin, clindamycin,
erythromycin, gentarrricin, nalidixic acid, and tetracycline. C. jejuni ATCC33560 was
used as a quality control strain since it was shown to produce repeatable antimicrobial
resistance profiles in microbroth dilution testing. Each plate was inoculated by adding
100 111 of the bacterial suspension using an autoinoculator, covered with a gas-permeable
seal, and incubated at 42EC at 5% CO2 for 48 hours. The minimum inhibitory
concentration (MIC) was identified by the minimum dilution at which no bacterial
growth occurred. The breakpoints used to categorize isolates as resistant or not resistant
(2 4 :g/ml for ciprofloxacin and _>_ 32 :g/ml for nalidixic acid) were those recommended
by the National Antimicrobial Resistance Monitoring System (N ARMS).

Fluorogenic PCR assay. A previous report describes identification of
fluoroquinolone-resistant C. jejuni by use of a fluorogenic PCR, which discriminates
between wild-type C. jejuni (susceptible to fluoroquinolone) and C. jejuni strains with
mutation in codon 86 of the gyrA gene (Wilson, et al., 2000). The DNA of C. jejuni were
extracted using QIAquick column (Qiagen), following manufacturer’s recommendations.
Final DNA products were diluted in sterile water to achieve a concentration of 1 :g/ml. In
brief, primers J L238 and J L239, along with probes TAQ2 and TAQ3, were used in 50 (1
PCR system. The fluorogenic assay PCR reaction mix contained the 1X Taqman buffer
(PE Applied Biosystem, Branchburg, NJ), 0.2 mM of each dNTP (0.4 mM dUTP), 0.5
pmol of each primer/mL, 200 nM of each fluorogenic probe, 0.05 U of Amplitaq Gold
polymerase (Perkins-Elmer)/mL, 0.01 U of Amperase UNG (Perkins-Elmer)/mL, 4.5

mM MgCl2, 0.05% gelatin, and 0.01% Tween20. Each reaction mix contained 10 ng of

149

chromosomal DNA. Initial denaturation was conducted at 95°C for 10 min, the annealing
and polymerization steps were combined at 60°C for 1 min, followed by denaturation at
95°C for 30 seconds. This process was cycled 40 times. Fluorescence emission detection
and allelic discrimination was done using an ABI Prism 7700 Sequence Detection System
(Perkins-Elmer). The wild-type control strain used was C. jejuni 81176, and a laboratory
strain of C. jejuni 81176 with a ciprofloxacin MIC of 16 :g/ml (which carry the Thr-86-

to-Ile mutation in gyrA) was used as the mutant control strain.

Statistical analysis. A Chi-square test was used to determine the significance
level of association between the proportion of C. jejuni with the Thr-86-to-Ile mutation
and ciprofloxacin or nalidixic acid resistance. The kappa statistic was computed to
indicate the level of agreement between resistance categorization using MIC values and

the presence of Thr-86-to-Ile mutation.

RESULTS

Of the 84 C. jejuni isolates tested, 60 were found to have the Thr-86-to-Ile
mutation by fluorogenic PCR assay (Table 7-1). All C. jejuni isolates with ciprofloxacin
MIC values > 2 :g/ml canied Thr-86-to-Ile mutation. When categorized by resistance, all
C. jejuni with resistance to ciprofloxacin carried the mutation (Figure 7-1). However,
there was one isolate of C. jejuni with no resistance to ciprofloxacin (MIC = 2: g/ml) that
carried the mutation. All C. jejuni isolates with a nalidixic acid MIC below 16 :g/ml did

not carry the Thr-86-to-Ile mutation. Of the 62 C. jejuni isolates with nalidixic acid MIC

150

_>_l6 :g/ml, two did not carry the mutation (Figure 7-2). When categorized by resistance,
all C. jejuni with resistance to nalidixic acid (MIC :32 :g/ml) carried the mutation.
There was very high agreement between ciprofloxacin resistance and the presence

of the Thr-86-to-Ile mutation (Table 7-2). The level of agreement was lower for nalidixic

acid resistance categories.

DISCUSSION AND CONCLUSION

The fluorogenic PCR assay for allelic discrimination used in our study has been
shown to be very specific, and faster than regular PCR or gene sequencing (Wilson, et al.,
2000). Assays for identification of C. jejuni could be performed using whole bacteria,
which would bypass the DNA extraction step (Padungtod, et al., 2002) and further speed
the process, but conditions for the PCR may require firrther optimization to take
advantage of this.

Previous studies have demonstrated the Thr-86-to-Ile mutation in C. jejuni in
human clinical isolates (Gibreel, et al., 1998; Ruiz, et al., 1998; Zimstein, et al., 1999).
Our study obtained Campylobacter fiom healthy animals, which may better represent the
general C. jejuni population at large. This study indicates that the Thr-86-to-Ile mutation
may be more prevalent in general C. jejuni population than previously report (Wilson, et
al., 2000).

The Thr-86 mutation in C. jejuni has been associated with ciprofloxacin resistance
(MIC 2 4 :g/ml ; Zimstein, et al., 1999). Our study found this mutation in C. jejuni with
ciprofloxacin MIC values as low as 2 :g/ml. This may be due to the fact that isolates

included in these studies were collected from sources that were not comparable (clinical

151

isolates versus isolates from healthy animals. The MIC values were generated using
different techniques (agar dilution (Ruiz, et al., 1998; Zimstein, et al., 1999) versus
microbroth dilution in this study), which may result in different MIC values for the same
bacteria (Caprioli, et al., 2000). Finally, there may be other mechanisms involved in
reducing the susceptibility of C. jejuni to fluoroquinolones, including mutations in parC
gene (Gibreel, et al., 1998) or the efflux pump (Charvalos, et al., 1995). Sequencing the
genes of our C. jejuni isolates may clarify which additional mechanism(s) are involved in
reducing fluoroquinolone susceptibility in C. jejuni isolated from food animals in
Thailand.

The presence of Thr-86-to-Ile mutation correlated very well with categorization of
C. jejuni using NARMS breakpoints for ciprofloxacin and nalidixic acid. As this study
demonstrated, the assay has a potential to be used as a screening tool for detecting
fluoroquinolone resistance in C. jejuni in samples from animals, humans, and food. Such
a screening tool will be very useful in areas where fluoroquinolones resistance C. jejuni
as a result of Thr-86-to-Ile mutation is prevalent.

In summary, our study showed high prevalence of C. jejuni with the Thr-86-to-Ile
mutation in the gyrA gene. This mutation confers resistance to ciprofloxacin at MIC _>_ 4

ug/ml, and correlates very well with resistance categories defined by the NARMS

breakpoints.

152

Table 7-1 MIC levels and mutations in 9er gene in C. jejuni

 

 

Ciprofloxacin Nalidixic acid Number with
MIC (pg/Ln!) MIC (pg/ml) Number tested mutation
0.03 2 2 0

4 3 0

8 1 0

0.06 4 7 0
64 1 0

0.12 4 6 0
8 2 0

16 1 0

0.25 4 1 0
2 64 1 1
4 16 3 3
32 5 5

64 7 7

128 l 1

8 32 4 4
64 21 21

128 5 5

16 16 1 1
64 4 4

128 7 7

64 64 1 1
Total 84 60

153

Table 7-2 Proportion of C. jejuni with mutation and resistance

 

Antimicrobial agents Ciprofloxacin N alidixic acid
Number tested 84 84
Number with resistance determined by in vitro
. . . 59 57

susceptrbrlrty test
Number of resistant C. jejuni with Thr-86

. 59 56
mutation
Number of resistant C.jejuni without Thr-86

. 24 23
mutation
Sensitivity (%) 100 98.2
Specificity (%) 96 70
Chi-square p value < 0.01 < 0.01

 

154

Figure ‘7-1 Number of Campylobacter isolates with the Thr-86-to-Ile mutation by MIC

for ciprofloxacin (total 84 isolates, 60 with mutation)

 

35

30

 

 

25

 

 

 

 

20

 

 

# Isolates

15

10

 

 

 

 

 

 

 

 

 

 

 

 

 

I T l f

0.03 0.06 0.12 0.25 2

MIC
13 N o Mutation

I Mutation

--———--- -—-—----q-—-—-_---h—--ih---db—-——+

Resistance breakpoint:

155

Figure 7-2 Number of Campylobacter isolates with the Thr-86-to-Ile mutation by MIC

for nalidixic acid (total 84 isolates, 60 with mutation)

 

40

35

 

 

30

 

 

 

 

25

20

 

 

# Isolates

 

 

 

15

10

 

 

_--.----‘----------ID---_---

    

 

 

 

 

 

 

 

 

 

2 4 8 16 E 32 64 128
MIC E
No Mutation :
I Mutation 1

Resistance breakpoint: 2

156

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Clinical infectious diseases. 32:1201-1206.

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Charvalos, E., Y. Tselentis, M. Hamzehpour, T. Kohler, and J. Pechere. 1995. Evidence
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158

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159

CONCLUSIONS

Results of work for each main objective:

I. Validate the use of a fluorogenic PCR assay to identifi' C. jejuni from field samples.
A rapid PCR-based 5' nuclease fluorogenic PCR assay for identifying C. jejuni was

applied to isolates from chickens from farm and slaughterhouse in Thailand. This assay

allowed identification of C. jejuni within one day after colonies appeared on selective

media, and yielded results comparable to convention tests (kappa = 0.75). When using

PCR-RFLP of 238 rRNA genes as a definitive confirmation of C. jejuni, the assay was

able to identify more isolates correctly than conventional test kits.

2. Determine the frequencies and the antimicrobial susceptibility level of
Campylobacter spp. isolated from food animals and workers at pig and chicken farms,
slaughterhouses, and markets.

The prevalence of Campylobacter spp. in pigs in northern Thailand were 70.3%,
45.6% and 20.9% at the farms, slaughterhouses, and markets, respectively. In chickens,
the prevalence of Campylobacter spp. were 73.4%, 50.6%, and 47.2% at farms,
slaughterhouses, and markets, respectively. The prevalence of Campylobacter spp. with
resistance to fluoroquinolones was higher than other antimicrobial agents in both pigs and
chickens. Multi-resistant Campylobacter spp. (resistance to more than one agent) were
seen in 79-97% of isolates fiom chickens, 96-100% in pigs, and 100% in farm workers.

The three antimicrobial agents to which Campylobacter isolates from pigs and chickens

160

showed the highest levels of resistance were tetracycline, nalidixic acid, and

ciprofloxacin in. Human isolates were also resistant to these three agents.

3. Compare the frequencies of Campylobacter spp. with resistance to antimicrobial
agents in food animals, food products and farm workers.

Resistance to all eight antimicrobial agents tested were observed in Campylobacter
spp. isolated from pigs, while resistance to six agents were observed in isolates fiom
chickens and three agents in isolates from humans. No resistance to chloramphenicol and
gentamycin were found in Campylobacter spp. isolated fi'om chickens. Resistant to
erythromycin, azithromycin and clindamycin was more prevalent in Campylobacter spp.
isolated from pigs than fiom chickens. The overall antimicrobial resistance profiles were
generally similar among Campylobacter spp. isolated from farms, slaughterhouses and

market in both pigs and chickens.

4. Determine the risk factors associated with the observed frequencies of
Campylobacter spp. and frequen cies of Campylobacter spp. with resistance to
antimicrobial agents isolated fiom various sources.

Smaller flock size of chickens and herd size of pig were significantly associated with
reduced probability of finding Campylobacter spp., but the magnitude of the effect was
low (95% CI for OR 0.97-1.0). Older chickens were less likely to be infected with

Campylobacter spp. than younger birds (OR=0.90).

161

5. Determine whether antimicrobial use in feed and treatment on pig and chicken
farms is associated with the fiequency'of antimicrobial resistance in Campylobacter.
On both pigs and chickens farms, the prevalence of resistant Campylobacter was
higher for those antimicrobial agents that were used on the farm in feed and/or for
treatment. No resistance or extremely low frequency of resistance was observed for those

antimicrobial agents not used on the farms.

6. Determine the association between mutation in gyrA gene of Campylobacterjejuni
and ciprofloxacin resistance.

The fluorogenic PCR developed in the laboratory at MSU can be used to detect the
Thr-86-to-Ile mutation in gyrA gene in C. jejuni from field samples. This mutation is

associated with decreased susceptibility to ciprofloxacin (MIC 2 2 ug/mL)

The work presented in this thesis was carried out in the field in Thailand, and in
laboratories in Thailand and the US. This study has shown high prevalence of
Campylobacter spp. in food animals through the production system in northern Thailand.
High proportions of these Campylobacter spp. were resistant to antimicrobial agents
commonly used for treatment of gastrointestinal infection in humans, including
ciprofloxacin and erythromycin. Most importantly, Campylobacter spp. isolated from
humans were resistant to the same agents that showed the highest prevalence of
resistance in isolates from food animals. This suggests that antimicrobial resistance in
Campylobacter isolates from humans is associated with the development of resistant

bacteria in food animals exposed to antimicrobial agents.

162

This study had several major strengths. We were able to follow the same animals
from the farm to slaughter and eventually to market during the second year of study,
which provided a strong basis for comparison of prevalence among different sampling
points. The use of enrichment media and microbroth dilution susceptibility testing
enhanced the validity of the study by improving the detectability level of the testing
protocol, and provided precise measurements of susceptibility. Enhancing the ability to
detect Campylobacter from field samples improved the estimates of prevalence
throughout the study. Reporting MIC values from antimicrobial susceptibility testing
provided greater detail for detecting changes in susceptibility, rather than the simple
classification of isolates as resistant or non-resistant. The weaknesses of the study were
the limited number of farms and slaughterhouses participating in the study, and the lack
of precise measurement of antimicrobial use on the farm. The limited number of facilities
used was due to the difficulty in setting up an adequate animal tracking system
throughout the production system. This limited the power of the study to clarify factors
associated with the observed prevalence, however it was felt that this weakness would be
made up in the improved animal tracking through the facilities that did participate in the
study. The lack of precise measurement of antimicrobial use on the farms is an inherent
problem on virtually all farms in developing countries, and limited the power of the study
to determine the association between antimicrobial use and the prevalence of
Campylobacter spp. with resistance to antimicrobial agents.

Although there were some limitations in this study, the information generated will

contribute to the global picture of the epidemiology of Campylobacter spp. and

163

antimicrobial resistance problems in developing countries. A more intensive study to
assess factors contributing to the development of Campylobacter spp. with resistance to
antimicrobial agents in developing countries, as well as further intervention studies
comparing resistance in farms that do and do not use antimicrobial agents, should be

conducted in the near future.

164

APPENDICES

165

APPENDIX 1

DATA COLLECTION FORM FOR PIG FARMS

Farm name:

 

Address:

 

Telephone

 

Contact person

 

Number of pigs:

 

Sows Boars Piglets F attening Other Total

 

 

 

 

 

 

 

 

 

Number of houses

 

Number of pigs per house

 

Number of workers per house

 

Use farm mix feed buy ready mix product

Use growth promoter/ probiotic/ prophylactic antibiotic during the past 12 month
No
Yes,

 

Product name Amount Type of Abx Group of animal Remark

 

 

 

 

 

 

 

 

 

 

 

 

166

APPENDIX 2

SAMPLE COLLECTION FORM FOR PIGS AT FARMS

Farm name

Date

 

 

 

Sample #

House #

Pen #

39

Breed

Remark

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

167

 

APPENDIX 3

SAMPLE COLLECTION FORM FOR WORKERS AT PIG FARMS

Farm name Date

 

 

 

Age Abx use during previous
Sample # House # QEUL Sex month

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

168

APPENDIX 4

DATA COLLECTION FORM FOR CHICKEN FARMS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Farm name
Address
Telephone
Contact person
Number of birds:
Broilers Number
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Week 7
Other
Total
Number of houses
Number of birds per house
Number of workers per house
Use farm mix feed buy ready mix product
Use growth promoter/ probiotic/ prophylactic antibiotic during the past 12 month
No
Yes,
Product name Amount Type oLfAbx Group of animal Remarks

 

 

 

 

 

 

 

 

 

 

 

 

169

APPENDIX 5

SAMPLE COLLECTION FORM FOR CHICKENS AT FARMS

Farm name

Date

 

 

Sample #

House #

Room #

Age

Breed

Type

Remarks

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

170

 

APPENDIX 6

SAMPLE COLLECTION FORM FOR WORKERS AT CHICKEN FARMS

Farm name Date

 

 

 

Age Abx use during previous
Sample # House # Qear) Sex month

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

171

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