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EVALUATION OF PROINFLAMMATORY CYTOKINES IN
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EVALUATION OF PROINFLAMMATORY CYTOKINES IN PIGS INFECTED
WITH CAMPYLOBACTER JEJUNI AND TRICHURIS SUIS

BY

Lakeisha Dianele Cunningham

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2007

ABSTRACT

EVALUATION OF PROINFLAMMATORY CYTOKINES IN PIGS INFECTED WITH
CAMPYLOBACTER JEJUNI AND TRICHURIS SUIS

By
Lakeisha Dianele Cunningham
Campylobacterjejuni is a leading cause of gastroenteritis worldwide. In
conventionally reared swine, C. jejuni is commonly found in the colon without
pathology, but infection and pathology result when the swine whipworm, T. suis, is
present. The central hypothesis of this dissertation was that C. jejuni induces natural host
resistance via proinflammatory cytokine responses following primary oral infection, but
this response is down-regulated in the presence of T. suis thus promoting C. jejuni
infection. Specific aims: 1) Evaluate secreted pro- and anti-inflammatory cytokines (IL-
8, IL-l-B, TNF-a, IL-4, IL-6, and IL-10) in the feces of pigs after single or dual challenge
infections with C. jejuni and T. suis, 2) Assess IL-8, IL-l-B and TNF-a production from
undifferentiated, crypt-like intestinal pig epithelial cells (IPEC-l cells) following C.
jejuni infection, and 3) Assess whether IL-8, TNF-a, or IL-l-B treatment of IPEC-l cells
have effects on C. jejuni invasion, adherence, and/ or transepithelial electrical resistance
(TER) across this mucosa] membrane. In aim 1, weaned piglets were used in short-term
(ST, 2 days) and long-term (LT, 23 days) challenge experiments. In the LT experiments,
there were 5 groups with two groups given 2500 T. suis orally. At 21 days post-T. suis
inoculation, some of the T. suis-infected pigs received C. jejuni, while the remaining pigs
received either C. jejuni alone, E. coli DHS-a, or sterile milk. On day 23, all pigs were
euthanized and gastrointestinal (GI) tissue samples taken to measure cytokine expression

by real time PCR. Campylobacterjejuni stimulated increased proinflammatory cytokine

expression in the jejunum while T. suis down-regulated these responses here and in the
proximal colon. T richuris suis contributed significantly to diarrheal disease in both single
and concurrent infections. Also, enzyme linked irnmunosorbent assay (ELISA)
conducted on supernatants from feces indicated that T. suis significantly decreased
expression of fecal IL-l—B during concurrent C. jejuni and T. suis infection. In the ST
experiment where pigs were inoculated simultaneously with either C. jejuni, T. suis, C.
jejuni and T. suis, E. coli DHS-a, or sterile milk none showed significant acute changes in
fecal IL-8, IL-l-B, TNF-a, IL-4, IL-6, or IL-10 expression by ELISA. Here, cytokine
responses were measurable using fecal ELISAs, but cytokine mRN A expression assays in
tissues were more informative. Also, we discovered that secondary invasion of certain
epsilon proteobacteria into GI tissues may affect cytokine responses. In aim 2, confluent
undifferentiated IPEC-1 cells were infected with C. jejuni and culture supematants
analyzed for IL-8, TNF-a, or IL-l-B expression using ELISA. Campylobacterjejuni
induced IPEC-1 cells to secrete TNF-a and lL-l-B, but IL-8 was constitutively secreted
and not inducible by C. jejuni infection. These data show a pattern of cytokine responses
indicative of an innate inflammatory response that we hypothesize to be responsible in
part for eliminating these bacteria from the host. In aim 3, the TER of confluent IPEC
cells was measured prior to and following treatment with recombinant swine IL-8, TNF-
a, or IL-l-B. None of these cytokines had direct effects on C. jejuni interactions with
undifferentiated, crypt-like intestinal epithelial cells. Collectively, these data provide
evidence for T. suis contributing to cytokine dysregulation during concurrent infection.
These immunomodulatory effects may promote C. jejuni infection of the intestinal

mucosal epithelium by limiting the natural host resistance to bacterial infection.

Copyright by
LAKEISHA DIANELE CUNNINGHAM
2007

DEDICATION

This dissertation is dedicated to my Lord and Savior, Jesus Christ;
my husband, Steven; my daughters, Jadyn and Kailyn and my mother, Carolyn Price.

ACKNOWLEDGEMENTS

There are several people I would like to acknowledge for making the completion
of this degree possible. First, I must acknowledge God for giving me this opportunity
and providing me with the strength to persevere and succeed. I sincerely and lovingly
thank my husband, Dr. Steven G. Cunningham; my daughters, Jadyn Elyse and Kailyn
Danielle; my mother, Carolyn; my father, George and my entire family for their never-
ending support, prayers, encouragement, and love throughout my graduate studies. I
truly thank my mentor, Dr. Linda S. Mansfield, for supporting and guiding me through
this journey of higher learning. I am also grateful to my committee members (Drs.
Richard Schwartz, Martha Mulks, John Linz, and Maria Patterson) for their support and
guidance throughout my graduate studies. I must also thank my labmates, Drs. Geetha
Parthasarathy and Kathryn Jones and Stacey Wilder, for their emotional support, their
assistance in conducting the experiments, and for their constructive criticism on my
writing. Dr. Julia Bell encouraged me along the way, shared her scientific research
expertise, and provided critical review on the writing of this dissertation; she believed in
me and will always be my dear friend. David Wilson also provided research assistance
and information pertaining to Campylobacterjejuni. Drs. Pawin Padtmgtod and Bo
Norby, provided veterinary assistance and advice on statistical analysis. I thank Drs.
Geetha Parthasarathy, Kathryn Jones, Pawin Padungtod, and Jeanie Gaymer along with
the other MSU Containment Facility staff and Stacey Wilder for their help with the
animal experiments and animal care. I also thank, Alice Murphy and Nicholas Barbu for
conducting the 16S rRNA PCR assays. I thank Alice Murphy and Sneha Dhital for

performing the ELISA to detect antibodies in the plasma of our pigs. Also, I thank Dr.

vi

Kathryn Jones for conducting the Real-time PCR and 23S rRNA RF LP analysis and
histology. I thank Dr. Geetha Parthasarathy for performing the ELISA to measure anti-
inflammatory cytokine concentrations in pig fecal samples. In addition, I thank Shirley
Xiao for performing the statistical analysis of my data. The studies for this dissertation
project were supported by National Institute of Health funding, and Drs. John Baker and
Vilma Yuzbasiyan-Gurkan also helped me to secure funding which supported the
completion of my graduate studies. I thank the entire Microbiology and Molecular
Genetics departmental staff and the entire National Food Safety and Toxicology Center
staff for all of their assistance. Additionally, I am genuinely thankful for Kanika
Williams and Mrs. Betsy Walker who provided supreme care for my children while I
worked late in the lab. I also thank DeMiracIe Woodson and Carolyn Butler for being
my prayer partners and my support angels throughout this journey. I am forever grateful

to all that contributed in helping me obtain this degree.

vii

TABLE OF CONENTS

List of Tables .................................................................................................................. ix

List of Figures ....................................................................................... x

Chapter 1

Literature review .................................................................................. 1
Campylobacter jej uni .................................................................... 1
T richuris suis ............................................................................ 36
Rationale ................................................................................. 53
References ................................................................................ 58

Chapter 2

T richuris suis-mediated cytokine dysregulation in

concurrent Campylobacterjejuni and T richuris suis infection ............................ 73
Abstract .................................................................................. 73
Introduction .............................................................................. 77
Materials and Methods ................................................................. 82
Results .................................................................................... 97
Discussion .............................................................................. 108
References ............................. . ................................................. 124
Tables and Figures ..................................................................... 132

Chapter 3

Evaluation of proinflammatory cytokine production

in intestinal pig epithelial cells infected with Campylobacterjejzmi ................... 160
Abstract ................................................................................. 160
Introduction ............................................................................. 162
Materials and Methods ................................................................ 166
Results ................................................................................... 173
Discussion .............................................................................. 177
References .............................................................................. 187
Tables and Figures ..................................................................... 193

Chapter 4

Summary and Conclusions ..................................................................... 212
References ............................................................................... 23 1

viii

LIST OF TABLES

Table 2.1. Experimental treatment groups for the
short (2 days) and long (23 days) term challenge experiments .......................... 132

Table 2.2. Inoculation timeline for
short term challenge experiments (2 days) ................................................. 133

Table 2.3. Inoculation timeline for I
long term challenge experiments (23 days) ................................................ 134

Table 2.4. Summary of Campylobacter detection and identification ................... 135
Table 2.5. Short term challenge experiment

16S RNA PCR analysis results for rapid identification of

Campylobacter and Helicobacter DNA in pig feces ...................................... 139
Table 2.6. Long term challenge experiment

l6S RNA PCR analysis results for rapid identification of

Campylobacter and Helicobacter DNA in pig feces ...................................... 140

Tables 2.7. Short term challenge: clinical assessment of diarrhea in
weaned, conventionally-reared swine infected with T. suis and/or C. jejuni ........... 145

Tables 2.8. Long term challenge: clinical assessment of diarrhea in

weaned, conventionally-reared swine infected with T. suis and/or C. jejuni ........... 146
Table 2.9. Fecal cytokine recovery rate ..................................................... 147
Table 3.1. IPEC-1 cells treatments for cytokine measurement (ELISA) ............... 193

Table 3.2 Recombinant swine cytokine pretreatment concentrations
for invasion and adherence assays ............................................................ 194

ix

LIST OF FIGURES
Figure 2.1. RFLP analysis of the Campylobacter 23S rRNA gene ...................... 136

Figures 2. 2 A and B. (A) Short term challenge experiment and
(B) long term challenge experiment ELISA results indicating
levels of plasma IgG antibodies detectable against C. jejuni protein antigen ............ 137

Figure 2.3. ELISA results indicating levels of plasma IgG antibodies

detectable against protein antigen of various Campylobacter species,

Arcobacter butzleri, and Helicobacter hepaticus,

suggesting IgG cross-reactivity .................................................................. 138

Figure 2.4. H&E stained sections from the colons of pigs

were examined for T. suis larvae ................................................................ 141
Figure 2.5. H&E section fi'om a C. jejuni infected pig ....................................... 142
Figure 2.6. H&E sections of LGCs from infected pigs ....................................... 143

Figure 2.7. Expression of mRNA from a panel of cytokines
was measured by Real Time PCR ............................................................... 144

Figure 2.8. Changes in IL-8 levels were quantified in the feces
of weaned piglets fi’om the short term challenge experiments. ............................. 148

Figure 2.9. Changes in TNF-u levels were quantified in the feces
of weaned piglets fiom the short term challenge experiments ............................... 149

Figure 2.10. Changes in IL-l-B levels were quantified in the feces
of weaned piglets from the short term challenge experiments ............................... 150

Figure 2.11. Changes in IL-4 levels were quantified in the feces
of weaned piglets fiom the short term challenge experiments ............................... 151

Figure 2.12. Changes in IL-6 levels were quantified in the feces
of weaned piglets fi'om the short term challenge experiments ............................... 152

Figure 2.13. Changes in IL-lO levels were quantified in the feces
of weaned piglets from the short term challenge experiments ............................... 153

Figure 2.14. Changes in fecal IL-8 levels were measured using ELISA at
various time points over a 23-day period ....................................................... 154

Figure 2.15. Changes in fecal TNF-a levels were measured using ELISA at
various time points over a 23-day period ....................................................... 155

Figure 2.16. Changes in fecal IL-l-B levels were measured using ELISA at
various time points over a 23-day period ..................................................... 156

Figure 2.17. Changes in fecal IL-4 levels were measured using ELISA at
various time points overa23-dayperiod.....................................................157

Figure 2.18. Changes in fecal IL-6 levels were measured using ELISA at
various time points over a 23-day period ..................................................... 158

Figure 2.19. Changes in fecal IL-lO levels were measured using ELISA at

various time points over a 23-day period ..................................................... 159
Figure 3.1. Standard curve for swine TNF-a used in the ELISA ......................... 195
Figure 3.2. Standard curve for swine IL-l-B used in the ELISA .......................... 196
Figure 3.3. Standard curve for swine IL-8 used in the ELISA ............................ 197

Figure 3.4. TNF-a secretion by IPEC-1 monolayers after exposure to
C. jejuni 33292 .................................................................................... 198

Figure 3.5. IL-l-B secretion by IPEC-l monolayers after exposure to
C. jejuni 33292 .................................................................................... 199

Figure 3.6 (A and B). The two graphs represent experiments conducted

at the same time. (A) Positive control: IPEC-l monolayer positively

induced to secrete IL-8 following phorbol myristate acetate (PMA 100 ng/ml)

and calcium ionophore (CI 100 ng/ml) exposure. (B) IL-8 secretion by IPEC-1
monolayers after exposure to C. jejuni 33292 ................................................ 200

Figure 3.7. IPEC-1 monolayers were pretreated with increasing amounts of
swine rTNF-a for 5 hours to determine the effect of swine rTNF-u on
C. jejuni 33292 invasion of intestinal epithelial cells. ....................................... 202

Figure 3.8. IPEC-1 monolayers were pretreated with increasing amounts of swine
rTNF-a for 5 hours to determine the effect of swine rTNF-a on
C. jejuni 33292 adherence to intestinal epithelial cells ....................................... 203

Figure 3.9. IPEC-1 monolayers were pretreated with increasing amounts of swine
rTNF-a for 5 hours to determine the effect of swine rTNF-a on
E. coli 0157:H43 DEC7A adherence to intestinal epithelial cells .......................... 204

xi

Figure 3.10. IPEC-1 monolayers were pretreated with increasing amounts of swine
rIL-l-B for 5 hours to determine the effect of swine rIL-l-B on
C. jejuni 33292 invasion of intestinal epithelial cells ......................................... 205

Figure 3.11. IPEC-1 monolayers were pretreated with increasing amounts of swine
rIL-I-B for 5 hours to determine the effect of swine rIL-l-B on
C. jejuni 33292 adherence to intestinal epithelial cells ....................................... 206

Figure 3.12. IPEC-l monolayers were pretreated with increasing amounts of swine
rIL-l-B for 5 hours to determine the effect of swine rIL-l-B on E. coli 0157:H43
DEC7A adherence to intestinal epithelial cells ................................................ 207

Figure 3.13. IPEC-l monolayers were pretreated with increasing amounts of
swine rIL-8 for 5 hours to determine the effect of swine rIL-8 on
C. jejuni 33292 invasion of intestinal epithelial cells ......................................... 208

Figure 3.14. IPEC-1 monolayers were pretreated with increasing amounts of
swine rIL—8 for 5 hours to determine the effect of swine rIL-8 on C. jejuni 33292
adherence to intestinal epithelial cells .......................................................... 209

Figure 3.15. IPEC-l monolayers were pretreated with increasing amounts of
swine rIL-8 for 5 hours to determine the effect of swine rIL-8 on
E. coli 0157:H43 DEC7A adherence to intestinal epithelial cells .......................... 210

Figure 3.16. Effect of swine rlL-8, rIL-l-B, and TNF-u on transepithelial electrical
resistance (TER) of IPEC-1 cells ................................................................ 211

xii

Chapter 1: Literature Review

I. Campylobacter jejuni
Basic microbiology

Campylobacterjejuni (C. jejuni) is a gram negative bacillus that has a
curved or spiral morphology. Its spiral shape and its polar flagellum at one or both ends
of the cell mediates a high degree of darting corkscrew motility (81). It is 6.0 pm long
and 0.2-0.5 um wide (81). The generation time of C. jejuni is approximately 90 minutes
(104). C. jejuni is nonsaccharolytic and requires enriched media and microaerophilic
conditions (3-15% 02, 3-5% C02) for adequate growth (81). Therrnophilic conditions at
42°C facilitate the best growth of these organisms; however, they can also grow at 37°C.
These organisms are sensitive to low pH (5 5), organic acids, NaCl concentrations
exceeding 2 %, and extended periods at temperatures between 10 and 30°C (104). In
unfavorable grth conditions, C. jejuni will convert to a viable, coccoid, nonculturable
state. It has been suggested that C. jejuni uses this ‘dormant’-like state as an adaptation
mechanism to survive in adverse environments(81). The estimated size of the genome is
1730 kb, based on the first two sequenced strains, and its DNA has a high AT content

(81,117).

Epidemiology

C. jejuni is one of the leading causes of acute gastroenteritis worldwide,
exceeding diseases caused by Salmonella spp., Shigella spp., or Escherichia coli
0157:H7 (6). It is estimated that there are 2.1 to 2.4 million cases of human

campylobacteriosis occurring each year in the United States, and >99% of these reported

infections with Campy/abacter are caused by C. jejuni (6, 7). In most cases, human
campylobacteriosis is a sporadic illness (7). Fecal oral transmission in food animals can
lead to contamination of meat. Documented sources for C. jejuni are improperly cooked
poultry, beef, pork, or turkey. Contaminated water, contact with pets, and international
travel are also sources of sporadic infection (7, 27, 81). Although the majority of cases
are sporadic, there have been reports of outbreaks associated with the consumption of
unpasteurized milk and contaminated water (1, 6, 27, 81, 131).

C. jejuni is considered to be a commensal in cattle, sheep, swine, fowl, dogs, cats,
and rodents (7). It is now confirmed as a human enteric pathogen. The coupling factor
relating C. jejuni to human disease is the transmission of C. jejuni to humans through the
consumption of foods of animal origin. Most cases of human campylobacteriosis are
associated with foodborne or waterborne transmission (20).

In developed/ industrialized countries, Campy/abacter gastroenteritis is most
typically characterized clinically by diarrhea, fever, and abdominal cramps. The most
common source of Campylabacter infections is the consumption and handling of chicken.
Studies conducted in the United States, Europe, and Australia indicated that 50-70% of
all Campylobacter infections have been attributed to the consumption of chicken (6). A
study conducted in the retail market reported a 98% isolation rate of C. jejuni from
chicken meat (7). Children (< 1 year of age) and young adults (age 15-44 years),
particularly males, are most commonly infected. The reported seasonal occurrence of C.
jejuni infection in the United States begins in May and peaks in August (6).

In developing countries studied, Campylobacter infections are hyperendemic.

Campylobacter infections occur commonly in both children and adults resulting in

asymptomatic infection. Infection with disease is more prevalent among young children
(<5 years of age) (6, 112). In developing countries the isolation rates of Campylobacter
from patients range from 5 to 20%; these estimates of incidence are from laboratories that
survey pathogens responsible for diarrhea. According to the World Health Organization
(WHO), the estimated incidence rate of campylobacteriosis is 40,000 to 60,000/ 100,000
in children <5 years old (33). In developing countries, environmental contamination and
contaminated foods are considered to be major sources of human infection (33). In many
of these settings it is common to have animals living in close proximity to people, and
this environment likely plays a significant role in the occurrence of campylobacteriosis.
In 2002, Coker et al. reported that the incidence of campylobacteriosis was
increasing worldwide, and the WHO turned its attentions to this concern. The increased
involvement of the WHO will stimulate public health awareness through strengthened
diagnostic facilities for campylobacteriosis and will promote the establishment of
national surveillance programs in countries where they do not exist (3 3). These efforts
are expected to play a significant role in understanding the global epidemiology of human

campylobacteriosis.

Disease

Symptoms of gastroenteritis associated with Campylobacter infection include
diarrhea, fever, and abdominal cramps. These symptoms commence within 2-4 days
following ingestion of C. jejuni (81). In most cases, illness is self-limiting, and the
duration of the illness is usually 1 week or less. C. jejuni-induced diarrhea occurs

following ingestion of as few as 800 organisms (25). In human clinical infection studies,

fresh blood, mucus, frank pus, and polymorphonuclear leukocytes were found in
diarrheic stool samples (25). However, symptoms can be severe, and death can ensue.
According to an estimated case/ fatality ratio for all C. jejuni infections, there is one death
per one thousand cases. Usually, C. jejuni-related deaths are associated with
immunocompromised individuals (6, 7).

Clinical manifestations of disease due to C. jejuni vary between developed and
developing countries. In developing countries, the clinical manifestations of
Campylobacter enteritis are less severe than in developed countries. Watery, nonbloody,
noninflammatory diarrhea is a common clinical characteristic of disease in developing
countries. In many cases the patients are underweight and malnourished (33). In
developed countries, such as the United States, disease is characterized by severe
inflammatory diarrhea with bloody stool, fever, and abdominal pain (33).

The optimal drug for treatment of Campylobacter infection is erythromycin.
Campylobacter continues to have quite a low rate of resistance to erythromycin despite
its use over many years. Erythromycin is safe, low in cost, easy to administer, and has a
narrow spectrum of activity (6). Macrolides such as azithromycin and clarithromycin,
are also effective against C. jejuni infections, but they are more expensive than
erythromycin and provide no clinical advantage (6).

Other disease manifestations produced by C. jejuni infections include bacteremia,
meningitis, proctitis, septic arthritis, septic abortion, carditis, pancreatitis, urinary tract
infection, and autoimmune disease(56, 124). In addition to the immediate effects caused
by acute Campylobacter infection, there are long-term sequelae associated with

campylobacteriosis. Studies have shown that reactive arthritis (ReA), Reiter’s syndrome,

Guillain-Barré, and Miller-Fisher syndrome may accompany or follow C. jejuni enteritis
(124). These post-infection sequelae result in serious health effects and have a significant
economic impact (105).

Reactive arthritis (ReA) is a non-purulent joint inflammation which is commonly
induced by enteric infections or urogenital tract infections (56). ReA is triggered by
agents such as Salmonella, Yersinia enterocolitica, Shigellaflexneri, Chlamydia
trachomatis and Campylobacterjejuni (39). Locht et al. observed that ReA patients that
had C. jejuni enteritis had more severe gastrointestinal symptoms and prolonged diarrhea
than patients who had enterocolitis only (94). Reactive arthritis is usually self-limiting,
but a 3-month course of Ciprofloxacin therapy decreases the symptoms for more severe
cases (104). The annual incidence of ReA following Campylobacter infection is 4.3 per
100,000 cases (56).

Reiter’s syndrome is another reactive arthropathy associated with C. jejuni
infection. This syndrome is thought to be an autoimmune response stimulated by
infection. It usually occurs 7-10 days after onset of diarrhea, and multiple joints can be
affected, particularly the knee joint. This syndrome triggers pain and possible
incapacitation which can last for months or become chronic (7). Reiter’s syndrome is
reported to occur in approximately 1% of patients with campylobacteriosis (7). The
pathogenesis of Reiter’s syndrome is not completely understood.

Guillain—Barré Syndrome (GBS) is another serious sequela of Campylobacter
infection. It was the first of the postinfectious autoimmune diseases identified as
occurring subsequent to C. jejuni colitis and is characterized by acute limb weakness and

loss of tendon reflexes (acute flaccid paralysis) (33, 148). C. jejuni serotype 0:19 are the

most prevalent strains associated with GBS worldwide (6, 33, 148). In the United States,
the mean annual incidence rate of GBS is 1.3 cases per 100,000 population (148).
Although C. jejuni infection can trigger GBS, the risk of developing GBS after C. jejuni
infection is quite small (approximately 1 case of GBS out of 1000 C. jejuni infections) (6,
7, 148). Approximately 20% of the patients with GBS are left with some long term
disability (7). GBS patients that suffer extensive axonal injury have a greater likelihood
of need for mechanical ventilation and an increased risk of irreversible neurological
damage (6). Approximately 5% of GBS patients die despite advances in supportive
respiratory care (7). It is now recognized that the risk of development of GBS does not
increase with the severity of C. jejuni infection. Additionally, many GBS-associated C.
jejuni infections are asymptomatic. Neurological symptoms of GBS typically occur l-3
weeks after the onset of diarrheal illness, and humoral irnmunopathogenic mechanisms
are believed to be involved (6). Similar to Fisher Syndrome (FS), it is believed that
molecular mimicry is a possible cause of GBS. Studies have identified the molecular
mimicry between GBS-associated C. jejuni lipo-oligosaccharides and peripheral nerve
glycolipids or myelin proteins, particularly the GM] ganglioside, as a mechanism for
anti-ganglioside antibody induction (6, 72, 159). It is strongly suggested that this
association plays an important role in the pathogenesis of Campylobacter-induced GBS
(6, 144). Li et al. demonstrated that C. jejuni-infected chickens spontaneously acquired
paralytic neuropathy associated with GBS (93).

Likewise, Fisher syndrome (FS), also known as Miller Fisher syndrome, is an
anatomically localized variant of Guillain-Barré syndrome (GBS) that is characterized by

acute onset of ophthalrnoplegia (paralysis of the eye muscles), ataxia, and areflexia (lack

of normal reflexes). It is considered a variant of GBS because some patients who present
with FS progress to GBS (148). The estimated annual incidence rate of FS is 0.09 per
100,000 population. C. jejuni is the most fiequently identified antecedent pathogen to

F S/GBS. It is believed that molecular mimicry is also a possible cause of both F S and
GBS. More specifically, studies have shown that anti-Gle IgG, an antiganglioside
antibody, is associated with F S (82, 114, 166). This is thought to occur because Gle is
enriched in the cranial nerves that innervate the extraocular muscles (166). Fisher
syndrome is typically a benign, self-limiting illness in comparison to GBS, and can be

treated successfully with plasmapheresis and intravenous immunoglobulin (114).

Model Systems

1). Animal models

There have been many attempts at establishing a suitable animal model system
that would mimic human campylobacteriosis (4, 10, ll, 19, 44, 100, 116, 129, 154).
However, the inability to fillly reproduce clinical symptoms of Campylobacter enteritis
has proven to be a hindrance in the progress of clarifying the biological significance of C.
jejuni virulence factors. Human campylobacteriosis is characterized by watery diarrhea,
abdominal pain, fever and the presence of blood and mucus in stools (6, 16, 33, 81, 95).
Various experiments conducted have used animals that are not anatomically and
physiologically similar to humans; therefore, requiring anatomical manipulation to
acquire disease (4, 44, 73). Also, some animal model systems are quite expensive to
maintain and/or have proven to yield inconsistent results (4, 73, 129). In the domestic

ferret model it has been shown that some form of specific, aspect of immunity is

responsible for at least partial resistance to colonization and disease production although
the mechanism has not been identified. Likewise, in this domestic ferret model study,
humoral immunity did not appear to play a role in preventing colonization but did protect
against enteric disease. Even though the use of the domestic ferret model proved to be
informative regarding C. jejuni colonization and immunity, further immunological
studies using this model are limited due to the lack of available immunologic reagents
and genome sequence (19). Furthermore, other animal models have been used to
specifically address certain sequelea associated with C. jejuni infection, such as, Guilain—
Barre' syndrome (GBS). C. jejuni-infected chickens have been shown to develop
spontaneous paralytic neuropathy associated with GBS (93). Overall, an adequate animal
model is needed to suitably assess the bacterial mechanisms and clinical manifestations
associated with C. jejuni infection.

Despite the prior attempts and criticisms, it is known that the pig is anatomically
and physiologically similar to the human, and studies have shown it to be a biologically
relevant and important animal to use to observe Campylobacterjejum‘ virulence and host
immune mechanisms. Newborn colostrum-deprived piglets that have been orally
challenged with an invasive strain of C. jejuni have been successfully used in
demonstrating the clinical manifestations associated with campylobacteriosis. The
piglets developed clinical symptoms and histopathological lesions similar to that seen in
human campylobacteriosis (10). Other swine model systems have proven to be of
suitable use as well. For example, Mansfield et. al showed that a synergistic relationship
between C. jejuni and T richuris suis (T. suis) enhanced disease and pathology in

gnotobiotic (germ-free) pigs (101). Likewise, additional swine disease model studies

demonstrated that the lyphoglandular complexes are important colonic sites for
irnmunoglobulin A induction against C. jejuni (100). Together, these findings have
provided significant support in using pigs as a suitable animal model for human
campylobacteriosis, and investigators should now be able to use this system to filrther
study virulence factors associated with this enteric disease.

To date, there are still unanswered questions although the swine disease model
has provided a better understanding of some host immunomodulatory responses triggered
by C. jejuni and T. suis infections. Therefore, a murine model system has been
developed to continue the advancement in understanding the mechanisms involved in C.
jejuni infection (99). Current studies have shown that C57BL/6 and congenic IL-IO
deficient mice infected with C. jejuni 11168 are stably colonized and exhibit a robust Th1
directed antibody response, but only the congenic IL-10 deficient mice exhibit significant
histopathological changes similar to responses seen in human when infected with
C. jejuni 11168. These data suggest that colonization of the mouse gastrointestinal tract
by C. jejuni 11168 is required but not adequate for enteritis development. Therefore,
C57BL/6 and congenic lL-lO deficient mice can serve as suitable models of C. jejuni
colonization and enteritis (99). Hence, the findings from these studies using the swine
disease model and the existing mouse model will permit further study to characterize the
immune responses that are possibly triggered by interactions between host factors of C.
jejuni and/or T. suis which may have an effect on the development of enteric

pathogenesis.

2). In vitro models

Extensive study of the mechanisms by which Campylobacterjejuni causes
disease has not only been investigated in viva, but various in vitro model systems have
been developed to facilitate the understanding of the mechanisms involved in bacterial-
host cell interaction (160). For example, Caco-2 cells is a cultured cell line that has been
used in many C. jejuni experiments to specifically observe bacterial interaction with host
cells that differentiate at confluency to mature enterocytes (137, 160). This cell line was
derived from a human colon adenocarcinoma and has been used in experiments to mimic
the intestinal epithelium. As these cells proliferate and differentiate in culture
(maturation process of the intestinal epithelium), they form a polarized epithelial
monolayer characterized by brush-border microvilli and tight junctions. When studying
differentiation and the regulation of intestinal functions, the Caco-2 model is one of the
most used models for in vitro study (64, 78). Likewise, INT 407 cells have been a widely
used cell line in C. jejuni/host cell interaction studies. This cell line is derived from
human small intestine and has been used experimentally to mimic the firnctions of human
undifferentiated intestinal epithelium in response to bacterial exposure (160). In
addition, primary swine intestinal cells have been used as a model for studying C.
jejuni/host cell interaction. It has been shown that clinical C. jejuni isolates (C. jejuni
F1474 and T13 193) invade primary swine intestinal cells at a significantly higher
frequency than an Eshcherichia coli control strain. Also, C. jejuni colonies recovered
fi'om these primary swine intestinal cells invade tissue cultured cells (INT 407) at a
significantly higher frequency than the parental strain (C. jejuni T13192) suggesting that

C. jejuni invasiveness may be an important in viva virulence attribute (11). Hence, these

10

findings, in part, have influenced the use primary swine intestinal epithelial cells in
ongoing C. jejuni/host cell interaction studies. For example, IPEC-1 cells (intestinal pig
epithelial cells) are cells derived from neonatal pig intestinal epithelium. These cells
have not been cloned, and this leads to some diversity of the population; however, these
cells can be induced to differentiate by culturing them for 12-14 days. It has been shown
that IPEC-l cells secrete IL-6, lL-lO and IL-18 following
C. jejuni exposure (Parathasarathy, dissertation, 2004 and Jones, dissertation, 2005).
Also, several other in vitro studies have demonstrated the roles of cytokines in C.
jejuni infection. Viable C. jejuni stimulates INT 407 cells to secrete IL-8, and this
response has been shown to be mediated via multiple mechanisms such as C. jejuni
adherence and/or invasion or C. jejuni cytolethal distending toxin (CDT) treatment (61,
63). Other proinflammatory chemokines, such as GROalpha, GROgamma, macrophage
inflammatory protein 1 (MCP-l), and gamma lP-10, are secreted from INT 407 cells
following exposure to viable C. jejuni and are believed to play a contributing role in the
observed host inflammatory responses (70). Likewise, it has been shown that viable C.
jejuni activates the mitogen-activated protein kinase pathways and the transcription
factor, NF-KB, which regulates the transcription of inflammatory cytokines and
chemokines (97). Viable C. jejuni also stimulates INT 407 cells to produce intracellular
IFN-y, IL-10, TNF-a, and IL-4 suggesting that viable C. jejuni is capable of generating a
dissociated Th1 and Th2 immune response (5). Likewise, C. jejuni-infected human
dendritic cells (DCs) trigger NF ~1cB activation, stimulates IL—l-B, IL-6, IL-8, TNF-q, IL-
10, IL-12 and IFN-y production, and induces DC maturation (69). Also, C. jejuni

interaction with cultured human monocytic cells elicits IL-1 -[3, IL-6, IL-8, and TNF ~01

ll

induction and triggers NF-KB nuclear translocation (77). In addition, C. jejuni has been
shown to invade avian macrophages and primary chick kidney cells and stimulate the
production of nitric oxide (NO), IL-l-B, IL-6, IL-8 and K60 (143). Therefore, it is
evident that C. jejuni-host cell interactions have been studied using multiple well.

established in vitro models.

Pathogenesis
Intestinal Colonization

Colonization is a key factor involved in bacterial survival in the different
microenvironments of the gastrointestinal tract of humans and animals and is necessary
but not sufficient for production of enteritis (146). Many commensal bacteria
asymptomatically colonize the intestinal tracts of animals used as a food source (146,
164). Several studies have demonstrated the importance of C. jejuni colonization to its
persistence (79, 98, 110, 149).

In order to survive as an intestinal commensal or foodborne pathogen, C. jejuni
must be able to adapt to and survive in different microenvironments especially those of
the gastrointestinal tract. This capability is likely controlled by a two-component
regulatory system (TCR), which is important for the coordinate regulation of gene
expression (28). It has been suggested that this novel TCR system plays an important
role in the growth and colonization of C. jejuni . This RacR-RacS (reduced ability to
colonize) system is believed to be involved in a temperature-dependent signaling
pathway. A mutation of the response regulatory gene, racR, reduced the ability of C.

jejuni to colonize the chicken intestinal tract and resulted in temperature-dependent

l2

changes in protein profile and growth characteristics (28). It is suspected that other TCR
systems may be involved in optimal in viva C. jejuni colonization. For example, a dccRS
two-component system (diminished Qapacity to golonize) was found to be important for
in viva colonization of immunodeficient limited flora (SCID-LF) mice and l-day old
chicks, but not relevant for in vitro growth or survival in epithelial cells (98). MacKichan
et al. demonstrated that mutations in the dccR response regulon and the dccS sensor
kinase reduced colonization in l-day old chicks and SCID-LF mice models and had no
significant effect on C. jejuni growth and/or survival in epithelial cells (98). In addition,
several genes encoding putative periplasmic and membrane proteins that are regulated by
this two-component system have been identified. After comprehensive analyses, the
genes involved in the RacR-RacS and dccRS two-component systems (racR and dccRS)
proved to be essential for optimal colonization (98). Likewise, it has been suggested that
C. jejuni outer membrane glycoproteins may act as adhesins thereby promoting
colonization (79). Mutation of the ngH gene involved in C. jejuni N-linked general
protein glycosylation showed reduced C. jejuni adherence and invasion of human
epithelial Caco-2 cells and significant colonization reduction in l-day old chicks (79). In
addition, chemotaxis and motility are believed to be significant virulence factors involved
in controlling the mechanisms of C. jejuni colonization of the intestinal tract (These
factors will be discussed in greater detail later in the literature review.) (149). Overall,
these studies have made significant advances to the knowledge needed for the
development of intervention strategies to control transmission of this foodbome
pathogen. However, despite progress, further studies are needed to better understand the

processes involved in C. jejuni colonization.

l3

Virulence factors

The characterization of C. jejuni virulence factors has been intensively studied
over the past few years because C. jejuni is known to be the most common cause of
gastroenteritis worldwide (6, 33). Significant progress has been made in identifying and
characterizing virulence factors of C. jejuni; however, the molecular mechanisms
involved in pathogenesis remain unclear. Herein, I will discuss a few of the virulence
properties that have been recognized and studied by researchers to elucidate the

pathogenic mechanisms of C. jejuni.

Motility and Chemotaxis

As mentioned in the colonization discussion, one of the best-characterized
virulence determinants of Campylobacters is flagella-mediated motility. Campylobacter
spp. flagella production is necessary for motility (81). It is believed that the spiral-shaped
cellular structure and the flagellum, in combination, give Campylobacters an
uncommonly high level of motility in viscous environments (81, 91). C. jejuni is motile
by the action of one or two polar flagella. It has two copies of the flagellin gene (flaA
and flaB). Two unique promoters control flagellin gene expression, and at least one of
the genes responds to environmental signals (147). It has been shown through targeted
mutagenesis that the flaA gene is the main gene for flagellin production and motility (75,
162). For instance, disruption of flaA produces a short, truncated and nonfirnctional
flagellum that has greatly reduced motility, lacks the ability to invade INT 407 cells, and

is unable to colonize the ceca of three-day-old chicks (110, 157, 162). However, when

14

there is a disruption of flaB, the flagellum length is nomial, and there is normal flagella
function, but slightly decreased motility as compared to the wild type strain (81).

Motility of C. jejuni is known to be phase variable, but the mechanism of this
variation remains unclear. Specific genes identified as the motility accessory factor (mat)
family of flagellin-associated proteins are related to flagellar biosynthesis and phase
variation. It has been suggested that these maf genes may confer on C. jejuni flexibility
in adapting to changing environmental conditions when flagellar expression and motility
are undesirable (80). The maf genes may also be involved in reversible expression of
flagella, which could be beneficial in evading the host immune response (1 l l). The
reversible expression of flagella in C. jejuni means that the genes involved in flagellum
synthesis and mobility can to switched on and off depending on the changing
environmental conditions (111). For instance, flagella genes may be initially switched on
to allow the flagellum to act as an adhesin and acquire attachment to the host cell.
However, once attached and colonization has been established, then flagella formation
may be switched off(111).

It has also been suggested that the regulation of C. jejuni motility is associated
with quorum sensing. C. jejuni produces functional autoinducer-2 (AI-2) cell-signaling
activity, and this AI-2 production is regulated by the luxS gene. AI-2 production allows
bacteria to assess the population density of other species of bacteria and communicate
with these other species of bacteria (75). Studies have demonstrated that inactivation of
the quS gene causes C. jejuni to become less motile than the wild type (43). Conversely,
quorum sensing has been observed to be related to C. jejuni motility. Quorum sensing in

C. jejuni influences flaA transcription and autoagglutination, which is known to be

15

closely associated to the existence of functional flagella in C. jejuni (75). Additional
evidence pertaining to the importance of the fla regulon has been acknowledged by the
recognition of a F lgS/F lgR two-component signal transduction system. The FlgS sensor
and the FlgR response regulator form this system, and it has been shown to be required
for the initial stages of chicken cecal colonization, but not for survival and persistence.
The signal that triggers this two-component system to turn on the fla regulon has not yet
been identified (161).

Chemotaxis is also required for effective colonization and is an important
virulence property. The ability Campylobacters have to detect chemical gradients along
with motility functions enable the cell to move up or down the gradient (81). The
importance of chemotaxis has been demonstrated in viva; chemically mutagenized or
non-chemotactic C. jejuni failed to colonize suckling mouse intestine (149). Two
adjacent genes have been identified to control chemotaxis, a methyl-accepting
chemotaxis protein (MCP) and a putative cytochrome c peroxidase. These were
identified using signature-tagged transposon mutagenesis of C. jejuni 81-176 followed by
testing for chick gastrointestinal tract colonization (59). It is suggested that MCP is
required for proper chemotaxis to a specific environmental element, and the putative
cytochrome c peroxidase may function to reduce periplasm hydrogen peroxide stress
during in viva growth. Loss of either gene function resulted in attenuation of growth
throughout the gastrointestinal tract of chicks (60). This particular study was one of the
first genetic screens involved in identifying the bacterial gene requirements necessary for

establishing a commensalistic relationship with a natural host. Also, it is clear from

16

recent studies in murine models that although colonization is not sufficient for disease

following oral C. jejuni infection, it is required for disease to be manifested (99).

Adherence

Bacterial adherence to target cells plays a critical initiating role in pathogenesis.
Bacterial adhesins interact with the intestinal epithelium and help pathogenic bacteria
resist peristalsis and the fluid flow of lumenal contents. Once bacteria are adhered to the
intestinal epithelium, the bacteria can form colonies, release enzymes and toxins, invade
cells, and/or alter intracellular function to their own advantage (68). Therefore, it is
suggested that adherence is an important determinant of virulence for various pathogenic
bacteria (50).

The interaction between adhesins and the host are believed to be a prerequisite for
bacterial penetration of the mucosal surface. Several Campylobacter spp. adhesins have
been identified and characterized since this genera was discovered. Various in vitro
studies have shown that some Campylobacter spp. adhere to cultured cells (106, 127,
141). In 1986, flagella were identified as a potential C. jejuni adhesin by comparing
adherence of flagellated and aflagellated variants to INT 407 cells (106). McSweegan et
al. showed that removal of the flagella by shearing the bacterial cells reduced adhesion.
Lipopolysaccharide (LPS) was also identified as another C. jejuni adhesin. In vitro C.
jejuni LPS specifically bound to epithelial cells and intestinal mucus gel (106). These
findings support the importance of flagella and LPS in initiating mucosal surface

adherence.

17

Other approaches have been used to determine the significance of campylobacter
adherence. For example, Yao and colleagues used an insertional mutagenesis method for
naturally transformable organisms to characterize non-adherent and non-invasive C.

jejuni strains. They generated a series of C. jejuni strain 81-176 mutants with a
kanamycin-resistant cassette placed in various regions of the open reading frame of the
flaA gene encoding flagellin. By this means, they identified strains that had defects in
motility, displayed reductions in invasion levels, and lacked the ability to adhere to the
target cells. They suggested that flagellin can serve as a secondary adhesin, and other
adhesins mediate a motility-dependent internalization process (162). In addition, these
generated mutants were used to identify a cheY gene that was responsible for the non-
adherent and non-invasive phenotypes observed in their experiments. Yao et al. also
showed that the diploid cheY strain was able to colonize mice, but was attenuated in a
ferret disease model (163). The results of the ferret disease model suggested that
virulence was reduced due to the presence of excess CheY. This also implied that the 2
copies of cheY may affect subsequent C. jejuni interactions with target epithelial cells in
the colon rather than affecting the ability of the organism to survive in the mucus lining
of the small intestine (163).

As mentioned, cultured cells and animal models have been used extensively to
demonstrate C. jejuni adherence to and invasion of epithelial cells (30, 168). Various
studies have led to the identification and characterization of other adhesins (76, 84, 122,
141). For example, a Campylobacter adhesion to fibronectin (CadF), a C. jejuni 37 kDa
protein, was identified as an adhesin. Various in vitro studies indicated that CadF was a

conserved outer membrane protein that mediated the specific binding of C. jejuni to the

18

extracellular matrix component, fibronectin (84). Likewise, other experiments
demonstrated the activity of another C. jejuni adhesin identified as a 43-45 kDa C. jejuni
major outer membrane protein (MOMP). The interaction between C. jejuni MOMP and
cultured INT 407 cell membranes showed significant binding activity, and this interaction
suggested that the MOMP firnctioned as an adhesin and mediated bacterial-host tissue
contact (141). leA, a C. jejuni specific novel surface-exposed lipoprotein, was another
identified adhesin that mediated adherence to host epithelial cells. Insertion and deletion
mutants of jlpA demonstrated that adherence to HEp-2 cells can be reduced compared to
parental C. jejuni TGH901]. Likewise, anti-GST-leA inhibits C. jejuni adherence to
HEp-2. Also, an irnmunoblotting assay showed that leA binds to HEp-2 cells, implying
that leA is a C. jejuni adhesin. From these findings, it has been suggested that
lipoproteins may play a role in bacterial infection because they induce tumor-necrosis
factor and cause macrophages to release interleukin-6. It has been suggested that J lpA
may stimulate host cytokine production because it is released into the culture medium.
However, this function remains to be established and could have multiple effects on the
immune system (76). Furthermore, in vitro and in vivo models were used to characterize
PEBl as an adhesin that enhances C. jejuni interactions with epithelial cells. To identify
the role of PEBl adhesion activity, pebIA was inactivated by allelic exchange, and used
to challenge cultured cells. The inactivation of the pebIA locus significantly reduced C.
jejuni adherence toHeLa cells, which suggested that PEBl was an important C. jejuni
adhesion (122). This study also provided information about the PEBI adhesin function in
C. jejuni invasion and colonization. PebIA locus inactivation significantly reduced C.

jejuni invasion of epithelial cells in culture and reduced the rate and duration of mouse

19

intestinal colonization as compared to the C. jejuni wild type isolate. Together, these
data indicated that PEBl plays an important role in bacterial-epithelial cell surface
interactions (122).

In summary, the identification and characterization of these adhesins have proven
to be beneficial in establishing a better understanding of C. jejuni host cell interactions.
According to these studies, adhesins play important roles in mediating C. jejuni
attachment to host epithelial cells. They also suggest that some adhesins may play a
significant role in virulence and host resistance and immunity. Further studies will

contribute to understanding the relationship between C. jejuni adhesion and infection.

Invasion

Campylobacter-host cell interactions have been described as intriguing and
mechanistically unique (160). Over the years, researchers have been persistently trying
to understand the mechanisms by which Campylobacter invade host cells. Both in vitro
and in vivo models have been used to establish the invasive potential of C. jejuni (10, 21,
23,29,46,57,61)

In vivo studies have shown that C. jejuni invasiveness is clearly correlated with
presentation of disease in the host (10, 21). In one particular study, newborn piglets were
orally inoculated with a strain of C. jejuni isolated from a patient. The clinical and
histopathological damage was similar to that observed in that patient and in other humans
with campylobacteriosis. I-Iere, internalized bacteria were only observed within the
intestinal cells of these C. jejuni-infected piglets, and there was disruption of the

microvilli in the infected area (10). In various studies, cultured mammalian intestinal

20

epithelial cells have been used to demonstrate the process of C. jejuni invasion (86, 87,
107, 135). For example, Konkel and colleagues identified a Campylobacter invasion
antigen (CiaB) protein that affects C. jejuni invasion and shows similarities to type III
secreted proteins (86). Further studies indicated that maximal invasion of eukaryotic
cells and Cia protein secretion depended on intact flagellar synthesis genes, thus
suggesting that Cia protein export occurs through the flagellar transport and/or assembly
apparatus. Collectively, these findings suggested that the flagellar type III secretion
pathway is required for C ia protein export (87). In addition, it has been suggested that
the Cia proteins are secreted during colonization, and they may play a significant role in
C. jejuni colonization (22). Konkel and colleagues also identified, CadF, a fibronectin—
binding protein that is required for the binding of C. jejuni to intestinal epithelial cells.
They also found that this interaction enhanced bacterial internalization (84). Monteville
et al. found that maximal C. jejuni entry required the binding of CadF to fibronectin
which thereby triggered host cell signaling events associated with bacterial uptake. In
this study, INT 407 cells were infected with a C. jejuni wild-type isolate and at 30 and 45
minutes post infection, there was an increase in the level of tyrosine phosphorylated
paxillin (a focal adhesion signaling molecule)(107). Likewise, ij29 an unknown
glutamine-rich protein, was another protein required for C. jejuni invasion, and it was
found inside INT 407 cells, suggesting that it may be secreted by a type IV secretion
system ( 12).

Additional studies have been carried out in order to determine if invasion is a
significant virulence factor in facilitating C. jejuni enteritis. Researchers have examined

the possible roles of microfilaments and microtubules in the C. jejuni invasion process

21

(23, 71, 89, 113). Treatment of intestinal epithelial cells (INT 407 cells) with
cytochalasin-D (a chemical that causes microfilament depolymerization) and colchicines
and nocodazole (chemicals that cause microtubule depolymerization) inhibited C. jejuni
invasion of INT 407 cells in a dose-dependent manner. There was a more pronounced
inhibitory effect of microfilament depolymerization on C. jejuni invasion than that of
microtubule depolymerization. Similar observations have been reported on Klebsiella
pneumoniae enterohemorrhagic E. coli, enteropathogenic E. coli and E. coli associated
with new born meningitis (23, 24). These overall findings suggest that the mechanisms
involved in C. jejuni invasion is associated with the combined effect of microfilaments
and microtubules of host cells (23).

C. jejuni vaccine strain 81-176 has two plasmids (pTet- an R factor plasmid
encoding tetracycline resistance and the other, pVir, encoding the cag pathogenicity
island usually found in Helicobacter pylori) that have been characterized and are believed
to affect the virulence of C. jejuni by encoding a type IV secretion system. Specific
mutations in some of the identified plasmid genes (comB3 and virBI I) have been shown
to significantly reduce invasion of INT 407 cells (13). Other analyses indicated that there
are four additional pVir genes (ij15, ij29, ij32, and ij49) which need further
characterization using mutational analyses followed by testing in animal disease models,
but they have been found to significantly affect C. jejuni invasion into intestinal epithelial
cells. These four genes encode proteins that are envisaged to be soluble and once
invasion occurs are located in the cytoplasm. Further studies are being done to elucidate

the possible effector protein roles of this putative type IV secretion system (13).

22

The C. jejuni invasion process is multifactorial and influenced by many cellular
processes (21). This invasion process appears to be C. jejuni strain dependent and host
cell-type dependent. Freshly isolated clinical strains also appear to be more invasive
(36). Invasion of the host cell is an important virulence mechanism of C. jejuni, and the

molecular mechanisms mediating host cell invasion are gradually being elucidated.

Intracellular survival

Intracellular survival is definitely a key factor in the pathogenesis of C. jejuni;
however, the mechanisms by which C. jejuni persist in the host cell have not yet been
defined. Various studies have attempted to demonstrate the entry process, intracellular
survival and location of C. jejuni (24, 35, 36, 85, 86). Using transmission electron
microscopy and the gentamicin killing assay, DeMelo et a1. observed cellular events and
intracellular survival of C. jejuni in Hep-2 cells (3 6). At one hour post infection, DeMelo
and colleagues noticed C. jejuni cell surface and microvilli interactions which possibly
imply a phagocytic-like mechanism preceding C. jejuni internalization (36). Also at one
hour post infection, there was notable evidence of host cytoskeletal rearrangement and
the presence of C. jejuni enveloped within endocytic vacuoles in the cytoplasm of the
host cell (23, 36, 83). Once internalized and about 9 hours postinfection, phagolysosome
fusion occurred, and lysosomes assembled and co-localized in the cytoplasmic matrix.
This lysosomal event could possibly explain the loss of C. jejuni viability and the change
in the morphological structure from spiral to coccal form at this stage (83). Konkel et al.
and Biswas et al. also did studies to firrther characterize C. jejuni internalization and

intracellular survival. They concluded that C. jejuni internalization of INT 407 cells was

23

affected by both, host microfilament and microtubule depolymerization (23, 24, 83). In
addition, Konkel et al. found that ammonium chloride and methylamine did not inhibit
endosomal acidification and did not affect C. jejuni internalization by INT 407 cells (83),
but Biswas et al. showed that monenesin significantly inhibited endosome acidification,
thereby reducing the number of intracellular C. jejuni (24). Biswas et al. also showed
that viable intracellular C. jejuni were reduced in number by treating INT 407 cells with
G-strophanthin and monodansylcadaverine which indicated interference of receptor-
mediated endocytosis (62).

It is known that C. jejuni has the ability to persist and multiply in epithelial cells
and phagocytic host cells (35, 36, 85, 123). Konkel et al. showed that intracellular C.
jejuni can elicit a cytotoxic effect on INT 407 cells causing deterioration of the cells (85).
The weakening effects on the cell monolayers are believed to represent a pathogenic
mechanism associated with C. jejuni enteritis (85). Likewise, Hickey et al. showed that
C. jejuni strain 81-176 is capable of replicating extensively within human monocytic cell
vacuoles. They also suggested that viable intracellular C. jejuni induce apoptotic death
via secreted C. jejuni cytolethal distending toxin (62). Similar to the findings of Guerry
et al., Siegesmund et al. showed that C. jejuni Cia-secreted proteins are required for
maximal apoptosis induction of monocytic cells in vitro (142). It has been suggested that
these secreted proteins are associated with a type 111 system of C. jejuni, and are
responsible for intestinal epithelial cell invasion, and possibly play a role in C. jejuni
enteritis (142). Siegesmund et al. also showed that the apoptotic death of C. jejuni-

infected monocytic cells is caspase-l-independent and is not dependent on the concurrent

24

secretion of IL-l-B, which is caspase-l dependent (142). However, it is unclear at this
time as to how macrophage apoptosis is associated with C. jejuni-mediated enteritis.
There have been several studies which have given insight to the possible
mechanisms of intracellular survival (35, 123). For instance, it has been demonstrated
how C. jejuni iron superoxide dismutase (SOD) enzyme contributes to intraepithelial cell
survival (123). This enzyme catalyzes the breakdown of superoxide radicals to hydrogen
peroxide and dioxygen and acts against oxidative stress in the bacterial cell thereby
contributing to the defense mechanisms of the bacterial cell (123). To date, only one sod
gene has been identified in C. jejuni, which is similar to other reported FeSODs. In one
particular study, Pesci et a1. constructed isogenic sod mutants which proved to be
significantly more sensitive to intracellular killing than wild-type C. jejuni strains thereby
suggesting that SOD plays a role in C. jejuni intracellular survival (123). Likewise, C.
jejuni expresses a single catalase enzyme identified as katA (35). It is believed that this
catalase also plays a significant role in C. jejuni intracellular survival. Day et al.
constructed katA mutants to address this hypothesis, and the findings of this study
suggested that catalase plays a significant role in C. jejuni intramacrophage survival by
providing resistance to hydrogen peroxide(35). Together, the findings of Pesci et al. and
Day et al. suggest that organism persistence inside host cells is in part due to bacterial

factors that combat reactive oxygen species and play a role in intracellular survival (35,

123) .

25

Lipopolysaccharide/Lipoolygosaccharide

In gram-negative bacteria, lipopolysaccharide (LPS) is a major constituent of the
outer membrane, and it is composed of three distinct structural components: lipid A,
which anchors the membrane; an oligosaccharide core; and the 0 antigen composed of
one or more glycosyl residues that covalently attach to the core (88). LPS molecules that
lack O-antigen units and have core oligosaccharides structures limited to 10 saccharide
units are referred to as lipooligosaccharides (LOS) (130).

It has been suggested that C. jejuni LPS and LOS are excellent targets for the
control of infectious diseases and for diagnostic purposes. In vitro and in vivo studies
have demonstrated the functions of LPS and LOS in C. jejuni virulence (55, 108, 128,
167). It is believed that C. jejuni LPS and LOS mimic host ganglioside structures,
specifically components of the perpherial nerve tissue (55). More specifically, LOS has
been shown to play an important role in the pathogenesis of gastroenteritis due to C.
jejuni. Phase variation of C. jejuni 81-176 LOS has been demonstrated in vitro and in
vivo, and the ability of C. jejuni to undergo phase variation in LOS biosynthesis genes
may provide selective advantages in causing gastrointestinal disease (5 5). It has been
suggested that phase variation of LOS biosynthesis allows a single bacterial strain to
produce a repertoire of LOS molecules that differ in core length and carbohydrate content
due to the addition of glycose groups at the nonreducing termini (128). In vitro, slip
strand mismatch recombination of cgtA, a gene that encodes N-acetylgalactosaminyl
(GalNAc) transferase, resulted in changes in the major core structure from GM; to GM;
ganglioside mimicry. Also, site-specific insertional inactivation of the cgtA gene of C.

jejuni LOS resulted in a mutant that mimics GM3 and enhances internalization and

26

invasion of intestinal epithelial cells in vitro thereby suggesting that changes in LOS
structure may directly affect the ability of C. jejuni to cause gastroenteritis (55). Also,
variation in the sialylation of C. jejuni LOS was demonstrated via site-specific
mutagenesis of the neuCI gene resulting in the loss of sialic acid in the LOS core,
increased irnmunogenicity of the core, and decreased resistance to normal human serum
(53, 55, 128, 167). Antigenic phase variation in ganglioside mimicry has also been
demonstrated in viva (128). In a volunteer experimental oral infection study, Prendergast
et al. found that the LOS of C. jejuni isolates underwent antigenic phase variation in
ganglioside mimicry during passage in vivo, and serological testing showed that
antiganglioside antibodies were not present following experimental C. jejuni infection or
following administration of 3 killed C. jejuni whole-cell vaccine (53, 108, 109, 167).
They concluded that their findings were consistent with in vitro reports that have shown
that C. jejuni possesses the genetic mechanisms for its LOS to undergo phase variation.
They also believe that these LOS phase variation responses could cause difficulties in
developing successful vaccines because C. jejuni can synthesize ganglioside mimics and
has the ability to convert between different LOS structures (128). These observations
also suggest that the ability of C. jejuni to undergo LOS phase variation may be
advantageous to bacterial persistence in the host. Hence, the changes that can occur in
the major core of C. jejuni due to bacteria] phase variation suggest that C. jejuni LOS
core structures are dynamic, and there are indications which imply that there may be
differences in the mechanisms by which C. jejuni strains cause diarrhea] disease (55).
Further study is needed to elucidate C. jejuni phase variation and how these variations

may play a role in C. jejuni-induced gastroenteritis.

27

Additionally, more studies have been conducted to elucidate the association
between C. jejuni LOS and GBS (109). It has been demonstrated that molecular mimicry
between human ganglioside and C. jejuni LOS play a relevant role in the pathogenesis of
GBS (53, 109). Nachamkin et al. found that est-II (or-2,3 and/or d-2,3/ 0,-2,8
sialyltransferase), cgtA (Bl,4-N-aetylgalactosyltransferase) and cgtB ([31,3-
galactosyltransferase)are LOS biosynthesis genes involved in C. jejuni LOS core
extension and appear to be critical to C. jejuni ganglioside mimic expression associated
with GBS-related C. jejuni isolates(21, 167). Also, based on LOS locus analysis and
knockout mutants, Godschalk et a]. suggested that specific classes of the LOS
biosynthesis gene locus or specific types of genes involved in sialic acid biosynthesis and
transfer may be crucial for the induction of neuropathogenic cross-reactive antibodies
(53). Various studies have presented evidence implying that the development of auto-
antibodies raised against distinct C. jejuni surface LPS and LOS is involved in the
pathogenesis of Guillain-Barre syndrome(GBS)(55, 108, 166). In rabbits sensitized with
C. jejuni LOS, anti-GM] IgG antibodies were detected and flaccid limb weakness
developed, which resembles clinical signs of GBS in humans. The pathological studies
of the sciatic nerve specimens of the paralyzed rabbits showed Wallerian-like
degeneration identical to that seen in GBS patients (167). In addition, immune naive
mice injected with C. jejuni LOS generated monoclonal antibodies (mAb) that reacted
with GM] and bound to human peripheral nerves. In general, these findings suggest that
carbohydrate mimicry between C. jejuni LOS and human gangliosides are important for

the development of GBS.

28

Overall, C. jejuni LPS and LOS appear to play an important role in the
pathogenesis of C. jejuni-mediated enteritis and autoimmune diseases such as GBS, and
further evaluation of these particular virulence determinants (LPS and LOS) may
elucidate specific mechanisms involved in C. jejuni’s ability to persist in host cells

causing pathology and disease.

Toxins

Campylobacter spp. that produce toxins have been isolated from various human
and animal sources throughout the world. In attempts to study toxigenicity, researchers
usually correlate toxin production with clinical presentation and in vitro analysis (156).
Since C. jejuni is one of the leading causes of acute diarrhea in humans in developed
countries and has been isolated from healthy and diseased young children in developing
countries, I have limited the discussion to this species (156). There are two types of
diarrhea induced by C. jejuni: l) a noninflammatory, watery diarrhea without leukocytes
or blood and 2) an inflarmnatory, bloody diarrhea containing leukocytes. In both of these
clinical presentations, diarrhea] disease caused by C. jejuni is believed to be associated
with toxin production.

Enterotoxins and cytotoxins are two classes of proteinaceous toxins that differ in
their primary mode of action. Enterotoxins are secreted proteins with the ability to bind
to a cellular receptor, enter the cell, and elevate intracellular cyclic AMP (CAMP) levels.
This elevation of CAMP causes changes in the cellular ion flux, which leads to excessive
secretion of fluid, resulting in watery diarrhea (156). Cytotoxins kill target cells by

inhibition of cellular protein synthesis and actin filament formation or by forming pores

29

in target cell membranes. Cytotoxins cause inhibition of cellular protein synthesis by
inactivating ribosomes by depurination of a nucleotide in the 28S rRNA, and this
inhibition leads to cell death. Also, these secreted proteins can damage the intestinal
mucosa and cause diarrhea by covalently modifying Rho proteins resulting in the
disruption of the actin cytoskeleton. Another cytotoxic mechanism can be directed
specifically towards nucleated cells. For instance, the cytotoxin forms pores in the
nucleated cell thereby inducing cytokine release, cytoskeleton dysfunction, secretion of
granule constituents, and generation of lipid mediators. These profound local and distant
effects in host tissues may lead to an overall diminution of the immune responses of the
host (156).

C. jejuni enterotoxin activity has been demonstrated in cell culture by the
elongation of Chinese hamster ovary (CHO) cells, rounding of mouse adrenal tumor cells
(Y-l), or by a measurable increase in intracellular CAMP in exposed cells (156).
Likewise, Ruiz-Palacios et al. showed that supernatant of a prototype virulent C. jejuni
strain causes the induction of fluid secretion in the rat ileal loop test (RILT), but not in
the rabbit ileal loop or the infant mouse assay (136, 156). It has been suggested that
enterotoxin production is responsible for the watery diarrhea form of campylobacteriosis,
as opposed to the other form with inflammatory, bloody diarrhea (156). However,
controversy surrounds C. jejuni enterotoxin activity because, to date, no gene encoding
this product has been found. Also, the inability to consistently reproduce some of the in
vitro and in viva assays used to detect enterotoxin activity continues to fuel the
controversy. These concerns will not be resolved until the enterotoxin structural gene has

been cloned, sequenced, and the firnctional experiments are proven to be reproducible.

30

C. jejuni cytotoxins are also not well characterized. Several studies have
evaluated the correlation between cytotoxin production and clinical manifestations. It has
been suggested that cytotoxin-producing Campylobacter strains are more commonly
isolated from patients with inflammatory diarrhea than other strains (156). Various cell
lines, bacterial culture conditions, and bacterial strains have been used to study
Campylobacter spp. cytotoxin production (156). Although diverse methodologies have
been used to classify Campylobacter spp. cytotoxin production, to date, some of the
research has not been reproducible (125).

However, to date, the cytolethal distending toxin (CDT) is the best characterized
C. jejuni toxin. In 1996, the cdt genes from C. jejuni strain 81-176 were cloned and
sequenced (125). Lara-Tejero et a]. discovered that the cdt genes are required to form a
tripartite holotoxin that promotes CDT activity (90). They found that 3 subunits, Cth,
CdtB, and CdtC, make up the C. jejuni CDT holotoxin, and all subunits are required for
complete toxic activity (90). It has been observed that Cth and CdtC bind the host cell
and comprise CdtB. The CdtB subunit has been shown to be the active enzymatic
subunit which is imported into the nucleus and functions similar to a DNAse 1 enzyme
(32, 88, 92). It is proposed that the CdtB subunit damages the DNA and inhibits the
dephosphorylation of Cdc2, a key kinase regulator of cell cycle progression (90). C.
jejuni CDT causes elevated intracellular CAMP levels, blocks the Gz/M phase of
eukaryotic cells prior to cell division, induces cytoplasm distension, and eventually leads
to cell death (32). This cell cycle arrest activity has been demonstrated in experiments
using a yeast model system that showed that CdtB induced G2 cell cycle arrest

accompanied by degradation of the host cell chromosomal DNA (58).

31

In addition to these findings, it has been suggested that C. jejuni CDT plays a role
in host cell immunomodulation. In vitro, CDT stimulates cultured human intestinal
epithelial cells to produce IL-8 (63). In an in viva challenge experiment, mutant C. jejuni
lacking the cdtB gene successfully colonized NFKB deficient mice producing gastritis but
caused much less severe disease than the isogenic parent wild type C. jejuni. This
finding suggests that CDT has a proinflamatory effect similar to CDT effects reported in
vitro (51). Also, IgG2a levels were lower in mice infected with the CDT mutant than the
mice infected with the wild type strain (51). These findings suggest that CDT plays a
significant role in stimulating an immune response and in developing disease.

Toxin production by Campylobacter spp. has been a very complex virulence
property to classify, and the difficulties with Campylobacter spp. toxin classification may
reflect significant strain variation in this species. There has been a continuous effort
towards developing in vitro and in viva methodologies to determine the modes of action
of putative toxins (165). Although there have been several reports of toxin producing

Campylobacter spp., the correlation with disease and the mechanisms of action require

firrther study.

Immune responses

The mechanisms by which C. jejuni elicit a host immune response are not well
understood. In developing countries, it is believed that healthy infants, children and
adults are constantly exposed to Campylobacter antigens in the environment, and
consequently, there is early development of serum antibodies to Campylobacter species

(33). However, most cases in developed countries occur later in life and are usually

32

primary, sporadic infections with more severe and prolonged clinical manifestations, and
the immune response related to disease is not clear (33). Therefore, the host immune
responses to campylobacteriosis differ among people in developing and developed
countries, and various studies have been conducted to determine the importance of C.
jejuni immune responses and host factors that affect the outcome of disease.

Humoral immunity is necessary to resolve C. jejuni infection. During C. jejuni
infection, the levels of all immunoglobulin classes rise. However, IgA is one of the most
important isotypes because it can cross the gut wall and immobilize organisms in the gut
(155). In a swine disease model, a significant efflux of immunoglobulin A (IgA) was
detected in the enlarged germinal centers of the lymphoglandular complexes (LGCs) of
C. jejuni-infected pigs (100). This immune response suggests that there are important
colonic sites worthy of further immunological consideration to elucidate mechanisms of
resistance or susceptibility to disease. In addition, Cawthraw et a]. detected significant
IgA antibody levels in the postinfection serum, saliva and urine of patients with C. jejuni
infections (31). Also, serum and salivary antibodies against flagellin were detected
suggesting that flagellin is an important surface antigen against which immunity is
directed (19). In a domestic ferret model system, humoral immunity does not appear to
play a role in preventing colonization but does protect against enteric disease. Taken
together, these findings suggest that a Th2-type immune response can in part play a
protective role during C. jejuni infection.

T-cell-mediated responses to C. jejuni infection appear to be of relevance as well.
Rhijn et a]. demonstrated peripheral blood 75 T cell expansion following the exposure of

patients to crude sonicates of five different C. jejuni serotypes (153). This expansion was

33

dependent on the presence of CDT/(18+ T cells in cultures or the addition of exogenous
IL-2 or IL-15, and 75 TCR mediated the C. jejuni-specific stimulation (153).

Also, several in vitro studies have demonstrated the roles of cytokines in C. jejuni
infection. Viable C. jejuni stimulates the secretion of IL-8 by INT 407 cells (human
intestinal epithelial cells), and this response is mediated via multiple mechanisms, 1) C.
jejuni adherence and/or invasion or 2) C. jejuni cytolethal distending toxin (CDT)
treatment (61, 63). Other proinflammatory chemokines, such as GROalpha,
GROgamma, macrophage inflammatory protein 1, MCP-l, and gamma lP-lO, are
secreted by INT 407 cells due to exposure to viable C. jejuni and are believed to play a
contributing role in the observed host inflammatory responses (70). Furthermore, it has
been shown that viable C. jejuni activates the mitogen-activated protein kinase pathways
and the transcription factor NF ~1<B which regulates the transcription of inflammatory
cytokines and chemokines (97). Viable C. jejuni also stimulates INT 407 cells to produce
intracellular IFN-y, IL-10, TNF-a, and lL-4 suggesting that viable C. jejuni is capable of
generating a dissociated Th1 and Th2 immune response (5). Likewise, C. jejuni-infected
human dendritic cells (DCs) trigger NFKB activation, stimulates IL-l-B, IL-6, IL—8,
TNF-a, IL-10, IL-12 and IFN- 7 production, and induces DC maturation (69). Also, C.
jejuni interaction with cultured human monocytic cells elicits IL-l-B, IL-6, IL-8, and
TNF-a induction and triggers NFKB nuclear translocation (77). In addition, cultured
swine intestinal epithelial cells secrete IL-6, IL-10 and IL-18 following C. jejuni
exposure (Parathasarathy, dissertation, 2004 and Jones, dissertation, 2005). Similar to
mammalian cells, avian primary chick kidney cells and avian macrophages express the

production of nitric oxide (NO), IL-l—B, IL-6, IL-8 and K60 due to C. jejuni invasion

34

(143). In vivo, C. jejuni also induces peritoneal phagocyte oxidative activity, increased
polymorphonuclear (PMN) cells from the spleen, and IL—2 production in infected
BALB/c mice (115, 1 l6). Altogether, these findings suggest that cytokines likely play
significant roles in C. jejuni-mediated infection.

Overall, the findings of these studies appear to suggest the cooperation of both
innate and adaptive host immune responses to resolve C. jejuni infection in viva. It is
suggestive that the inflammatory immune responses induced by viable C. jejuni play an
important contributing role in directing host immune responses towards generating and/or
modulating protective immunity for the benefit of the host. However, in some cases
depending on the susceptibility of the host, inflammatory responses can benefit bacterial
survival during immune assault. Therefore, it is imperative that further study is done to
elucidate the dissociated immune responses induced by viable C. jejuni to determine how
the responses play a role in immunopathogenetic events associated with inflammatory

diarrhea due to C. jejuni infections.

35

II. T richuris suis
Basic parasitology

T richuris suis (T. suis) is a member of the nematode superfamily Trichuroidea. A
distinguishing characteristic of this phylum is the stichosome esophagus which consists
of stichocytes that have been shown to be involved in feeding and attachment(l7, 74,
151). There are five genera associated with this superfamily: T richinella, T richuris,
Capillaria, T richosomoides, and Anatrichosoma. There are also related species of
interest: T richuris ovis (T. ovis)-cattle, sheep and goat; T richuris discolor (T. discolor)-
cattle; T richuris suis (T. suis)-swine; T richuris vulpis (T. vulpis)-dog; Trichurisfelis (T.
felis)-cat; T richuris muris (T. muris)-mouse; T richuris trichiura (T. trichiura)-humans
and non-human primates; T richuris Ieporis (T. Ieporis)-rabbits. These parasites are

known as whipworms and are normally found in the host intestine (126).

Morphology

T. suis is a swine whipworm helminth that has a thread-like appearance featuring
a slender anterior end which attaches to the mucosa of the pig’s cecum and has a thick
posterior end which protrudes freely into the lumen. This whipworm is about 5-8 cm
long and is dioecious (exists as separate sexes). The male nematode is smaller than the
female, and the male has a distinct curved spicules at its posterior end. T. suis eggs are
barrel shaped with a thick shell that consists of three outer layers and one thin inner
vitelline membrane with bipolar plugs. The external surface and the body cavity lining

of T. suis are covered by the cuticle. It serves as a tough, flexible, protective covering

36

that is resistant to most digestive enzymes. Beneath the cuticle is the hypodermis layer
which aids in regulating the body wall permeability. This hypodermis layer gives rise to
the bacillary band which displays glandular activity. Directly underneath the hypodermis
lies muscle cells consisting of a large vacuolar cytoplasmic body called the stichosome,
which enfolds a narrow capillary-like esophagus. The alimentary tract of the parasite is
complete with a mouth, buccal capsule (not always present), and an esophagus that
empties into the intestine and onto the anus. The nervous system consists of dorsal and
ventral nerve cords that surround the esophagus. There have been no findings of a
circulatory or respiratory system. However, it is believed the bacillary band is utilized
for oxygen exchange between the host’s tissues and the nematode’s internal organs via
diffusion through pseudocoelomic fluid. The male reproductive system consists of testis,
vas deferens, ejaculatory duct, spicules, and spicular muscles. The female reproductive
system consists of the vagina, uterus, oviduct, and ovary. The life span of T. suis usually

does not exceed 4-5 months (17, 74).

T. suis life cycle

There are five larval stages in the life cycle of T. suis which accounts for the
development of the egg into a mature adult. The prepatent period is 41—45 days, and the
life span of this whipworm usually does not exceed 4 to 5 months (17, 74, 151). T. suis
female reproduces 3000 to 5000 eggs per day following copulation. The eggs are
unsegmented, barrel shaped with a thick shell and are operculate at each end. The female
lays the unfertilized eggs, and they are passed out of the pig via the feces, and the

infective L1 larvae began to develop inside the egg. The development of the egg is

37

 

influenced by temperature, and studies have shown that the optimal temperature for the
development of the infective L1 larvae is 34°C. By day 19, infective larvae are formed,
and a characteristic feature of infective Ll larvae is the presence of an oral spear (stylet).
The infective L1 larvae are ingested by the pig, and continued development of the larvae
to the adult stage, which occurs in the mucosa of the cecum and colon. The hatching of
the L1 larva began as early as 9 hours post-infection, and there is continued hatching for
up to 3 days in the small intestine, cecum, and colon. The oral stylet is used by the L1
larvae to pierce the surrounding membrane and emerge from the shell. The L1 larvae
burrow into the mucosa via the Crypts of Lieberkiihn and penetrate the epithelial and
goblet cells lining the crypts. As the larvae continue to grow, by day 7, they begin to
invade the lamina propria cells. On day 10, the first molting (shedding of the cuticle
form the body) occurs, and the L1 develops to the L2 larvae stage. The L2 larvae
continue to grow and body organs differentiate as the L2 larvae migrate deeper towards
the mucosal epithelium that begins the histotrophic phase of the T. suis life cycle. This
molting process continues throughout the developing stages. Around day 16, the L2
larvae undergo another molt to become L3 larvae. At this stage, the most posterior end of
the L3 larvae protrude into the gut lumen, and the body length is increased significantly.
The reproductive system is also developing at this time and further differentiation of
body organs is occurring. On day 20, molting occurs again, and the L3 larvae develop to
the L4 stage, and the reproductive tract can be distinguished in some larvae. The entire
posterior end of the larvae is free in the lumen while the anterior end is embedded in the
mucosa. By day 28, the larvae lay in a shallow tunnel on the mucosal surface covered by

mucosal epithelial cells. The last molting occurs around days 32 and 37; at this time, the

38

L4 larvae develop into the adult stage L5 larvae. The sexual organs are completely
developed, and the females appear larger than the males. By day 41, eggs are seen in the

uterus, vagina, and feces of the female permitting the life cycle to start again (26).

Epidemiology

T. suis are most prevalent throughout the world in places with warm, humid
climates (52). These whipworm nematodes cause severe disease, and a 1991 study
reported that approximately 45% of swine farms nationwide were infected with T. suis.
Moderate to heavy infection can cause serious economic losses due to scours, which
results in diarrhea, reduced carcass weight, and even death. A controlled trial experiment
showed that the specific economic losses were 26 lbs. of pork and 2 dead pigs out of 12
animals (26). Severe T. suis infection also affects the swine industry by forcing an
extension in the pig’s growth and processing time by fifteen extra days before being able
to sell at the market (26). The incidence of T. suis in swine herds is of great economical
relevance, and studies have been conducted to inform the swine industry and the general
public of the consequences of T. suis infection. A study conducted to determine the
health status of a feral swine population in Kansas, USA indicated that 10% of the pigs
were infected with T. suis (52). Also, a survey of gastrointestinal pig parasites on free-
range, organic and conventional pig farms in the Netherlands indicated that T. suis is
quite prevalent on various farms. Results showed that T. suis was found on 37.5% of the
free-range farms, 36.4% of the organic farms, and 11.1% of the conventional farms. This
study suggested that T. suis infection in pigs on farms with outdoor facilities is higher

than pigs on conventional farms. The infection was most commonly seen in the sows on

39

 

the free range farms (50%) and organic farms (30%) (40). Likewise, on intensive farms
in Guangdong Province, People’s Republic of China, of the 3636 pigs sampled, 189
(5.2%) were infected with T. suis, and T. suis was the most common nematode causing
infection in the breeding, young and adult pigs. There was no strategic anti-parasite
treatment regime implemented on these farms (158). Further studies are needed to better
address preventive strategies associated with T. suis infection, economical loss, and

public health awareness.

Pathology

Human Pathology. Gastrointestinal nematode infections affect people throughout
the world and tend to be chronic with high reinfection rates (49). T richuris infections in
humans are symptomatic in developing countries, and most patients in developed
countries are asymptomatic unless exposed to heavy infection. Heavy infections in
humans cause chronic diarrhea with bloody stool. Other symptoms include abdominal
pain and distention, nausea, vomiting, flatulence, headache, weight loss, malnutrition and
anemia. Untreated infections in some children can lead to clubbing of the fingers and
growth retardation (151).

Animal Pathology. T richuris infections also affect livestock production
worldwide and cause economic losses for farmers (26). The economic loss in the swine
industry is due to serious T. suis infections that cause ‘scours’ (severe mucohemorrhagic
diarrhea) which is associated with increased weight loss, unthrifliness, anemia,

dehydration and death in heavy infections (26).

40

T. suis is found in the cecum and colon of swine (18). Usually it is the younger,
weaned pigs that acquire catarrhal enteritis with serious pathology (121). Trichuriasis is
commonly called ’21-day scours’ by swine producers because of the severe, bloody
diarrhea associated with the infection that usually occurs 21 days after pigs are exposed
to a T richuris contaminated lot (17, 26, 74). The histotrophic phase of T richuris
infection lasts for thirteen days in the mucosa, and onset of clinical signs are associated
with the reemergence of third-stage larvae into the cecal lumen (17, 74). Much of the
pathology associated with this helminth infection is due to the burrowing activities of the
larval and adult stages in host colonic tissues and secretion of proteolytic enzymes and
proteases (17, 74). However, following the initial mucosal damage by the parasite,
secondary invasion by opportunistic gut microflora may result; thereby, amplifying the
severity of the infection (17, 74, 101, 103, 138). The parasite maintains a syncytial
environment (firses with the cells) in the cecal epithelium with its anterior end within the
host epithelium and its posterior portion free in the cecal lumen (17, 74). Beer et al.
suspected that these syncytial tunnels created by T. suis burrowing contributed to the
secondary invasion by gut microflora, which contributed to the overall pathology (17,
138).

Likewise, Mansfield and Urban also conducted an in vivo experiment describing a
porcine model of mucohemorrhagic diarrhea that demonstrated the overgrowth of
opportunistic bacteria during subclinical T. suis infection (103). The findings of this
study suggested that secondary bacterial infection likely plays a contributing role in
inducing pathology during T. suis infection. Results also indicated that antibiotic therapy

relieved the pathology observed (103). In this study, pigs infected with T. suis only and

41

receiving no antibiotics exhibited mucopurulent lesions in the distal colon surrounding
the lymphoglandular complexes. This observation led to the isolation of 3 opportunistic
bacteria, including a tissue invasive bacterium, Campylobacterjejuni (103). The
pathology observed supported the early study of Rutter and Beer who observed secondary
bacteria] invasion while studying the synergistic relationship between T. suis and the
microbial flora of the large intestine. They believed this synergistic relationship caused
swine dysentery (138). With this in mind, Mansfield et a1. conducted another study using
gnotobiotic (germ-free) pigs to examine the hypothesis that disease and pathology is
induced by the synergistic effect of T. suis and C. jejuni in the colon. The pigs infected
with both T. suis and C. jejuni, showed severe clinical signs and pathology; whereas, the
T. suis only and C. jejuni only-infected pigs showed no clinical signs or pathology (101).
This confirmed that secondary invasion by bacteria can contribute to the clinical
syndrome seen during this helminth infection.

While studying the pathogenesis caused by the interaction between T. suis and C.
jejuni (the secondary opportunistic bacterium), Mansfield et al. also detected the presence
of the histopathologic lesions in the colon of infected pigs (100, 101, 103). Lesions were
seen in the proximal and distal colon. At the site of worm attachment in the proximal
colon, there was thickening of the intestinal layers and crypt and absorptive cell
destruction. There was also an infiltration of inflammatory cells in the lamina propria
that included lymphocytes, macrophages, neutrophils, eosinophils and plasma cells. In
the distal colon, the lymphoglandular complexes (LGCs) in the submucosa were enlarged
mainly due to enlarged B cell-dependent germinal centers, and some lymphocytes,

macrophages and a few eosinophils were present (100, 101, 103).

42

T. suis Excretory/Secretory Products (ESP) and antimicrobial activity

There have been several T. suis ESP identified and characterized, and it is
believed that these T. suis products may be involved in nematode tissue invasion,
migration and feeding of the larvae and adults. It has also been suggested that ESP could
possibly be facilitating or directly inducing the pathology seen during infection. T. suis
ESP was collected while isolating and maintaining T. suis in culture. These ESP

components will be discussed in detail in the following section:

1. Zinc metalloprotease

Hill et al. collected large quantities of a T. suis 45 kDa zinc metalloprotease from
the culture fluids of adult worms. This protease functions at an optimal pH of 7 and has
been localized to the parasite stichosome. It degrades fibrinogen and elastin, which is
indicative of its possible role in feeding and/or tissue penetration. The secreted protease
may also be associated with submucosa] connective tissue migration in the cecum and

colon of pigs, but firrther studies are needed to define its function (65).

2. Phenol oxidase

The association of phenol oxidase production with T. suis ESP was established
during the adult worm culturing process. A brown pigment was observed in female T.
suis after 1 day of culturing under aerobic conditions. Early work showed that this

enzyme is associated with the tanning (oxidative process) of the eggshell proteins or with

43

eggshell synthesis. This observation was mamly noted in the female worms, and the
firnction of this enzyme suggested that it may be needed to enhance persistence of ova in
the environment. Phenol oxidase was found to be localized anteriorly in the parasite in
the proximal part of the uterus posterior to the stichosome. It was clearly associated with
the female reproductive tract, and the enzyme activity was also triggered when the female
worm was removed from an anaerobic environment in the cecum into an aerobic
environment during the culturing process. Egg shells of the parasite also had evidence of
phenol oxidase activity and the authors postulated that this enzyme plays a crucial role in
parasite survival in the environment. They also suggested that the phenol oxidase could
be a target for control measures to reduce the risk of T. suis infection. Inhibition of
phenol oxidase activity could increase the sensitivity of the T. suis eggs and allow
unfavorable environmental conditions to prevent normal egg shell hardening thereby

positively effecting swine production facilities (47, 48).

3. Pore-forming protein

A major 47 kDa pore-fomiing protein (also referred to as TT47) has been
identified in T. trichiura ESP, the human whipworm. Likewise, its counterpart, TM43,
has been described in the T. muris mouse model. Studies have shown that the protein
induces ion-conducting pores in phospholipid bilayers. Therefore, it is hypothesized that
these proteins facilitate invasion of the host gut by T richuris thereby enabling the worm
to form a syncytial tunnel in the epithelium thus promoting survival in the host.
Although this pore-forming protein has not been identified in T. suis ESP, it is still

noteworthy to recognize the possible role it plays in T richuris infection (37, 38).

44

4. Thio] protease

A thiol protease was identified in the cultured fluids of T. suis. The optimal pH of
this protease is between 5.5 and 8.5, and its function at this time is unknown. The
secreted protease was also detected in the extracts of adult worm gut tissue implying that

the protease may be involved in nutrient digestion and absorption (67).

5. Specific diagnostic antigen

Diagnosis of T. suis infection in swine has been problematic because the
symptoms associated with trichuriasis are quite similar to other common swine
gastrointestinal disorders. However, a 20 kDa ES glycoprotein has been identified in the
cultured fluids of adult T. suis, and it is specifically diagnostic for T. suis infection in
pigs. Western blots or enzyme-linked immunoabsorbent assays (ELISA) conducted
using this antigen had no crossreactivity when conducted with sera from pigs infected
with Ascaris suum, T richinella spiralis, 0esophagostomum dentatum, or T oxoplasma
gondii. Interestingly, the antigen was also diagnostic for T. vulpis infection in dogs, but
did not crossreact with sera from dogs infected with Toxocara canis, Ancylostoma

caninum, or Strongyloides stercoralis (66).
6. Secretory serine protease inhibitor

A 6.6 kDa secreted protease inhibitor was identified in T. suis extracts and culture

fluids. Studies indicated that it has specificity for trypsin and chymotrypsin. It has been

45

suggested that the inhibitor may function in irnmunomodulation of host effector

mechanisms by inactivating immune cell proteases involved in the inflammatory process

(133).

7. Secretory chymotrypsin/elastase inhibitor
A 6.437 kDa protease inhibitor was purified from adult T. suis with specificity for
chymotrypsin, pancreatic and neutrophil elastase, chymase and cathepsin G. It too has

been suggested to function in irnmunomodulation of host effector mechanisms (134).

8. ESP antimicrobial activity

Cultured T. suis ESP fi'om adult parasites demonstrated antimicrobial activity.
Bacteria including Campylobacter jejuni, Campylobacter coli, Escherichia coli and
Staphylococcus aureus were sensitive to this activity in a dose-dependent manner. The
activity was heat stable and showed resistance to pronase E and trypsin digestion. It is
believed that the antimicrobial activity may play a role in T. suis survival during infection
in the bacteria rich environment of the colon (3). In addition to characterizing this
activity, Abner et a]. demonstrated a dose-dependent cytotoxic response in ESP-treated
cultured intestinal epithelial cells. Intestinal epithelial cells were exposed apically and
basolaterally to T. suis ESP. Both treatment routes caused the transepithelial electrical
resistance to be significantly reduced. This response suggested that T. suis ESP can
induce mechanical damage at the site of worm attachment. Also, when swine intestinal
epithelial cells were pretreated with ESP and then exposed to C. jejuni to examine the

effects of ESP on bacterial invasion, it was found that intracellular C. jejuni were

46

significantly reduced. The antimicrobial activity was believed to be partially responsible
for this response (2). Other studies also showed that T. suis ESP stimulated the
production of IL-6 and Il-10 in undifferentiated and differentiated cultured swine
intestinal epithelial cells (118). These findings suggest that T. suis ESP may play a role

in immunomodulation and development of enteropathy in the live host.

Immune Response

Recently, gastrointestinal nematode infections and the host factors involved in
immunity to nematodes have been studied more thoroughly. These studies are becoming
especially relevant since certain nematode eggs (specifically T. suis ova) are being tested
for possible use in treatment of Crohn’s disease, and clinical studies have shown that
exposure to T. suis reduces disease activity in patients with Inflammatory Bowel Disease
(IBD) (41, 102, 145). There are few reports specifically pertaining to immune responses
to T. suis. However, this area of study is evolving and more research is being conducted
using T. suis-infected swine as a model system for human trichuriasis because of the
similarities in human and pig digestive anatomy and physiology and parasite species
(119). Studies have shown that swine infected experimentally and naturally with T. suis
stimulate cytokine mRN A gene expression in local tissues of the proximal colon and in
mesenteric lymph node (MLN) cells. At 35 days post-infection, IL-10 mRNA was
significantly expressed in the MLN of the experimentally infected pigs as compared to
the control pigs, and IL-12 gene expression was undetectable. However, 1L-10 and IL- 1.2
mRNA was significantly expressed in the MLN of pigs naturally infected with T. suis

contaminated soil. This study demonstrated the ability to quantitate local cytokine gene

47

expression in mesenteric tissue using a competitive polymerase reaction (cPCR) assay
(102). Studies have also been conducted to demonstrate the development of acquired
immunity to T. suis infection in pigs. In one study, pigs were given a single challenge
infection with T. suis and groups were killed each week for 7 weeks in a time course
design. Pigs developed Th2 predominant adaptive responses as measured by mRN A
expression of cytokines in the proximal colon that resulted in expulsion of the adult
worms by 49 days after infection. These pigs also had a significant decrease in Th1
associated cytokines in these tissues. In another study, trickle inoculation was carried out
by exposing pigs to multiple small doses of T. suis eggs over an eight week period
(administering 250 infective eggs twice weekly), and during this time of observation,
acquired immunity to T. suis developed in the infected pigs. The pigs given trickle
infections in this study also displayed a noticeable decrease in worm fecundity and
reduced weight gains along with the development of a more feeble resistance level than
in pigs given a single challenge infection (119). Overall, these findings are evident of the
significant progress being made in the advancement of T. suis immunological studies in
spite of the limited reagents for investigating swine immune responses.

Swine experiments are quite expensive to conduct in laboratories and the
availability of porcine reagents are limited. Therefore, most immunological studies have
thus far been conducted in mice where T. muris is the infectious agent. The well-defined
T. muris-mouse model system has been extensively used to study trichuriasis and host
immune responses. Various studies have demonstrated that the immune responses
elicited by T. muris infection are driven by a pattern of cytokines derived from two

distinct CD4+ helper T cell subsets (42, 49, 54, 152). Generally during chronic

48

gastrointestinal helminth infections, CD4+ helper T cells are driven towards a polarized
Th2 response that results in worm expulsion. The Th2 response is associated with the
secretion of IL-4, IL-5, lL-9, IL-10, and IL-13 and the promotion of eosinophilia,
mastocytosis, and the production of IgE and IgG] which promotes resistance to nematode
infection thereby triggering worm expulsion (54). Conversely, a Th1 response has also
been shown to inhibit the protective immunity to T. muris infection, and induction of
IFN-y, IL-2, IL-12, lymphotoxin, and the production of IgG2a are characteristics of this
Th1 response that promotes worm survival. There are various factors which influence the
polarized CD4+ T cell response and the course of an infection. Studies have shown that
antigen dose, trickle infections, and co-infections play a role in determining the dominant
immune response to intestinal nematodes (15, 103, 119, 120). These findings are also
supported by the in vivo studies of Else et a]. Here, Else et a]. demonstrated parasite
expulsion in a mouse strain that was normally susceptible to T. muris by the deletion of a
key cytokine gene of the Th1 pathway. They experimentally infected interferon gamma
(IFN-y) deficient AKR mice, which are normally susceptible, with T. muris and showed
that these mice expelled the parasite proving a role for IFN-y in the establishment of
chronic helminth infection. In addition, they blocked IL-4 firnction in normally resistant
mice (BALB/ K) and inhibited IL-4 dependent-T. muris resistance to infection. However,
when IL-4 was conversely administered to the susceptible AKR mice late in the infection,
the adult worms were expelled (42). This study shows that the use of mouse strains with
different genetic backgrounds has led to the conclusion that genetic variability plays a
role in influencing the CD4+ T cell—directed immune response (42). Furthermore, there

have been similar in vivo helminth model experiments conducted which included

49

Heligmosomoides polygyrus and Nippostrongylus brasiliensis, and similar findings were
identified: 1. Prolonged parasite survival is associated with Th1 responses and 2. Host
protection is associated with Th2 responses (49, 152).

TNF-a is also involved in the mechanisms of protective immunity to T. muris
infection. Studies have shown that administering or blocking TNF-a does effect the
outcome of host protection, but has no effect on the magnitude of the Th2 responses,
thereby suggesting that TNF-a may play a role in regulating Th2 effector function (8).
Likewise, BALB/c lL-4 knockout (KO) mice were treated with anti-TNF-u mAb or rat
IgG intraperitoneally, and worm expulsion increased considerably by day 35 with 43%
worm clearance. Th2 responses were not altered (IL-5, IL-9, IL-10 and IL-13 production
remained). Prior results had indicated that blocking IL-l3 with a soluble IL-l3R fusion
protein in the absence of IL-4 completely abrogated host protection (14). Therefore these
additional findings confirmed that IL-13 plays a role in worm expulsion in the absence of
lL-4 and TNF-u. This study suggests that IL-4 is not independently promoting T. muris
resistance, but IL-13 and TNFor are required as well (8).

Further immunological studies provided the evidence that support the role of IL-
18 in susceptibility to gastrointestinal nematode infection. IL-18 is a pleiotropic cytokine
and has different roles depending on where and when it is induced. It plays a role in
negatively regulating Th2 cytokine secretion in T richuris infection. More specifically,
IL-18 plays a role in downregulating IL-l3, thereby promoting host susceptibility to T.
muris infection, and this response is independent of IL-12 and IFN-y (59).

Likewise, IL-10 is critical in the polarization of Th2 responses to T. muris

infection and survival. Mice lacking IL-10 displayed significant morbidity and mortality

50

possibly due to the overproduction of systemic IF N-y and TNF-a. It appears that IL-10 is
an irnmunoregulatory cytokine, and plays a critical role in nematode resistance (140).
However, finther evaluations are needed to determine the mechanisms involved in
possible IL-lO dependent down regulation of Th1 and Th2 responses associated with T.
trichiura infection (150). Early results in studies on immune regulation in infectious
diseases suggest that T—regulatory (T-reg) cells also produce significant amounts of IL-10
and lead to downregulation of detrimental proinflammatory responses.

It has been observed that the well-defined T. muris-mouse model system has
provided critical information towards firrther understanding the mechanisms involved in
the host immune response to T richuris infection. On the other hand, there have been very
few experiments done in humans pertaining to T. trichuris infection and host immune
responses. However, it is importance to discuss the information that is known whether it
be obtained through the results of laboratory experiments or epidemiological observation.
It has been shown that children chronically and heavily infected with T. trichiura develop
a dysentery syndrome. In the colons of these infected children, a local immediate
hypersensitivity response is elicited resulting in severe growth retardation and bloody
diarrhea with a high presence of mucus cells. IgE is secreted in response to this infection
along with significant mast cell production, but these responses have not been shown to
play a significant role in worm expulsion (34). Furthermore, a study was conducted to
observe the effects of age and infection intensity on cytokine and antibody production in
humans infected with T. trichiura. These studies indicated that the intensity of T richuris
infection was affected by age, and younger children (ages 4-7) were more heavily

infected than adults. Also, adults that were less heavily infected showed a greater IgE

51

response than infected children, thereby implying an association of significant IgE
production with the level of protection. Therefore, T richuris host resistance is age-
related. This phenomenon was also demonstrated and explained in T. suis-infected pigs,
and controlled experimental evidence was presented (12]). Here, there was positive
correlation between lgG2 production and infection intensity, but no significant
relationship demonstrated between cytokine production and age or infection intensity
(45). Unfortunately, at this time no reagents were available for measuring IgE production
in swine.

Overall, most T richuris studies have been conducted using the well established T.
mum's-mouse model system. This has really promoted advances in understanding the
mechanisms involved in clinical trichuriasis. Likewise, the T. suis-swine disease model
system has also contributed to the growing knowledge of T richuris-host interaction.
With the use of these models and other resources, chronic gastrointestinal nematode
infection will be better understood and more preventive measures can be taken to control

the outcome of these infections.

52

III. Rationale

Because of the increasing health and economical impact that food-bome diseases
are causing throughout the world, greater efforts are being executed towards elucidating
the mechanisms of pathogenesis related to the source of food-borne illnesses (96). The
human gastrointestinal tract is colonized by approximately 500 species of commensal
bacteria which can cause disease when normal anatomic or immunologic defenses are
abrogated (146). Bacteria that normally reside in cattle, swine, and poultry as
commensals that sometimes cause invasive infection in animals and humans (6, 7, 33).
There have been reports of overlaps between serotypes of C. jejuni found in humans,
poultry, and cattle which suggest that foods of animal origin may play a significant role
in transmitting C. jejuni to humans (7). C. jejuni is a food-borne pathogen that has been
identified throughout the world as one of the leading causes of human gastroenteritis (6,
7). There have been significant advances in understanding the mechanisms by which C.
jejuni causes disease, but few immunological studies have been pursued. This is
especially true of those using the naturally occurring animal model systems and cultured
primary intestinal epithelial cells. C. jejuni is commonly found in the colon of
conventionally-reared pigs without signs of pathology. However, infection and
pathology result in the presence of the swine whipworm, T. suis (103). We hypothesized
that the severe pathology observed during this interaction mimics the clinical disease of
Campylobacteriosis in swine and humans. Therefore, to address this hypothesis, 1
studied Th-l cytokine responses of primary swine intestinal cells afler single or dual

challenge infections with C. jejuni and T. suis infection.

53

Preliminary studies suggested that T. suis infection may play a significant role in
C. jejuni infection in weaning-aged swine (100, 101, 103). Additional in vivo studies
using this existing swine model contributed to the understanding of Th2 cytokine
responses following dual infection (Parathasarathy, dissertation, 2004). In addition, a
primary swine intestinal epithelial cell line was also used to observe the expression of
Th2 cytokines induced by C. jejuni and/or T. suis ESP (Parathasarathy, dissertation,
2004). Likewise, several other studies were done to observe C. jejuni colonization and
localization in the distal colon and to measure localized cytokine expression following
rectal or oral C. jejuni and Tsuis challenges (Jones, dissertation, 2005).

Hence, this existing swine disease model has provided a better understanding of
some host immunomodulatory responses triggered by C. jejuni and T. suis infections.
However, there are still unanswered questions, and the model described herein will
permit further study to characterize the immune responses that are possibly triggered by
interactions between host factors of C. jejuni and/or Tsuis which may have an effect on

the development of enteric pathogenesis.

Hypothesis

The central hypothesis of this work is that C. jejuni interaction with the intestinal
mucosa] epithelium stimulates an acute proinflammatory cytokine response. A related
hypothesis is that T. suis exposure alters this proinflammatory cytokine response thereby

promoting C. jejuni infection.

54

 

Short-term goals

1.

4.

To determine whether C. jejuni and/or T. suis infections induce a Th1
proinflammatory cytokine response in vivo

To determine if cultured swine intestinal epithelial cells secrete
proinflammatory cytokines (IL-8, TN F -a and IL-l-fl) following C. jejuni

exposure

. To determine if recombinant swine IL—8, TN F -a or IL-l-B affects C. jejuni

adherence, invasion, and/or the transepithelial electrical resistance (TER) of
undifferentiated swine intestinal epithelial cells with persistent exposure, in
vitro.

To determine if there is a significant, synergistic proinflammatory cytokine
response triggered by C. jejuni and T. suis infection

In summary, the goal of this dissertation work was to determine the

proinflammatory cytokine response (IL-8, TNF-u and IL-l-B) to C. jejuni and T.suis

infection, and determine whether IL-8, TNF-or and IL-l-B expression plays a significant

role in promoting campylobacteriosis in swine that were dually infected with C. jejuni

and Tsuis.

Chapter 2 summarizes in vivo studies on the proinflammatory cytokine responses

(IL-8, TNF-d and IL-l—B) elicited by C. jejuni and T. suis infection in swine. Four to

five-week-old pigs were orally infected with C. jejuni, T. suis embryonated eggs, or both.

There were 2 experimental challenges; they were conducted as a short-term challenge

(Day 0-Day2) or as a long-term challenge (Day 0-Day 23). Experimental human studies

have shown that pro- and anti-inflammatory cytokine levels can be detected in stools of

55

patients with Shigella infection or with inflammatory bowel disease (9, 132, 139). These
findings support the hypothesis that the host response to invasion, injury, or infection is
driven by the release of a series of cytokines. Therefore, we measured the secreted
proinflammatory cytokine levels from the feces of pigs orally infected with C. jejuni, T.
suis embryonated eggs, or both. The potential roles of these cytokines with significant
levels of expression are discussed.

Chapter 3 summarizes in vitro studies on the proinflammatory cytokine responses
(IL-8, TNF-u and IL-l-B) from swine intestinal epithelial cells infected with C. jejuni.
This chapter also summarizes the specific roles of lL-8, TNF-u and IL-l-B in C. jejuni
adherence and/or invasion of a primary swine intestinal epithelial cell line that mimics
the basal epithelial crypt cells where Campylobacter resides. Studies have shown that IL-
8, TNF-a and IL-l-B are proinflammatory cytokines secreted by cultured intestinal
epithelial cells following bacterial stimulation. Also, it is known that cytokines are likely
to filnction as a network and rarely as isolated mediators. It is suggested that during
infection, cytokine networks are likely the main controlling elements in both
inflammation and immune reactions. The cytokine networks are also believed to control
the interactions of Th1 and Th2 lymphocytes . However, in order to elucidate the
proinflammatory mechanisms of the cytokine networks, it is also relevant to understand
the isolated cytokine mediator responses due to infection. Therefore, we measured
secreted IL-8, TNF-a and lL-l-B protein from C. jejuni-infected, undifferentiated swine
intestinal epithelial cells (IPEC-l). We also pretreated these cultured primary cells with
recombinant swine IL-8, TNF-a and IL-l-B to determine whether these cytokines play a

significant role in C. jejuni adherence, invasion, and/or the transepithelial electrical

56

resistance (TER) with persistent exposure. The nature of the responses from
undifferentiated IPEC-l cells and the potential roles of IL-8, TNF-u and IL-l-B in C.
jejuni adherence and/or invasion are discussed.

Chapter 4 summarizes the previous chapters and elaborates on the proposed swine
disease model and the possible mechanisms of pathogenesis with C. jejuni and T. suis in

swine. It also covers important firture directions for this research area.

57

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72

 

Chapter 2: “T richuris suis-mediated cytokine dysregulation in concurrent
Campylobacterjejuni and T richuris suis infection”

Cunningham, L. D.; Parthasarathy. G; Jones, K.; Murphy, A. and Mansfield, L. S.;

Department of Microbiology and Molecular Genetics, Michigan State University, East

Lansing, Michigan.
Abstract

Interactions between helrninths and bacteria have been documented to induce

gastrointestinal disease in swine. It has been shown that infections with T richuris suis
and Campylobacterjejuni in swine produce a mucohemorrhagic enteritis which appears
clinically similar to human campylobacteriosis. The mechanism by which this
synergistic relationship causes disease and pathology is unclear. Cytokine dysregulation
is believed to play a significant role in this dual infection. We hypothesized that C. jejuni
interactions with the intestinal mucosa stimulates an acute proinflammatory cytokine
response, and that concurrent T. suis infection alters this pro-inflammatory cytokine
response by inducing anti-inflammatory cytokines, thereby promoting C. jejuni infection.
To test this hypothesis and determine if changes in mRN A and protein expression of pro-
and anti-inflammatory cytokines are associated with the pathological lesions observed in
this infection, we measured cytokine expression in the jejunum, proximal and distal colon
tissues and feces of infected pigs using Real-Time polymerase chain reaction (PCR)
analysis and enzyme-linked immunoabsorbent assays (ELISA). We used the following
experimental designs: (1) a short term challenge experiment (day 0—day 2) and (2) along
term challenge experiment (day O—day 23). For both experiments (short and long term
challenges), four-week-old, weaned pigs were divided into 5 treatment groups: Group 1,

C. jejuni; Group 2, C. jejuni and T. suis; Group 3, T. suis; Group 4, noninvasive E. coli

73

DH5-a; and Group 5, sterile milk (uninfected control). In the short term challenge
experiments, on day 0, fecal samples were collected from all pigs, and the separate
groups of pigs were then orally inoculated with either C. jejuni (2.0 x 108 cfu), T. suis
(2500 embryonated eggs), C. jejuni and T. suis, noninvasive E. coli DHS-u (2.0 x 108 cfir)
or sterile milk (uninfected control). Fecal samples were collected again on days 1 and 2
post-inoculation. In the long term challenge experiments, fecal samples were collected
from all pigs on day 0, and the pigs in Groups 2 and 3 were given 2500 embryonated T.
suis eggs orally while all other pigs received sterile milk. On day 21, fecal samples were
first collected. Then, pigs assigned to Groups 1 and 2 were inoculated orally with 2.0 x
108 cfu of c. jejuni, and the pigs in Group 4 were given 2.0 x 108 Chi ofE. coli DHS-u,
while the remaining pigs were given sterile milk. On day 22, fecal samples were
collected fiom all pigs. On day 23, fecal samples were taken for ELISA analysis of
cytokine expression, all pigs were euthanized, and tissue samples were taken for Real-
Time PCR analysis of cytokine expression.

Adult T. suis reside in the proximal colon and dominated immune responses in
this site. The presence of T. suis in the colon also influenced responses in the jejunum.
In the tissue samples taken on day 23 of the long term challenge experiments, T. suis
stimulated increased expression of IL-13 mRNA in the proximal colon, while expression
of IL-8, MCP-1, and IL-12 were decreased in pigs infected with T. suis alone or
concurrently with T. suis and C. jejuni. Pigs given C. jejuni alone produced few
responses here in the proximal colon. In the distal colon, increased expression of IL-l-B,
IL-6, IFN-y and IL-13 was measured in response to T. suis alone, while pigs dually

infected with T. suis and C. jejuni had no significant response similar to that seen in pigs

74

given C. jejuni alone. In the jejunum, C. jejuni infection stimulated increased expression
of MCP-l, TNF-a, IL-12p40, IFN-y, IL-4, and IL-10, while pigs infected with C. jejuni
and T. suis concurrently had no measurable proinflammatory cytokines and showed
decreased expression of GM-CSF. These data suggest that Campylobacters or
Campj.~lobacter-like bacteria stimulate a proinflammatory response mainly in the jejunum
and that T. suis down regulates proinflammatory responses in that site in concurrent
infections with C. jejuni. T. suis also down regulates proinflammatory responses in the
proximal colon, both in single infections and in concurrent infections with C. jejuni. At
48 hours after C. jejuni challenge, pigs showed few responses in the colon to this
bacteria. Conversely, T. suis dominated responses in the colon in the long term
experiments that lasted 23 days, but had little effect in the short term experiments when
pigs were sacrificed 48 hours afier challenge.

Also, we measured IL-8, TNF-a, IL-l-fi, IL-4, IL-6, and IL-10 concentrations in
the fecal supernatants of pigs in all groups in the short- and long-term experiments. IL-8,
TNF-u, IL-l-B, IL-4, IL-6, and IL-10 were detected in the feces of all pigs in both
experiments. However, IL-8, TNF-u, IL-l-B, IL-4, IL-6, and IL-10 were not
significantly affected by the administered treatments in the short term challenge
experiments. In the long term challenge experiments, neither IL-8, TNF-a, IL-4, IL-6 nor
IL-10 expression was affected in response to the treatments. However, IL- 1 -B was
significantly decreased in the pigs given T. suis alone by 23 days post-infection. In
addition, the pigs dually infected with C. jejuni and T. suis had moderately decreased IL-
l-B expression. In both experiments, we found that the pigs given C. jejuni, T. suis, or T.

suis and C. jejuni had clinical diarrhea post-infection. The pigs that were given T. suis

75

alone or T. suis and C. jejuni had more frequent, more severe diarrhea than the pigs that
received C. jejuni. Some of the C. jejuni-infected pigs had mild diarrhea. The pigs given
E. coli DHS-u or milk only showed no signs of disease.

In addition, we found that the two distinct methodologies (Real-time PCR and
ELISA for quantitating fecal cytokines) for assessing innate and adaptive responses did
not show comparable results for cytokine expression in this study. The measurements of
cytokines in fecal supernatants were less sensitive and more prone to problems with
degradation of the measured product than the RT-PCR applied to tissues. However,
despite the issues with the ELISA assay when applied to fecal supernatants, some
changes in cytokine production could be observed. Using ELISA, RT-PCR, clinical
assessment and gross and histopathology, we were able to conclude that T. suis infection
likely contributes to the down regulation of pro-inflammatory cytokines in singly or
dually-infected pigs and that T. suis is mame responsible for the frequent, severe
diarrhea observed in pigs infected concurrently with C. jejuni and T. suis or T. suis only.
The overall findings suggest that T. suis interaction with the intestinal mucosa is likely
mediating the cytokine dysregulation observed and may be playing a role in promoting a
more conducive environment for secondary infection by Campylobacter species, other
related Campylobacter-like bacteria, or other opportunistic enteric bacteria during a

natural, concurrent infection.

76

Introduction

Throughout the world Campylobacterjejuni is known to be one of the leading
bacterial causes of diarrhea] disease in humans (6). The enterocolitis caused by this
infection commonly occurs following the consumption of improperly cooked pork,
poultry or beef; unpasteurized cow’s milk; or contaminated water (6, 72). C. jejuni, C.
coli, and C. lari can be found as comrnensals in the intestinal tracts of animals and thus
can serve as sources of food contamination (6, 7, 26, 72). Recent studies have shown that
there is a significant incidence of C. jejuni in the intestinal tract of pigs raised on
integrated commercial hog farms. The mean incidence of C. jejuni was 76.8% in gilts,
87% in pregnant sows, and 31.7% in newborn piglets (78). This finding suggests that
pigs are significant reservoirs of C. jejuni.

By 1991, approximately 45% of the US swine farms were infected with T. suis, a
whipworm intestinal nematode primarily found in the cecum and proximal colon (Bliss,
1991). Manifestations associated with infection include weight loss, dysentery, anemia,
and anorexia (18). Typically, pigs with light T. suis infections present mild clinical signs
and can be treated with anthelminthics to induce nematode expulsion (Jones et al.,
unpublished data). However, pigs with severe natural infections often die (24). These
conditions contribute to economic losses in the swine industry. Reports from 1991
indicated that pork producers directly and indirectly lost approximately $1 billion dollars
annually as a result of swine internal parasites (24). It was suspected that much of the
pathology associated with this T. suis infection was due to syncytial ttmnels created by

larvae burrowing into the intestinal epithelium, which contributed to breakdown of the

77

epithelial barrier and secondary invasion by intestinal microflora, thereby amplifying the
severity of the clinical signs (18). Later findings supported this hypothesis by
demonstrating a synergism between T. suis and the microbial flora of the large intestine,
causing dysentery in pigs (65). This phenomenon has greater significance because it is
also known that the human whipworm, T. trichiura, affects an estimated one billion
people globally, causing chronic disease (73).

Since pigs are similar to humans with respect to digestive anatomy and
physiology, they can be used as a suitable animal model for studying bacterial and
helminth infections that cause disease in humans. There have been several attempts to
develop an animal model suitable for the study of immunological responses due to
Campylobacter infection. Unfortunately, some of these models have proven less suitable
for mimicking human campylobacteriosis. Some of these reported models using ferrets,
rabbits, hamsters, dogs, chickens, and mice were not suitable because the investigators
modified the gut flora in order to facilitate C. jejuni infection. Not only are some of these
animal models altered so that they do not reflect natural infection, they are also expensive
and lack anatomical and physiological similarities to the human colon (5, 12, 21, 43, 62,
65, 73). Unlike other studies, the pig C. jejuni/ T. suis model system established by
Mansfield and colleagues reliably reproduced the clinical symptoms associated with
human Campylobacter infection (50). They have confirmed through several studies that
C. jejuni is commonly found in the colon of conventionally reared pigs without signs of
pathology. However, in the presence of T. suis, infection and pathology result (49-51).
With the increasing awareness of concurrent bacterial/nematode interactions, the

identification and characterization of the mechanisms by which these enteric pathogens

78

concurrently cause disease will be beneficial from both animal and human health
perspectives. Studies will enable researchers to address such questions as why some
animals or individuals develop disease due to such synergisms and others do not. For
example, in one case, a recent study has shown that concurrent enteric helminth infection
in mice can reduce helicobacter-induced gastric atrophy (34). Other investigators are
also now suggesting that T. suis is a safe and effective treatment of inflammatory bowel
disease, ulcerative colitis, and Crohn’s disease (69, 70).

The mechanisms by which C. jejuni causes disease in humans are not clearly
understood. However, it is believed that host cytokines play a pivotal role in eliciting
immune responses during enteric infection. A localized acute inflammatory response is
typically associated with Campylobacter enterocolitis and is believed to play an
important role in the clinical symptoms elicited by enteric invasion, injury, or infection.
Bacterial-host cell interactions have been studied using multiple well-established in vitro
models, and various bacterial and host cell components have been identified as playing
significant roles during infection (38, 39, 53). However, the in vitro model systems do
not completely mimic the true physiologic conditions and the responses seen in animal
model systems or, more importantly, mimic the clinical manifestations associated with
human campylobacteriosis. Therefore, at this time it is necessary to use, both a cultured
intestinal epithelial cell model and an animal model for more thorough studies of the
overall effects of cytokine regulation in this disease.

Experimental human studies have shown that pro- and anti-inflammatory cytokine
levels can be detected in stools of patients with inflammatory bowel disease (57, 66). It

has also been shown that significant levels of proinflammatory cytokines (TNF-or, IL-1-

79

B, IL-lra, IL-6, IL-8, and GM-CSF) have been detected in stool samples from patients
with Shigella infection (57, 63). The findings of this Shigella study showed that the
correlation of cytokine levels with severity of disease was significant; cytokine levels
were higher in the stools of patients with severe disease as compared to the stools of
patients with mild disease. These findings showed that an array of cytokines, particularly
pro-inflammatory cytokines, play a role in initiating and amplifying acute mucosal
inflammatory response following epithelial barrier invasion, injury, or infection. Hence,
these reports showed that measurement of stool cytokines can be a reliable and
noninvasive means of understanding the disease status in the intestine (66).

We hypothesized that C. jejuni infection elicits an innate immune response by up-
regulating pro-inflammatory cytokines in weaned, conventionally-reared pigs, but during
concurrent T. suis infection, anti-inflammatory cytokines are triggered that can down-
regulate pro-inflammatory cytokines, thereby promoting C. jejuni infection. Therefore,
the aim of this study was to detect and quantify pro- and anti-inflammatory cytokine
mRNA levels in intestinal tissue samples and protein levels in the feces of pigs infected
with C. jejuni and T. suis and to determine if the two cytokine analyses (ELISA on fecal
supernatants and RT-PCR on tissues) were comparable and useful in correlating cytokine
dysregulation with disease. We also documented clinical signs such as the occurrence of
diarrhea during the infection as it related to the severity of disease. This study was
designed based on previous evidence suggesting a synergistic effect involved in these
infections and the differential development of pathology induced by C. jejuni and T. suis
in the colon of naive, germ-free pigs (49, 50). The mechanism by which this concurrent

infection causes disease similar to human campylobacteriosis is unknown. However, we

80

do know that T. suis causes direct mechanical damage by invading the basal crypt
epithelial cells of the proximal colon and that C. jejuni invades through the epithelium
and proliferates in the lamina propria and submucosa surrounding the site of worm
attachment (50). It is believed that these modes of action play a significant role in the
infection and observed disease in these dual infections. Also, it is suspected that
cytokine-mediated damage triggered when these pathogens breach the epithelium may
also play a significant role in the pathology associated with these infections. Therefore,
this investigation was important in determining the role of pro- and anti-inflammatory

cytokine responses in concurrent infections with C. jejuni and T. suis in pigs.

81

Materials and Methods
Animals

In this study, the pigs were divided into 5 treatment groups (Table 2.1). In two
replicate experiments each, there were 4 or 6 pigs per treatment group in the short term
challenge experiments (2 days) and 5 or 6 pigs per treatment group in the long term
challenge experiments (23 days). The four-week-old, weaned, conventionally-reared
Duroc/Yorkshire/Landrace piglets were obtained from the Michigan State University
Swine Farm. They were transported to the University Research Containment Facility in
sterile dog cages and housed in 5 adjoining rooms in identical large isolation units to
reduce cross contamination. Feed and water were provided to the pigs ad libitum, and
they were handled under identical management conditions in accordance with university
and National Institutes of Health guidelines for humane animal use; protocol number

06/01-096-00.

Bacterial strains, growth conditions, and experimental inoculum preparation for
swine challenge

C. jejuni 33292 was grown at 37°C, 5% C02 on Bolton agar (Oxoid Inc.,
Basingstoke, Hampshire, England) plates containing 5% sheep’s blood. E. coli DHS-oc
was grown on Bolton agar plates without sheep’s blood at 37°C. The bacteria] were
grown for 18—20 hours, harvested with RPMI 1640 medium (Sigma Chemical Co., St.
Louis, MO) and suspended in sterile milk at a final concentration of

1 x 108 cfu/ml.

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Bacterial strains and growth conditions for enzyme-linked immunosorbant assay
(ELISA) and 16S rRNA PCR analysis

For ELISA, C. jejuni NCTC 11168, C. lari, C. coli, C. upsaliensis, C.
hyointestinalis, and Arcobacter butzleri were grown on Tryptose soy agar (TSA)(Oxoid
Inc., Ontario, Canada) plates with 5% sheep’s blood at 37° C in Gas Pak jars with a
CampyGen pack for 48 hours. Helicobacter hepaticus was grown on Tryptose soy agar
(TSA)(Oxoid Inc., Ontario, Cananda) plates with 5% sheep’s blood for 5 days in a Gas
Pak jar without catalyst at 37° C evacuated to -20 mmHg and then filled with a gas
mixture, N2, H2, and CO2 (80: 10:10). The bacteria were harvested fi‘om agar plates by
resuspending the organisms in sterile 10 mM phosphate buffered saline (PBS). As
described below, protein was extracted from the bacterial suspensions using the Pierce
(Rockford, IL) B-PER® midi-scale protein extraction protocol according to the
manufacturer’s instructions and later used in the ELISA.

For 16S rRNA PCR analysis, Campylobacter DNA extraction was performed as
described by Ausubel et al (8). The Helicobacter hepaticus DNA was kindly provided
by Dr. Vincent Young at the Food Safety and Toxicology Center at Michigan State

University, East Lansing, MI.

Embryonated T. suis eggs inoculum

Adult whipworms were isolated fi'om the colonic mucosa of experimentally
infected pigs, and embryonated eggs were collected and prepared as described
previously(40). T. suis-infected pigs were anesthetized via intramuscular injection of 4.4
mg/kg Telazole/2.2 mg/kg xylazine (MSU Veterinary Pharmacy, East Lansing, MI) and

euthanized with an intravenous injection of 86 mg/kg sodium pentobarbital (Fatal Plus®,

83

Vortech Pharmaceuticals, Dearborn, MI). Adult worms were harvested from the
proximal colon using forceps and cultured at 37°C, 5% CO2 for approximately 10 days or
until the worms showed no sign of movement. Throughout the culturing process,
medium with eggs was collected and washed by centrifugation 2 or 3 times at 10,000 rpm
for 5 minutes to remove excess debris. The T. suis eggs were suspended in sterile MilliQ
water, placed in a vented tissue culture flask, and incubated at 34°C, 5% CO2 for nineteen
days to induce embryonation. On day 19, porcine bile extract (Sigma Chemical Co., St.
Louis, MO) was added to the flask at 10 ug/ml and incubation was continued for an
additional 3 to 5 days. Each day the eggs were examined microscopically for the
maturation of the L1 stage nematode, which was detected as movement of the larva

inside the egg. The development and movement of the worm indicated that
embryonation had taken place. The embryonated eggs were collected and centrifuged at
10,000 rpm for 10 minutes, resuspended in sterile water in tissue culture flasks, and

stored at 4°C in the dark until inoculation.

Animal inoculation

All pigs were inoculated with treatments as indicated in Table 2.1 by oral gavage
using a sterile 12 gauge steel ball feeding needle placed over the back of the tongue.
Prior to the experimental challenge, at each time point, and immediately prior to
euthanasia, all pigs were rectally swabbed to test for the presence of C. jejuni. The rectal
swabs were transported in Cary Blair medium (Difco Laboratories, Detroit, M1) on ice to
the laboratory for Campylobacter isolation and identification. Also, at each time point,

some of the collected fecal material was microscopically examined to detect the presence

84

of T. suis eggs. All inocula were prepared using nonfat dry milk reconstituted in sterile
MilliQ water. T. suis embryonated eggs were examined microscopically and enumerated
using a hemacytometer. The inoculum for the pigs that received T. suis was 2500
embryonated eggs suspended in sterile milk in a total volume of 2.0 ml. Eighteen to
twenty hour cultures of C. jejuni 33292 or E. coli DH5-awere harvested fiom plates with
RPMI 1640 and suspended in sterile milk at a final concentration of 1 x 108 cfu/ml. The

pigs that received C. jejuni 33292 or E. coli DH5-a were inoculated with 2.0 x 108 cfir of

bacteria in a total volume of 2.0 ml. Control pigs were inoculated with 2.0 ml of sterile

milk.

Experimental design

These experiments were conducted as a short term challenge (2 days) or a long
term challenge (23 days). For each of the experiments, the pigs were divided into 5
treatment groups for inoculation (Table 2.1): Group 1, C. jejuni; Group 2, C. jejuni and
T. suis; Group 3, T. suis; Group 4, E. coli DHS-a; and Group 5, uninfected milk control.
At the end of each experiment, all pigs were euthanized with an intravenous injection of
86 mg/kg sodium pentobarbital (Fatal Plus®, Vortech Pharmaceuticals, Dearborn, M1) on
day 2 or 23 following fecal sampling. Following euthanasia on day 2 (from 2nd repetition
of short term challenge only) or day 23, blood and plasma samples were obtained fiom all
pigs and stored at -80°C until assayed. Also, following euthanasia on day 23 of the long
term challenge only, tissue samples were taken fi'om the jejunum, proximal colon, and

distal colon of all the pigs and stored at -80°C until assayed.

85

Short term challenge (2 days)

Four pigs per group were used in the first experiment, and 6 pigs per group were
used when the experiment was repeated for a total of 10 pigs per group. On day 0, prior
to inoculation, feces were collected from all animals. Next, the pigs in each group were
inoculated with 2.0 ml of the designated treatment. At 24 and 48 hours post challenge,
fecal samples were collected fiom all animals. All pigs were euthanized at 48 hours post-
challenge following fecal sampling. Blood samples were obtained after the pigs were
euthanized to measure total anti-Campylobacter IgG in the plasma (samples taken only
from the 2nd repetition of experiment). Table 2.2 illustrates the inoculation and fecal

sampling protocol.

Long term challenge experiment (23 days)

Five pigs were used in the first experiment, and 6 pigs per group were used when
the experiment was repeated for a total of 11 pigs per group. On day 0, prior to
inoculation, feces were collected from all experimental animals. Next, each of the pigs in
group 2 and 3 received 2500 T. suis embryonated eggs, and the pigs in groups 1, 4, and 5
were given sterile milk. On day 21 post-inoculation, fecal samples were collected from
all animals. Then, each of the pigs in groups 1 and 2 was inoculated with 2 x 108 cfu C.
jejuni, and the pigs in group 4 were given 2 X 108 cfu E. coli DH5-a. The pigs in group 5
received sterile milk. On days 22 and 23, feces were collected from all experimental
animals. All pigs were euthanized on day 23 following fecal sampling, and tissue
samples were collected as described below from the jejunum, proximal colon, and distal

colon of all the pigs. Blood samples were also obtained after the pigs were euthanized to

86

measure total anti-Campylobacter IgG in the plasma. Table 2.3 illustrates the inoculation

and fecal and tissue sampling protocol.

Sample collection

All fecal samples were collected in sterile 25 ml polystyrene tubes and placed on
ice to be transported to the laboratory for processing using the fecal extraction procedure
described below; fecal samples were stored at -80°C until analysis. Blood was collected
by venipuncture into sterile red top tubes, and serum was removed and stored at 4°C and
centrifuged; plasma was stored at -80°C until analysis.

“To eliminate cross contamination, a separate set of sterile dissection instruments
was used for each group. For sample collection, the abdomen was opened by a midline
incision and the small and large intestines were exteriorized. Full thickness samples of
the jejunum, proximal colon, and distal colon were collected using sterile scissors and
forceps. One set of samples was fixed in 3.7% formaldehyde for histology. A second set
was placed in cryovials and snap frozen in liquid nitrogen for nucleic acid isolation.”

(Jones, dissertation, 2005, reprinted with permission)

Isolation and identification of C. jejuni from feces and tissue samples

Several assays were used to determine if pigs were free of C. jejuni prior to
inoculation and to monitor the presence of the inoculated strain post-inoculat ion. The
isolation and identification of C. jejuni from feces was conducted using previously
described methods (31). Briefly, prior to inoculation and immediately prior to

euthanasia, all pigs were tested for the presence of C. jejuni by culture of feces from

87

rectal swabs. The rectal swabs were streaked onto Preston selective agar (Oxoid Inc.,
Basingstoke, Hampshire, England) and incubated at 42°C, 5% CO2 in humidified air for
48 hours. Isolated colonies were tested by PCR using primers specific for the quinolone
resistance-determining region (QRDR) of the gyrA gene of C. jejuni (75).

"Tissue samples were also taken from each segment of the intestine of each pig to
test for the presence of Campylobacter organisms. Total DNA was isolated from the
tissue using a Qiagen DNEasy tissue kit (Qiagen, Valencia, CA) following the
manufacturer’s protocol. The DNA was quantified spectrophotometrically (Beckman
model DU530, Fullerton, CA), and PCR was used to amplify the C. jejuni QRDR target
from 500 ng of the total DNA using primers JL238 and JL239 (75). Electrophoresis,
ethidium bromide staining, and UV illumination were used to visualize the results as
previously described. A Restriction Fragment Length Polymorphism (RFLP) assay
developed to detect and distinguish C. jejuni, C. coli, C. lari and C. upsaliensis by
analyzing a portion of the 23S rRNA gene was used to identify thermophilic
Campylobacters in the animals (31). The target DNA was amplified using primers
THERMI and THERM4. Electrophoresis, ethidium bromide staining, and UV
illumination were used to visualize the results as previously described” (31)(Jones,

dissertation, 2005, reprinted with permission).

ELISA for measuring total IgG in pig plasma
We developed an indirect ELISA protocol to measure total IgG in the plasma of
the pigs to determine if there were cross-reacting antibodies against the challenge-dose C.

jejuni or C. jejuni-related bacteria (epsilon proteobacteria). The C. jejuni antigen was

88

prepared using the Pierce (Rockford, IL) B-PER® midi-scale protein extraction protocol
as given by the manufacturer, and the Bradford assay was used to determine and optimize
the protein concentration. The antigen concentration was adjusted by diluting the antigen
1:5000 to a final concentration of 1.9 ug/ml in 10 mM phosphate buffered saline (PBS),
pH 7.4. 96-well Nunc MaxiSorp plates (Fisher Scientific, Hanover Park, IL) were coated
with the antigen/coating solution at 100 ul/well and stored overnight at 4°C. Following
overnight incubation, the antigen/coating solution was removed, and blocking buffer (3%
BSA solution in PBS (10 mM, pH 7.4) containing 0.05% Tween 20) was added to the
plates at 200 ul/well and incubated overnight at 4°C. Plates were washed 4 times with
wash buffer containing 100 mM PBS, pH7.3, 0.05% Tween-20. The pig plasma samples
served as the primary antibody. All plasma was diluted 1:10 in blocking buffer, added to
the plates at 100 ul/well, and incubated at room temperature for 1 hour. Plates were
washed 4 times with wash buffer containing 100 mM PBS, pH7.3, 0.05% Tween-20.

The conjugate antibody (Biotin-SP-conjugated AffmiPure F (ab’)2 Fragment Goat Anti-
Swine IgG (H+L); Jackson ImmunoResearch Labs, Inc., West Grove, PA) was diluted
1:20,000 in blocking buffer and used as a secondary antibody at 100 ul/well and
incubated at room temperature for 1 hour. Plates were washed 4 times with wash buffer
containing 100 mM PBS, pH7.3, 0.05% Tween-20. The optimal titers for the primary
and secondary antibodies were determined fi'om preliminary experiments. The color
reaction was produced using extravidin peroxidase (Sigma, St. Louis, MO) diluted
1:2000 in blocking buffer and incubated for 1 hour at room temperature at 100 ul/well.
Plates were washed 4 times with wash buffer containing 100 mM PBS, pH7.3, 0.05%

Tween-20. TMB substrate (Sigma, St. Louis, MO) was warmed to room temperature,

89

added to plates (100 ule) and incubated at room temperature for ~10 minutes or until
color changed. Immediately, the color reaction was stopped by adding 100 ul/well of 2N
H2SO4. A microplate reader (EL800 Universal Microplate Reader, Bio-Tek instruments,
Winooski, Vermont) was used to measure the absorbance at 450 nm, and the total IgG in
each sample was quantified using KCjunior® software (Bio-Tek instruments, Winooski,
Vermont).

The ELISA for measuring total IgG in pig plasma was repeated as described
above using protein extracted from C. jejuni and various related bacteria (C. lari, C. coli,
C. upsaliensis, C. hyointestinalis, Arcobacter butzleri, and Helicobacter hepaticus). The
extracted protein from these bacteria was used as bacteria] antigen in this assay to
determine if the pigs were exposed to C. jejuni-related bacteria prior to the experiments.
The plasma samples tested were taken from 8 experimentally C. jejuni-infected pigs
(randomly chosen from the short and long term challenge experiments) to represent the
known positive control. Plasma samples from two newborn piglets (< 6 hours old) fi'om
the MSU swine farm were used as negative controls. We also used plasma samples from
6 control pigs that were given milk only throughout the challenge experiments; these pig
plasma samples were also randomly chosen from the short and long term challenge

experiments.

Identification of Campylobacters and Helicobacters by 16S rRNA PCR amplification
Species-specific identification of Campylobacters and Helicobacters using PCR
assays have been described previously (47, 64). The PCR assay for identifying

Campylobacter species is described as follows: all reactions were performed in a total

90

volume of 25 ul containing 25 ng genomic DNA; 20 mM Tris-HCL (pH 8.3); 50 mM
KCl; 2.5 mM MgCl2; 200 uM (each) dATP, dCTP, dGTP and dTTP; 0.4 uM of each
primer; and 0.625 units of T aq DNA polymerase. DNA was amplified in a thermocycler
for 25 cycles. The following parameters were used: denaturation, 94°C, 1 min;
annealing, 55-65°C depending on the primer pair, 1 min; extension, 72°C, 1 min. The
PCR products were separated by electrophoresis on agarose gels stained with ethidium
bromide and visualized by UV light (47). The PCR assay for identifying Helicobacter
species is described as follows: all reactions were performed in a total volume of 50 u]
containing: 1.25 pg of template DNA (unless otherwise stated), 3.0 units of T aq
polymerase, 10 mM Tris-HCL (pH 8.3); 50 mM KCI; 1.5 mM MgCl2; and 1 11M of each
oligonucleotide primer. The time and temperature parameters were as follows: samples
were heated at 94°C for 5 min, and the DNA was amplified for 35 cycles under the
following conditions: denaturation, 94°C, 2 sec; annealing, 60°C, 2 sec; primer
extension, 72°C, 30 sec. The PCR products were separated by electrophoresis on agarose

gels stained with ethidium bromide and visualized by UV light (64).

Histology

“Tissues were examined microscopically to detect T. suis larvae, to evaluate
histological changes, and to localize specifically stained Campylobacter organisms. Fixed
tissue samples were embedded in paraffin and 5 11m sections were cut and adhered to
charged slides. Slides were routinely dehydrated in progressively higher concentrations

of ethanol, deparaffmized and rehydrated. For histological evaluation, slides were stained

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with Mayer's hematoxylin and eosin and cover slipped.“ (Jones, dissertation, 2005,

reprinted with permission).

Real-time PCR

“ T richuris suis resides in the cecum and proximal colons of pig (19), while C.
jejuni can colonize throughout the small and large intestine of pigs (10, 50). Therefore, to
determine the effect of infection with T. suis, C. jejuni, or both on local immune
responses in areas where these pathogens are present, we took samples from the jejunum,
proximal colon, and distal colon. mRN A expression of 15 cytokines, chemokines, and
iNOS was measured from each tissue using a real time PCR assay (29). Total RNA was
isolated from frozen tissue samples from each pig using Trizol reagent (GibcoBRL Life
Technologies). Briefly, 3mm3 tissue samples were homogenized in Trizol using a
Polytron® tissue homogenizer (Kinematica, Cincinnati, OH). RNA was extracted with
chloroform, precipitated with isopropanol, and washed with ethanol. Pellets were air
dried, then resuspended in DEPC treated H20 (GibcoBRL Life Technologies). DNA
contamination was removed by treatment with approximately 27 Kunitz units of DNase I
for 15 minutes. Samples were repurified on a Qiagen RN Easy column following the
manufacturer’s protocol (Qiagen, Valencia, CA). RNA concentration and integrity were
determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
For each sample, 10 ug of total RNA was reverse transcribed with random primers using
a Stratagene First Strand RT-PCR kit (Stratagene, La Jolla, CA). First strand cDNA was
used as the template in real time PCR reactions. Real time PCR primers and probes

designed using the Primer Express (Applied Biosystems, Foster City, CA) software

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package was used to analyze 14 cytokines (Chapter 2, Table 1). The 18S rRNA probe
was obtained from Applied Biosystems and was used as a constitutively active gene for
normalization. PCR was performed using a commercially available kit (Brilliant kit,
Stratagene, La Jolla, CA) on an ABI PRISM 7900HT (Applied Biosystems).
Amplification conditions were as follows: 50°C for 2 minutes; 95°C for 10 minutes; 40
cycles of 95°C 15 seconds and 60°C for 1 minute. Fluorescence signals measured during
amplification were processed post-amplification. Threshold cycle (Q) values were
determined using values of 0.2 for TET, 0.1 for VIC, and 0.05 for FAM reporter dyes”

(Jones, dissertation, 2005, reprinted with permission).

ELISA for fecal cytokine analysis

IL-8, IL-l-B, TNF-or, IL-4, and IL-10 were measured in the supernatants of the
fecal samples using swine sandwich ELISA kits according to the manufacturer’s
instructions (BioSource International, Inc., Camarillo, CA). Indirect ELISA was
performed to measure secreted IL-6. The swine IL-6 cytokine and primary anti-swine IL-
6 antibody (rabbit polyclonal, IgG isotype) were purchased from BioSource International,
Camarillo, CA. The secondary biotinylated antibody (anti-rabbit IgG (whole molecule)),
Bovine Serum Albumin (BSA), extravidin peroxidase, and tetramethyl benzidine (TMB)
were purchased from Sigma Chemical Co., St. Louis, MO. The fecal supernatants were
used to coat the 96-well plates and stored overnight at 4°C. Each plate also contained IL-
6 protein standard controls (in duplicate), at 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0
ng/ml, in RPMI 1640 without phenol red and PBS. Following the overnight incubation,

supernatants were discarded and the plates were blocked with 3% BSA solution (in

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Phosphate Buffered Saline (PBS, 0.01 M) containing 0.1% Tween 20) and incubated for
90 minutes at 37°C. Primary rabbit anti-swine IL-6 antibody (1/100 titer) was used to
capture the IL-6 protein (3 7°C, 90 minutes) and a goat anti-rabbit antibody conjugated
with biotin (1/5000) was used as a secondary antibody at 37°C, 90 minutes). The optimal
titers for the primary and secondary antibodies were determined fiom preliminary
experiments (data not shown). The color reaction was produced using extravidin
peroxidase (l ug/ml, 45 minutes at room temperature) and a ready-to-use peroxide-
containing TMB substrate (30 minutes, room temperature). The color reaction was
stopped with 6N H2SO4. Plates were washed after each step with PBS containing 0.1%
Tween-20. A microplate reader (EL800 Universal Microplate Reader, Bio-Tek
instruments, Winooski, Vermont) was used to measure the absorbance at 450 nm, and the
concentration of IL-6 protein in each sample was determined using KCjunior® software

(Bio-Tek instruments, Winooski, Vermont).

Fecal extraction procedure for cytokine measurement

The fecal extraction procedure for cytokine measurement has been described
previously (63). The feces were collected fiom the anus of pigs into sterile 25 ml
polystryrene tubes. Five to ten grams of the fecal sample was diluted 1:3 in sterile
phosphate buffered saline (PBS, pH 7.2) containing trypsin inhibitor from soybeans (1
mg/ml) and phenylrnethylsulfonyl fluoride (PMSF, 1 mg/ml) and centrifilged at 16,500
rpm at 4°C for 15—30 minutes. Supematants were collected and stored at -80 °C until
further use in ELISA. The PBS was obtained from Gibco BRL, Rockville, MD. The

trypsin inhibitor and PMSF were obtained from Sigma Chemical Co., St. Louis, MO.

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Fecal cytokine recovery

In order to control for breakdown of cytokines in the fecal material, a cytokine
recovery assay was performed. This was done by spiking the fecal samples fi’om
uninfected pigs with known quantities of commercial recombinant cytokine before
mixing the samples in the fecal extraction procedure as discussed above. The cytokines
were added to the fecal contents at the following concentrations: 50 pg/ml, 100 pg/ml,
250 pg/ml, 500 pg/ml, and 1000 pg/ml of IL-8, TNF-a, IL-1-|3, IL-4, IL-6, or IL-10. The
fecal supernatants were tested by ELISA to estimate the recovery rates of the cytokines

added.

Statistical analysis

“The cytokine mRN A expression data for each tissue was analyzed separately
using Statview 5.0 for Macintosh (Abacus Concepts, Berkeley, Calif). The C, value for
the 18S ribosomal subunit was subtracted from the C. value for each cytokine message to
normalize for RNA content prior to statistical analysis. One-way analysis of variance
(ANOVA) was performed to determine the statistically significant differences between
treatments. A Fisher’s least squares difference test was performed to determine
differences between infected and control animals at each time point. P values of 0.05 or
less were considered statistically significant” (Jones, dissertation, 2005, reprinted with
permission).

The total IgG ELISA data were analyzed using descriptive statistic routines in

Windows Excel.

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The cytokine protein expression data were analyzed using Statistical Analysis
Software (SAS) version 9.1 (SAS Institute, Inc., Cary, NC). Statistical analysis was
performed using the mixed model analysis with repeated measurements. The fixed
effects in this model were the: experiment, treatment, day, and all of the possible
interactions between these 3 fixed factors. The random effect in this model was the
individual animal. The short (2 days) and long (23 days) term challenge experiments
were each conducted twice. The mixed model analysis allowed the experimental data
from the two short term challenge experiments to be combined for analysis and likewise
for the two long term challenge experiments. The data could be combined because the
main effect of the interaction term with the experiment was insignificant (P > 0.05).
Cytokine levels are expressed as mean (in picograms per milliliter) i standard error of the

mean (SEM). Differences were considered statistically significant if P _<_ 0.05.

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Results
C. jejuni isolation and detection

We were unable to isolate Campylobacterjejuni from the fecal cultures of any
pigs before or after infection (Table 2.4). To enhance sensitivity of detection, we used
PCR assays to determine the presence of C. jejuni in the tissue samples taken separately
from the jejunum, proximal colon, and distal colon. Polymerase chain reaction (PCR)
using specific primers for the QRDR of the gyrA gene of C. jejuni did not detect C. jejuni
in any tissue samples from any pig group, except in 1 out of 6 pigs in the T. suis-infected
group (Table 2.4). However, Campylobacter 23S rRNA RF LP analysis suggested that C.
jejuni, C. coli, and an unidentified Campylobacter species that gave a unique RFLP
pattern were present in our animals (Figure 2.1). These findings in pigs handled
rigourously as specific pathogen free by various methods lead us to firrther explore these

results.

Immunologic recognition of C. jejuni

To confirm C. jejuni infection in experimentally inoculated pigs and to
discriminate between C. jejuni and related epsilon proteobacter infection in preinoculated
and control pigs, we pursued ELISA measurements of bacteria specific IgG. Figures 2.2
A and B show that IgG antibodies in the plasma of all the pigs in each treatment group
from the 2nd repetition of the short term challenge experiments (days 0, l and 2) reacted
with C. jejuni antigen (samples were not available item the first independent
experiment). IgG antibodies in the plasma of most pigs in the treatment groups fiom the

long term (days 0, 2], 22 and 23) challenge experiments (from 2 independent

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experiments) also reacted with C. jejuni antigen. The ELISA was conducted using the
plasma samples collected 2 days post C. jejuni inoculation on day 2 (short term
challenge) and day 23 (long term challenge) of the experiments. Given that we detected
high levels of anti-Campylobacter IgG antibodies in the plasma of our pigs and that it has
been shown that the induction of an I gG response requires more than 2 days post-
Campylobacter infection (about 8—14 days)(23), our data suggest that the pigs had
established humoral immune recognition of C. jejuni before being experimentally
infected. Therefore, we also wanted to distinguish whether these IgG antibodies
measured in the pig plasma were C. jejuni specific or resulted fi'om cross-reactions with
other Campylobacter spp. or other environmental Campylobacter-like bacterial antigens.
After randomly selecting plasma samples from some of the pigs in the short (days 0, 1
and 2) (2nd repetition only) and long term (days 0, 21, 22 and 23; from 2 independent
experiments) challenge experiments, we found that IgG antibodies in the plasma of 8 pigs
randomly chosen fiom the C. jejuni-infected groups (positive control) and 6 pigs
randomly chosen from the pig groups that received milk alone reacted with C. jejuni, C.
lari, C. coli, C. upsaliensis, C. hyointestinalis, A. butzleri, and H. hepaticus antigen by
ELISA (Figure 2.3). Conversely, plasma samples taken fiom 2 newborn piglets

(< 6 hours old) at the MSU swine farm (negative control) did not elicit any IgG response
against Campylobacter spp., A. butzleri, and H. hepaticus antigen. In these studies of our
experimental pigs, the greatest IgG response was directed against H. hepaticus antigen (3
related epsilon proteobacteria), suggesting that these bacteria or closely related

Helicobacters were present in reasonably high numbers in the pigs prior to inoculation

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with C. jejuni. Once again, these results stimulated further studies to distinguish whether

the pigs carried a Helicobacter spp.

Recognition of Campylobacter spp. and Helicobacter spp. in feces

In order to confirm the previous ELISA findings, we performed subsequent
species-specific PCR assays to identify Campylobacter spp. or Helicobacter spp., which
could have possibly had an effect on the cytokine immune response of the pigs. We
conducted Campylobacter 16S rRNA PCR analysis and found that Campylobacter
species were undetectable in the fecal samples fiom any pig group before or after
bacterial infection (Tables 2.5 and 2.6). However, after Helicobacter 16S rRNA PCR
analysis, we found that Helicobacter DNA could be detected in the feces of some of the
pigs from the uninfected control group, suggesting that they were likely exposed to
Helicobacter species (Campylobacter-like bacteria) prior to any experimental exposure to
C. jejuni (Tables 2.5 and 2.6). The presence of these closely related bacteria likely
contributed to the cross-reacting IgG antibodies detected in the plasma of the pigs by
ELISA. The IgG cross-reacting antibodies may have contributed to the immune response
against the experimentally-administered C. jejuni, thereby having an effect on the
outcome of the cytokine responses elicited throughout the study. In the pig, Helicobacter
spp. are found mame in the stomach and small intestines of swine so larger effects are

expected in these compartments.

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Histopathology

“Haemotoxylin and Eosin (H&E) stained sections of jejunum, proximal colon,
and distal colon of each pig were examined microscopically for the presence of T. suis
larvae and histological changes due to infection. T. suis larvae were seen in proximal
colon sections from 5 of 6 pigs in group 3, and 5 of 6 pigs in group 4. No larvae were
seen in groups 1 or 2 (Figure 2.4).

Control animals (Group 1) had no proliferative, inflammatory, or degenerative
lesions in any of the tissues examined and were therefore used as a normal comparison
for all infection groups. All lesions were confined to the colon. Colonic lesions due to C.
jejuni or T. suis infection were similar to those described in germ free piglets (50). Pigs
in group 2 (C. jejuni only) developed focal mild crypt distension with sloughed epithelial
cells, mucus and bacteria in the crypts in the distal colon (Figure 2.5A) and LGCs (Figure
2.6A). There was no significant inflammatory infiltrate into the lamina propria on day 23
after inoculation. Pigs infected with T. suis only developed mild to moderate colitis with
goblet cell hyperplasia and crypt distension with mucus in superficial lamina propria.
Lesions were found only in the areas where T. suis larvae were located. A mixed
inflammatory infiltrate was present in the lamina propria and included lymphocytes,
plasma cells, neutrophils, and eosinophils (Figure 2.5 B). Where present, crypts of LGCs
were not distended (Figure 2.6 B). Pigs infected with both C. jejuni and T. suis developed
mild colitis with goblet cell hyperplasia and crypt distension with mucus in superficial
lamina propria in the areas where T. suis larvae were located. A mixed inflammatory

infiltrate in the lamina propria included lymphocytes, plasma cells, neutrophils, and

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eosinophils. Entrapped crypts of the LGCs were distended with mucus and few sloughed

epithelial cells” (Figure 2.6 C). (Jones, dissertation, 2005, reprinted with permission)

Clinical observation

All piglets were observed prior to infection and prior to fecal sampling to assess
clinical signs. Diarrhea was the main clinical sign and was assessed as any evidence of
liquid feces ranging from soft stool to pipe stream diarrhea. The results are summarized
in Tables 2.7 (short term challenge experiment) and 2.8 (long term challenge
experiments). The occurrence of diarrhea was also indicated by subjective evaluation of
collected loose feces and large amounts of loose fecal discharge in the cages of the pigs.
Over a 2-day period (short term challenge), the occurrence of diarrhea was assessed on
days 0, l and 2 during fecal sampling. Pigs given C. jejuni only had 2 episodes of
diarrhea, and the dually infected pigs that received C. jejuni and T. suis had 5 episodes of
diarrhea. There were no signs of clinical diarrhea in the uninfected pigs, the T. suis only
infected pigs, or the noninvasive E. coli DHS-a infected pigs. The occurrence of diarrhea
was also assessed on days 0, 21, 22 and 23 of fecal sampling for the long term challenge
experiments and more diarrheic episodes were seen. Diarrhea was most prominent in the
pigs infected with both T. suis and C. jejuni (19 episodes) and the pigs given T. suis only
(20 episodes). Pigs given C. jejuni only had 6 episodes of diarrhea over the observation
period. Pigs that were uninfected or received noninvasive E. coli DHS-a had no signs of

diarrhea.

101

Cytokine expression following Real-time PCR analysis

“We measured expression of mRN A fi'om 15 cytokines, chemokines, and iNOS in
each segment of intestine sampled in an effort to determine if T. suis or C. jejuni had a
significant effect on cytokine expression, and to determine if the affected cytokines were
indicative of a specific response.

Cytokine expression patterns were distinct in each segment of the intestine tested
(Figure 2.7). In the proximal colon of the T. suis only and dual infection groups, IL-13
expression was increased 3 to 4 fold while IL-12 and IL-8 expression was decreased 2 to
4 fold when compared to uninfected controls. In the dual infection group, expression of
IL-5 and MCP-l were each decreased 2 fold compared to controls. These data indicate
that T. suis stimulates IL-13 in its local environment while decreasing expression of
proinflammatory cytokines.

In the distal colon of pigs infected with T. suis only, expression of IL-l-B, IL-6,
IFNy, and IL-13 was increased 3 to 4 fold over controls. Infection with C. jejuni only or
dual infection with C. jejuni and T. suis did not significantly affect expression of any
cytokines, chemokines, or iNOS in the distal colon. These data indicate that a
predominately proinflammatory response has been initiated in the distal colons of pigs
infected with T. suis only.

In the jejunum, expression of MCP-1, TNFa, IL-12p40, IFNy, IL-4, and IL-10
were increased 3 to 5 fold in the C. jejuni only group when compared to controls.
Expression of GM-CSF was decreased 5 fold in the dual infection group when compared

to controls. These data indicate that Campylobacter species present at low levels in the

102

tissues or related Campylobacter-like bacteria stimulate a predominately
proinflammatory response in the jejunum, while T. suis may be down regulating

proinflammatory cytokines.” (Jones, dissertation, 2005 reprinted with permission)

Cytokine rate recovery from feces

Table 2.9 shows the recovery rate of exogenously added recombinant IL-8, IL-1-
[3, TNF-a, IL-4, IL-6, and IL-10 present in the feces. In general, these exogenously added
recombinant cytokines were recovered from feces at low rates (IO-20% recovery). These
findings suggest that the low recovery rate may have an impact on accurately measuring
cytokine expression in the feces of the pigs. Additionally, preliminary experiments
showed that when water was experimentally evaporated from either solid or loose fecal
samples, there were no significant changes in the weight of the samples. Thus, these
results suggested that the consistency of the feces would not likely have an effect on

adequately measuring changes in fecal cytokine levels (data not shown).

Cytokine Secretion—short term challenge (2 days)

IL-8, IL-l-B, TNF-a, IL-4, IL-6, and IL-10 concentrations were measured in the
feces of all piglets throughout the experiment. However, there were no significant
changes in IL-8, IL-l-B, TNF-d, IL-4, IL-6, and IL-10 expression due to any of the
administered treatments (IL-8, P e 0.56; IL-l-B, P = 0.27; TNF-a, P = 0.15; IL-4, P =
0.26; IL-6, P = 0.45; IL-10, P = 0.79). The results are shown in Figures 2.8 and 2.9 and
2.10 and 2.11 and 2.12 and 2.13, respectively. These results represent the combined data

of two independent experiments.

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Cytokine secretion—long term challenge (23 days)
IL-8 secretion

IL-8 was detected in all fecal samples throughout the experiment (23 days). The
results are illustrated in Figures 2.14. By day 23, pigs infected with C. jejuni alone
showed a significant increase in IL-8 expression compared to levels on day 0 (pre-
inoculation; P = 0.02); however, this increased expression was not statistically significant
compared to the milk only control group on days 0, 21, 22 and 23 (P > 0.05). The T. suis
only infected group showed a significant decrease in IL-8 expression by day 23 compared
to levels on day 21 (P = 0.01) and day 22 (P = 0.01), but this decrease was not
significantly different (P > 0.05) compared to day 0 and the milk only control group on
day 23. Also, E. coli DHS-a did not induce any changes in IL-8 expression. Therefore,
the overall changes of IL-8 expression due to the administered treatment over a period of
time were insignificant with a P value of 0.08. These results represent the combined data

of 2 independent experiments.

TNF-a secretion

TNF-a was detected in all fecal samples. The results for these experiments are
shown in Figure 2.15. The effects of each treatment on TNF-u expression over a period
of time showed moderate changes within the treatment groups. The pigs infected with C.
jejuni showed a significant increase in TNF-a expression from day 0 to days 22 and 23,
and the E. coli DHS-a group showed a significant increase in TNF-a expression fiom day
0 to day 22, but these increases in TNF-a expression were not significantly different in

comparison to the milk only control group. From day 0 to day 23, the pigs that were

104

given milk only showed a significant increase in TNF-a. From day 21 to day 22, the T.
suis group showed an increase in TNF-u and a decrease from day 22 to day 23, but there
were no significant differences compared to day 0 or to the milk only control group. In
addition, the pigs infected with C. jejuni and T. suis showed a significant increase in
TNF-a expression from day 0 to day 22 and then showed a decrease by day 23; however,
these changes were not significant compared to the milk control. Hence, the overall
changes in TNF-a expression due to the administered treatment over 23 days were not

significant in comparison to the milk only and day 0 control groups with P values > 0.05.

IL-l-B secretion

IL-l-B was detectable in all stool samples throughout the experiment (23 days).
The results are shown in Figure 2.16. By day 23, the pigs infected with T. suis alone
showed a significant decrease in IL-l-B expression compared to day 0 and the milk only
control group on days 0, 21, 22 and 23. This result indicates that infection with this
helminth was successfirl, and triggered down-regulation of a pro-inflammatory cytokine.
The T. suis group also showed significantly decreased IL-l-B expression on day 23
compared to the pigs that received C. jejuni. The dual infected group and the E. coli
DHS-a group showed a significant decrease in IL-l-B expression by day 23 compared to
day 0, but the decrease was not significant when compared to the milk only group.
Furthermore, the significant decrease in IL-l-B expression implies that T. suis suppressed
the IL-l-B response. These results represent the combined data from 2 independent

experiments.

105

IL—4 secretion

IL-4 was detectable in all stool samples throughout the experiment (23 days). The
results are shown in Figure 2.17. The effects of each treatment on IL-4 expression
showed moderate changes within the treatment groups. By day 23, T. suis-infected pigs
showed a significant decrease in IL-4 expression compared to IL-4 levels on days 0, 21,
and 22. Also, pigs given C. jejuni, E. coli DH5-a, or C. jejuni and T. suis showed a
decrease in IL-4 expression by day 23 compared to levels on day 0 within each of the
treatment groups. However, the overall changes of IL-4 expression were not statistically
significant compared to the milk only control group (P 2 0.05). These results represent

the combined data of 2 independent experiments.

IL-6 secretion
IL-6 was detectable in all stool samples throughout the experiment (23 days).
There were no significant changes in IL-6 expression due to the administered treatments

(P > 0.05). The results are shown in Figure 2.18.

IL-10 secretion

IL-10 was detectable in all stool samples throughout the experiment (23 days).
The results are shown in Figure 2.19. There were some moderate changes in IL-10
expression within the treatment groups throughout the experiment. Pigs given C. jejuni,
T. suis, or C. jejuni and T. suis showed increased IL-10 levels by day 22 compared to day
0. Also, E. coli DHS-u-infected pigs showed an increase in IL-10 expression by day 22

that continued through day 23. These moderate changes in IL-10 expression within the

106

treatment groups were not significant when compared to the IL-10 levels detected in the

pigs given milk only (P > 0.05).

107

Discussion

In our Campylobacter swine disease model, there have been repeated
observations suggesting that there is enhanced disease and pathology in the gut mucosal
epithelium and LGCs in response to a concurrent infection with C. jejuni and T. suis (49,
50)(Jones, dissertation, 2005). In this study, clinical signs and histopathology also
support an interaction between these two pathogens. The most severe clinical signs and
histopathological lesions were observed in dually infected pigs. Lesions were similar to
that seen previously with germ free pigs with major changes observed in the proximal
colon surrounding the worms and in the distal colon in the LGCs (50). It is thought that
cytokine dysregulation plays a significant role in this response. In the present study, we
hypothesized that weaned piglets infected with C. jejuni demonstrate an innate protective
immune response via pro-inflammatory cytokine up-regulation and simultaneous T. suis
infection mediates the down-regulation of the pro-inflammatory cytokine response,
thereby promoting C. jejuni infection.

In this study, we used standard culture procedures, PCR assays, and
immunological testing to determine whether the weaned piglets used in our experiments
had been exposed to C. jejuni, other Campylobacter species, or Campylobacter-like
organisms (epsilon proteobacteria) at the MSU swine farm facility prior to experimental
infection. The piglets apparently had low level exposures to Helicobacter spp. We must
consider the possibility that the piglets were also exposed to related Campylobacter
species or other environmental Campylobacter-like bacteria from the sow. This was an

important factor because it may have caused control pigs to have falsely elevated

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cytokine responses elicited following exposure to Helicobacter. Also after experimental
infections, we attempted to use standard culture methods to identify C. jejuni prior to and
throughout the experiment, but in spite of our best efforts, culture alone was not sensitive
enough to detect C. jejuni. Others have also found it difficult to recover C. jejuni from
feces by culture alone and have used PCR assays and irnmunohistochemical examination
to detect C. jejuni (20, 79). Because of problems detecting C. jejuni in pigs after
inoculation, some researchers in recent work used culture of supernantants from 5-10
gram samples of feces to enhance the sensitivity (Moller-Nielson, personal
communication). To increase the specificity and sensitivity of detection in our studies,
we used C. jejuni specific gyrA Real-time PCR (TaqMan) for detection and RF LP
analysis of the Campylobacter 23S rRN A gene for thermophilic Campylobacter species
differentiation. TaqMan analysis of post infection tissue samples fi'om necropsy gave
negative results for most pigs, but a positive result for C. jejuni in distal colon samples
fiom 16% (1 of 6) of the pigs that received T. suis-only. In follow-up analysis of post
infection samples from necropsy, 23S rRNA RF LP analysis gave positive results for C.
jejuni in jejunum samples fi'om the dually infected pigs (16%, 1 of 6) and in proximal
colon samples from 16% (1 of 6) of the pigs that received T. suis-only. Likewise, C.
jejuni was detected in proximal colon samples from 66% (4 of 6) of the dually-infected
pigs. RF LP analysis also showed positive results for C. coli and a possible
Campylobacter spp. that gave a unique banding pattern that was not consistent with C.
jejuni, C. lari, C. coli, or C. upsaliensis in jejunum, proximal and distal colon samples.
These RFLP findings conflicted somewhat with the gyrA TagMan assay results and

suggested that multiple Campylobacter species could have been present in the colon of

109

these pigs. RF LP results also implied that the pigs may have been exposed to
Campylobacter spp. or Campylobacter-like organisms (epsilon proteobacteria) prior to
the experiment. Subsequent immunological testing only partially clarified the issue. It
demonstrated the presence of IgG antibodies against C. jejuni protein antigen in the
plasma of all pigs in the short and long term challenge experiments. In addition, we
detected significant levels of IgG antibodies that reacted with antigen from other
Campylobacter species and Campylobacter-like bacteria (Arcobacter butzleri and
Helicobacter hepaticus) in the plasma of some of the pigs given C. jejuni (a total of 8
pigs randomly selected from the short and long term challenge) or milk only (a total of 6
pigs randomly selected from the short and long term challenge). These findings further
suggest that these pigs may have had a previous exposure to an epsilon proteobacterium
possibly C. jejuni or other closely related member of the group with similar outer
membrane proteins. It has been shown in humans that IgA and IgG are produced in
response to C. jejuni infection, and serum and salivary IgG antibody responses are
detectable up to 1 year post-infection while long-term IgA antibody responses are not
detectable ( 14, 27). The greatest IgG reaction was obtained with H. hepaticus protein
antigen, thereby suggesting that this was the main contaminating bacterium that was
present in these pigs. It also suggests that the IgG antibodies detected in the plasma of
the pigs cross-reacted with the protein antigen of other Campylobacter species and
Campylobacter-like bacteria. Because of the short time period between initial bacterial
inoculation (short term challenge-day 0; long term challenge-day 21) and sampling (short
term challenge-day 2; long term challenge-day 23), the experimentally inoculated pigs

would not have enough time to trigger an IgG response against the administered C. jejuni.

110

As in C. jejuni-infected humans, it has been shown that IgG antibody titers generally
peak at approximately 8-14 days postexposure, but in some cases may not peak until 3 to
4 weeks postexposure, and remain elevated and persist at high levels for several weeks or
months in patients (23, 45). Taken together, it appears that the pigs had been exposed to
the putative contaminating bacterium (epsilon proteobacteria), possibly a Helicobacter
spp., at birth or shortly after birth at the MSU swine farm prior to the experiment.

Indeed, these ELISA results show increased levels of IgG antibodies reacting with
multiple bacterial protein antigens, including C. jejuni, other Campylobacter species, A.
butzleri, and H. hepaticus, and could not distinguish between the occurrence of cross
reactivity and infection with multiple epsilon proteobacteria. Because no published work
exists comparing homologies between outer membrane proteins of bacteria within this
group, we cannot distinguish between these possibilities based on these assays. In a final
attempt to distinguish the identity of the early bacterial exposure in these pigs, we tested
fecal supernatants for the presence of other Campylobacter species and Helicobacter
species using 16S rRNA PCR analysis. Here we found that almost all pigs in the short-
and long-term challenge experiments tested negative for the presence of Campylobacter
species pre- and post-infection. However, several pigs tested positive for the presence of
Helicobacter species pre- and post-infection. This data provides the best evidence to
suggest that the pigs were exposed to Helicobacter spp. prior to the experiment and that
the cytokine responses measured were likely affected by this prior bacterial exposure.
This also suggests the possibility that the effects seen actually represent host resistance to
secondary bacterial infection. In this scenario, it is likely that these pigs acquired

immunity after early exposure to an epsilon proteobacteria such as Helicobacter, and

111

elicited a humoral immune response which provided partial protective immunity to
subsequent C. jejuni challenge. For example, it is possible that systemic IgG arising from
early exposures may have bound to the experimentally administered C. jejuni or to shed
C. jejuni surface material and limited C. jejuni-enterocyte interactions which is the main
mechanism for increased inflammatory cytokine production in bacterial infections (38,
39, 42).

In the short-term challenge experiments, there were no significant changes in
secreted IL-8, IL-l-B, TNF-a, IL-4, IL-6, or IL-10 expression due to any treatment when
compared to the uninfected pigs that received milk only. We hypothesized that C. jejuni
exposure would trigger an increase in the expression of pro-inflammatory cytokines, such
as IL-8, IL-l-B, and TNF-a, and simultaneous T. suis infection would stimulate increased
production of anti-inflammatory cytokines, such as IL-4, IL-6, and IL-10. Based on
literature and previous findings in our laboratory, we believe that the anti-inflammatory
cytokine response can down regulate the pro-inflammatory cytokine response and thereby
facilitate C. jejuni infection. Consequently, the dysregulation of cytokine expression due
to this concurrent infection could cause immunomodulatory effects that may contribute to
the observed disease and pathology, which is similar to that seen in campylobacteriosis.
However, because the data here provide some evidence suggesting that these pigs were
exposed to Helicobacter spp. or a Campylobacter-like bacteria prior to the experiment, it
is reasonable to believe that the administered C. jejuni was a secondary bacteria]
challenge. Therefore, in the groups of pigs that received bacteria alone (C. jejuni only or
E. coli DHS-a only), the cytokine expressions measured were likely a result of host

resistance to secondary bacterial infection. In the short- term challenge experiment, we

112

found that C. jejuni alone did not induce the expected increase in secreted pro-
inflammatory cytokine expression in feces. There were also no significant changes in
cytokine expression in the pigs that received E. coli DHS-u only. Likewise, in a previous
study, Withanage et al. demonstrated that there were no significant changes in the
expression of proinflammatory cytokines or chemokines, such as IL-IB, IL-8, or K60, in
the gastrointestinal tract of chickens following secondary Salmonella infection.

However, there was notable expression of MIP family chemokine and IL-6 in the ileum
and cecal tonsils (76). Additionally, in our study, we found that there were increased
levels of IgG in the plasma of almost all the pigs regardless of treatment that reacted in
the C. jejuni ELISA. It has been shown in previous studies that significant levels of
circulating IgG antibodies specific for Salmonella were high in infected chickens
following primary infection and remained high with little change following secondary
infection (15, 17, 76). In addition, significant levels of specific IgG antibodies have been
detected in patients with Campylobacter enteritis, and IgG levels have been reported to
be detectable in most patients up to a year post infection (23, 27). Therefore, our results
appear to be similar to previous reports that suggest that humoral responses may play a
significant role in protective immunity during homologous and heterologous re-challenge
(16). The current finding also raises speculation about the possibility that there may be
cross-reactive humoral immune responses to Campylobacter species or other episilon
proteobacteria that are associated with protective immunity. As for the two groups of
pigs that received the T. suis in the short-term challenge (day O-day 2), the results were
not surprising. Studies have shown that clinical signs mostly begin 13-14 days following

T. suis infection. The severe, bloody diarrhea associated with swine trichuriasis from the

113

20th day onwards is due in part to the burrowing activities of the larval and adult stages in
host colonic tissues and secretion of proteolytic enzymes and proteases (ESP). During
this phase of T. suis development, the posterior end of the helminth fi'eely protrudes into
the lumen while the anterior body is embedded in the mucosa (18). At this phase, the
third stage larvae have just moulted to the fourth larval stage. Following the direct
mucosal damage by the helminth, localized lesions have been observed surrounding the
site of worm attachment. It is at this time that resident bacteria have been demonstrated
to amplify the severity of the infection (3, 4, 56, 77). The evidence provided by these
reports suggests that T. suis acts synergistically with C. jejuni in producing the
pathogenesis of swine mucohemorrhagic colitis. Thus, it is reasonable to believe that
results fiom the long-term challenge experiments (23 days) would likely reflect the
immune response patterns of natural infections more than that seen in the short-term
challenge experiments (2 days).

In the long-term challenge experiments (day 0-day 23), we found that T. suis
infection significantly decreased secreted IL-l-B by 23 days post-inoculation. Also, on
day 23, IL-l-B was down-regulated in the pigs that received T. suis alone compared to the
pigs that only received C. jejuni. Furthermore, the dually infected pigs showed
moderately decreased IL-l-B expression by day 23, but the response was not significant.
The significant decrease seen in IL-l-B expression in response to T. suis alone is
characteristic of most helminth infections. In general, helminth infections elicit a Th2
response which induces the production of IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 during
infection (33, 74). This pattern of cytokine expression is characteristic of a Th2 response

which predominantly limits Th1 cytokine-induced inflammation (Th1 cytokines- Il-12,

114

IFN-y, IL-8, TNF-a, and IL-l-B) and protects the host by causing worm expulsion,
reducing worm fecundity and enhancing host resistance mechanisms (33, 44). Tissue
samples were also taken fi'om the pigs in the long-term challenge experiments on day 23
following fecal sampling, and using these samples, Real Time PCR analysis showed
increased IL-l3 mRN A (Th2 cytokine) in the proximal colon of T. suis-infected pigs and
decreased IL-8 and IL-12 expression (Th1 cytokines) (Jones, dissertation, 2005). This
supports the hypothesis that T. suis stimulates Th2 anti-inflammatory cytokine production
which can down-regulate the production of Th1 pro-inflammatory cytokines. We have
also found in our laboratory that intestinal pig epithelial cells stimulated with T. suis ESP
secrete IL-6 and IL-10 (60). Therefore, it is possible that the down regulation of IL-l-B in
response to T. suis alone is likely due to IL-4, IL-10 and IL-13 production in response to
the helminth infection. In vitro and in vivo studies have demonstrated IL-4, IL-10 and IL-
13 inhibition of intestinal inflammatory responses such as Thl-type cytokine production
(22, 46, 48, 68). Taken together, our fecal cytokine ELISA results suggest that T. suis
likely plays a significant role in the down-regulation of IL-l-B production, and this
response may play a contributing role in altering host conditions which promote
secondary bacterial infection. However, these results need to be confirmed in fiirther
studies because of the low cytokine recovery rate (IO-20%) determined for these fecal
ELISAs. Several factors may have contributed to the low cytokine recovery rate: protein
degradation associated with freezer storage, the loss of protein during the fecal
preparation due to the cytokines binding to the solid fecal matter, or protein degradation
due to protease activity. Because of the drawbacks associated with measuring cytokines

in feces using ELISA, we are unable to confidently support the future use of ELISA for

115

this purpose and believe that our results fiom this analysis are notable, but must be
confirmed with further experimentation. Nevertheless, in the long-term challenge
experiments, cytokine mRN A expression measurements in the tissues of these weaned
piglets were more informative. We observed significant changes in cytokine expression
in different intestinal compartments based on treatment group, specifically the jejunum,
proximal and distal colon. T. suis resides in the proximal colon while C. jejuni has been
found in all of these sites. We found that T. suis infection predominantly influenced
cytokine expression in the proximal colon with some effects in the distal colon. C. jejuni
mainly affected the cytokine expression in the jejunum with minimal changes in the
colon. In the proximal colon, T. suis stimulated increased IL-13 expression (3 to 4 fold)
in the pigs that received T. suis alone or those that were dually infected, and IL-8 and IL-
12 expression was decreased 2 to 4 fold in both groups when compared to the uninfected
control pigs that received milk only. MCP-1 expression was also decreased in the dually-
infected pigs. This is similar to other results that show that during intestinal nematode
infection, IL-l3 plays a significant role in affecting Th-2-cell-mediated responses which
are associated with goblet cell hyperplasia and worm expulsion (11, 37, 52). Th-2
responses are associated with the production of anti-inflammatory cytokines such as IL-4,
IL-5, IL-6, IL-9, IL-10 and IL-l3, and these anti-inflammatory cytokines can down
regulate Th-l responses (IFN-y, IL-2, IL-12, lymphotoxin, and IgG2a production) which
subsequently have a diminishing effect on pro-inflammatory cytokine responses (IL-8,
TNF-a, MCP-1, IL-1, and IL-6) (25, 28, 59). These types of cytokine modulations can
generally be seen during concurrent bacterial-helrninth infections. Previously reported

data from our laboratory showed that pigs rectally challenged with C. jejuni showed

116

increased IL-8 expression at 1 and 24 hours post-inoculation suggesting the importance
of IL-8 production in early host resistance to C. jejuni in immunologically naive swine
(Jones, dissertation, 2005). It has also been shown that C. jejuni stimulates cultured
intestinal epithelial cells (INT 407) to secrete IL-8 and MCP-l (38, 42). Likewise,
Helicobacter pylori stimulates cultured gastric epithelial cells to secrete IL-8 and MCP-1
(35, 58, 61). In another enteric bacteria with similar pathogenesis, it has been
demonstrated in a rabbit ligated intestinal loop model of Shigellaflexneri infection that
IL-8 appears to play a critical role in controlling the spread of infection (67). Because
bacteria have been shown to invade and proliferate in the lamina propria and submucosa
surrounding the site of worm attachment in previous studies (50), we expected to see
increases in proinflammatory cytokines like IL-8 in the proximal colon of pigs given C.
jejuni alone. However, no changes in IL-8 were observed in either study design
associated with C. jejuni infection alone in this tissue. This may have been affected by
the suspected early exposure to Helicobacter spp. in these studies or perhaps the
inoculated strain of C. jejuni invaded mame the small intestines based on the
proinflammatory responses measured there. Based on the tissue RT-PCR results it is also
possible that T. suis contributed to decreased IL-8 expression and may have facilitated
bacterial invasion and proliferation in the lamina propria and submucosa, similar to what
is observed in the Shigella model. IL-13 production due to T. suis infection likely
contributes to the decreased expression of IL-8, given that it has been demonstrated that
the production of IL-8 fi‘om intestinal epithelial cells can be inhibited by IL-13 and IL-4

(48).

117

We also found that expression of both IL-12 subunits was significantly decreased
in the proximal colon of pigs given T. suis. IL-12 is predominantly produced by
monocytes, macrophages, and dendritic cells and plays a role in inflammation and
acquired immune responses (36, 71). IL-12 drives the development of Thl-type immune
responses by stimulating T cells to produce IFN-y, and in vitro and in vivo studies have
shown that IL-12 plays a significant role in developing specific immunity against
intracellular pathogens (36, 71). However, IL-l3 has been shown to down-regulate IL-12
production (54). Increased expression of IL-13 has been documented in resistant mice
strains infected with T. muris suggesting its critical protective role in nematode infection
(1 1). Taken together, decreased expression of both IL-l2 subunits in the proximal colon
of T. suis-infected pigs can be attributed to the immunomodulatory effects of IL-l3.

Similar to the decreased expression of pro-inflammatory cytokines in the
proximal colon, IL-5 expression was significantly decreased in the dually infected pigs.
In general, IL-5 is up-regulated along with other classical Th2 cytokines during helminth
infections, and IL-5 has been shown to characteristically induce eosinophil production
and activation (32). However, experimental studies have shown that C. jejuni intestinal
colonization is reduced in Balb/c ByJ female mice that are orally pretreated with IL-5,
suggesting that IL-5 may play a protective role for the host (13). It is possible that C.
jejuni elicits the down regulation of IL-5 as a mechanism to evade host immune
responses and establish infection. In the proximal colon of the pigs that received C.
jejuni only, IL-5 expression was decreased, but the decrease was not statistically
significant. Even so, the decreased expression of IL-5 observed in the dually-infected

pigs and the C. jejuni-infected pigs suggests that this cytokine may play a significant role

118

in the pathology associated with C. jejuni infection in vivo and that firrther studies into
this hypothesis are warranted.

Unlike the decreased pro-inflammatory cytokine response observed in the
proximal colon, IL-l-l3, IL-6, IFN-y and IL-13 were up-regulated in the distal colon of
the pigs infected with T. suis only. We speculate that this immune response is likely due
in part to the effects of T. suis ESP in the distal colon. The up-regulation of IL-l-B, IL-6,
IFN-y is indicative of an inflammatory response. It has been demonstrated in vitro that
ESP treatment of differentiated IPEC-1 cells causes a dose dependent cytotoxic response
and significantly decreased transepithelial electrical resistance (TER)(1, 2). Also,
cultured differentiated and undifferentiated IPEC-l cells secrete IL-6 following ESP
exposure (Parthasarathy, 2005). Therefore, it is reasonable to consider that the cytotoxic
effects of ESP on intestinal epithelial cells can stimulate an inflammatory cytokine
response in the distal colon. Another possible reason for the inflammatory response
observed in the distal colon is secondary bacterial infection that is augmented by T. suis
infection. In the distal colon of T. suis-infected pigs in previous studies, several resident
bacterial species, including C. jejuni, C. coli, an unidentified Campylobacter species, and
E. coli, have been cultured fi'om abscessed lymphoglandular complexes (51). It has been
shown that C. jejuni and C. coli stimulate cultured human INT-407 intestinal epithelial
cells to secrete IL-8 (39). Therefore, it is possible that bacterial invasion of the distal
colon tissues in this study initiated an inflammatory response and was responsible for the
histopathological changes in the LGCs. It has been shown that some components of the
ESP consist of antibacterial agents, and a possible scenario is that the antibacterial effects

of ESP could kill residential bacteria thereby making the host environment more

119

conducive to facilitating the proliferation of pathogenic bacteria. Once the ESP
antibacterial activity has killed the residential bacteria, these pathogenic secondary
bacteria could more effectively interact with the intestinal epithelium and stimulate these
cells to produce proinflammatory cytokines. Also, the effects of ESP produced by 4th
stage larvae that have traveled to the distal colon could contribute to increased expression
of IL- 1 3. It has been shown that T. suis-infected pigs have serum antibodies as early as
21 days post inoculation that recognize a 20kDa glycoprotein isolated fiom ESP collected
from adult worms cultured in vitro (41). This indicates that an immune response is likely
triggered by the larval stages present in pigs 21 days post-T. suis infection. Altogether,
these data suggest that ESP may have direct effects on intestinal epithelial cells and could
account in part for the increased cytokine production in vivo at sites distal to worm
attachment.

In the jejunum, the pigs infected with C. jejuni only showed increased expression
of MCP-l, TNF-u, IL-12p40, IFN-y, IL-4, and IL-10. Although we did not confirm the
presence of C. jejuni or other Campylobacters in this tissue by culture, results from the
histopathology, immunological testing (IgG ELISA), and the 23S rRNA PCR analysis
suggest that it is still likely that the inoculated strain of Campylobacter triggered this
response. C. jejuni is notoriously difficult to recover from colonized pigs without disease
because of low bacterial numbers and the preferred predilection site that is strictly at the
base of mucosal crypts. In vitro, C. jejuni induces INT 407 cells to produce intracellular
MCP-l, TNF-a, IFN-y, IL-4, and IL-10 (42), and Helicobacter pylori induces gastric
epithelial cells to produce IL-8 and MCP-1 (35, 55, 58, 61). Thus, the cytokine changes

observed could be due to Campylobacter interaction with the intestinal epithelial cells in

120

the jejunum. Helicobacter responses, if present, would be expected to be similar based
on previous studies (34). In the jejunum of the dually-infected pigs, GM-CSF expression
was decreased 5 fold compared to the uninfected milk controls, suggesting that the T. suis
down-regulated pro-inflammatory cytokine expression similar to the response seen in the
proximal colon.

Furthermore, clinical diarrhea was a prominent sign of disease throughout the
experiments. During fecal sampling in both experiments, diarrhea was observed in pigs
that received C. jejuni only, T. suis only or both. However, the dually infected pigs and
the T. suis only-infected pigs had more frequent, more severe diarrhea than the other
treatment groups, thereby implying that T. suis is mainly responsible for causing the
observed diarrhea. Additionally, diarrhea was mostly observed in the long term
challenge experiments where T richuris infection had progressed for 21 days. As has
been reported frequently in literature reports on T richuris- induced disease, diarrhea
usually begins around 21 days post-infection and is commonly called the “21 day scours”
(24). In both experiments, some of the C. jejuni-infected pigs had mild diarrhea, which is
commonly seen in campylobacterosis (10). In a germ fiee Campylobacter swine disease
model experiment of similar design, Mansfield et a]. previously reported that some of the
C. jejuni-infected pigs showed clinical signs of mild diarrhea less severe than that in
dually-infected or T. suis only-infected pigs (50). Uninfected and E. coli DHS-a infected
pigs showed no signs of diarrhea. The observation of severe diarrhea in pigs infected
dually or with T. suis alone suggests that T. suis contributed significantly to diarrhea]

disease.

121

Additionally, the histopathologic lesions observed in the tissues of pigs infected
with T. suis alone or dually infected with T. suis and C. jejuni were consistent with what
has been reported in the literature(]0, 50). The lesions in the tissues from the C. jejuni-
infected pigs were mild, and there was no crypt distention or goblet cell hypertrophy.
The lesions observed in the tissues of T. suis-infected pigs showed signs of mild colitis
with crypt distention and increased thickness of all the layers of the colon. However, the
lesions in the tissues of dually-infected pigs showed the greatest signs of disease with the
greatest crypt distention and increased thickness of all layers of the colon, and greater
infiltration of immune cells. Also, in the groups of pigs that were dually-infected or
received T. suis alone, histological evaluation (H&E staining) confirmed the presence of
T. suis in the tissues (Figure 2.4). Altogether, these results confirmed that all the pigs
inoculated with T. suis became infected, and we were able to correlate the presence of the
worm with cytokine changes seen in the colon.

In summary, we found it necessary to use multiple diagnostic tests to detect and
identify Campylobacter species or other epsilon proteobacteria in the feces and intestinal
tissues of pigs, and despite this, definitive diagnosis was not possible. However, the data
presented here suggest that throughout the intestine, T. suis appears to mediate most
changes in pro- and anti- inflammatory cytokine expression during concurrent T. suis and
C. jejuni infection, and it is possible that the immunomodulatory effects associated with
these changes play a role in promoting a favorable host environment for secondary
bacterial infection. One possible scenario is that T. suis interacts with the resident
bacteria and down regulates the pro-inflammatory cytokine response which is generally

elicited by bacterial infection, and the immunomodulatory effects of these pro-

122

inflammatory cytokines are diminished which could thereby facilitate bacterial invasion
of intestinal cells and tissues causing cellular destruction and disease, as seen in human
campylobacteriosis. Although our hypotheses cannot be unequivocally accepted, the data
presented in this study provide some evidence to support this scenario. The occurrence
of frequent, severe diarrhea in pigs infected with C. jejuni and T. suis or T. suis alone is a
clear sign of disease and shows that T. suis contributes significantly to this syndrome.

We were also able to detect pro- and anti- inflammatory cytokines in the feces of all the
pigs regardless of treatment using ELISA, in agreement with data reported in the
literature (9, 30, 57, 63, 66). However, measuring cytokine expression in feces by ELISA
was found to be insufficient to directly address our hypothesis; unfortunately, this
noninvasive method for investigating fecal cytokine expression during infection does not
appear to be useful. In this study, the use of RT-PCR to measure cytokine expression in
the tissues served as a more accurate and reliable method to address the overall
hypothesis. Future studies will be conducted to describe the roles of these pro- and anti-

inflammatory cytokines in this concurrent infection.

123

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13]

Table 2.1. Experimental treatment groups for the short (2 days) and long (23 days) term
challenge experiments.

 

 

Treatment Treatment
Groups
Group 1 C. jejuni (2 x 108 cfu)
Group 2 C. jejuni (2 x 108 cfil)and
and T. suis (2500 embryonated eggs)
Group 3 T. suis (2500 embryonated eggs)
Group 4 E. coli DHS-a (2 x 108 cfir)
Group 5 Uninfected
milk control (2 ml)

 

132

Table 2.2. Inoculation timeline for short term challenge experiments (2 days): 4-week
old weaned, conventionally reared pigs were divided into 5 treatment groups with a total
of 10 pigs per group. The inoculations were administered via oral gavage in 2.0 ml of
sterile milk. The pigs were given 2 x 108 cfu C. jejuni, 2500 T. suis embryonated eggs,
both, 2 X 108 cfir E. coli DHS-a, or milk.

 

 

 

Day 0 Day 1 Day 2
l. Rectal swabbing Rectal swabbing 1. Rectal swabbing
and fecal sampling and fecal sampling and fecal sampling
of all animals of all animals
2. Inoculation 2. All pigs were
protocol: euthanized and
necropsied.

Group 1: C. jejuni
Group 2: C. jejuni and T. suis
Group 3: T. suis

Group 4: E. coli DHS-a

 

 

 

 

Group 5: Milk

 

133

Table 2.3. Inoculation timeline for long term challenge experiments (23 days): 4-week
old weaned, conventionally reared pigs were divided into 5 treatment groups with a total
of 1] pigs per group. The inoculations were administered via oral gavage in 2.0 ml of
sterile milk. The pigs were given 2 x 108 cfu C. jejuni, 2500 T. suis embryonated eggs,
both, 2 x 108 cfu E. coli DHS-a, or milk.

 

 

 

 

 

 

 

Day 0 Day 21 Day 22 Day 23
l. Rectal swabbing 1. Rectal swabbing Rectal 1. Rectal swabbing
and and fecal sampling swabbing and
fecal sampling and fecal fecal sampling
sampling of all animals
of all animals
2. Inoculation 2. Inoculation 2. All pigs were
protocol: protocol: euthanized and
necropsied.
Group 1: Milk Group 1: C. jejuni Tissue samples
were taken from
Group 2: T. suis Group 2: C. jejuni all pigs.
Group 3: T. suis Group 3: Milk
Group 4: Milk Group 4: E. coli
DHS-a
Group 5: Milk
Group 5: Milk

 

 

I34

Table 2.4. Summary of Campylobacter detection and identification. Campylobacters
were detected and identified fi'om pigs by fecal culture, PCR for the C. jejuni QRDR on
intestinal lumen contents obtained from each intestinal region and tissue DNA, and RFLP
analysis of the thermophilic CampJvlobacter 23S rRN A gene using tissue DNA. Samples
were taken from the pigs in the 2n repetition of the long term challenge experiments only
(Jones, dissertation, 2005, reprinted with permission).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Campylobacter
23S rRNA RFLP
QRDR (tissue DNA)
Unidentified
Fecal Tissue Campylobacter
Infection Culture DNA DNA C. jejuni C. coli sp.
Control 0/6 0/6 0/6 0/6 5/6 2/6
Jejunum C. jejuni 0/6 0/6 0/6 0/6 2/6 5/6
T. suis 0/6 0/6 0/6 0/6 3/6 2/6
Dual 0/6 0/6 0/6 1/6 4/6 1/6
Control 0/6 0/6 0/6 1/6 4/6 5/6
Proximal C. jejuni 0/6 0/6 0/6 1/6 2/6 4/6
colon T. suis 0/6 0/6 0/6 1/6 4/6 1/6
Dual 0/6 0/6 0/6 4/6 4/6 2/6
Control 0/6 0/6 0/6 0/6 0/6 6/6
Distal C. jqiuni 0/6 0/6 0/6 0/6 0/6 6/6
colon T. suis 0/6 0/6 1/6 0/6 2/6 3/6
Dual 0/6 0/6 0/6 0/6 2/6 6/6

 

 

 

 

 

 

 

 

 

135

 

Figure 2.1. RFLP analysis of the Campylobacter 23S rRNA gene. Panel A: AluI digest
of proximal colon samples fi'om infected pigs . Lane I: 100 bp DNA ladder; Lane 2: C.
jejuni; Lane 3: C. coli; Lanes 4 through 15: DNA from infected pigs. Panel B: Tsp509l
digest of proximal colon samples from infected pigs. Lane 1: 100 bp DNA ladder; Lane
2: C. jejuni; Lane 3: C. coli; Lane 4: C. hyoilei; Lanes 5 through 16: DNA from infected
pigs. Samples were taken from the pigs in the 2"d repetition of the long term challenge
experiments only (Jones, dissertation, 2005, reprinted with permission).

136

 

 

1.4
1.2
o 1
In
* 0.8
:5 0.6
0 0.4 :
0.2 l
0 I
Milk C. jejuni C. jejuni T. suis E. coli
control and T.
suis
Treatment groups
B
0.8
0.7
0.6
O
in 0.5
Q‘
. 0.4
Q
0 0.3
0.2
0.1
0
Milk C. jejuni C. jejuni T. suis E. coli
control and T.
suis

Treatment groups

Figures 2. 2 A and B. (A) Short term challenge experiment and (B) long term challenge
experiment ELISA results indicating levels of plasma IgG antibodies detectable against
C. jejuni protein antigen. IgG levels were measured in the plasma of all pigs fi'om each
treatment group. The data represent the mean i SEM.

I37

N
l
l
l
l
i
l
l
l
l

 

l‘

 

_s
'0)

 

 

l

 

iv

 

 

.0
co

 

O. D. 450

 

in fie were Emmi raini- 1‘!

 

 

---
”ha/g, F) ----
6) ---
new 6) ----

O o o
o N b a)
_-
Hm:

ease reassess se.§e s
E§°Q§ g§3e§o§agg tags» 5
~Q.@8\_ 0 SGgQOE (3737803 0
'Q 0 <50 94’ s 33 as
(50 we 8 9° 3.3
$19 -' Q
3.3 as it“ s
a 3:6 .753:
00 §~Q

Figure 2.3. ELISA results indicating levels of plasma IgG antibodies detectable against
protein antigen of various Campylobacter species, Arcobacter butzleri, and Helicobacter
hepaticus, suggesting IgG cross-reactivity. IgG levels were measured in the plasma of 8
C. jejuni-infected pigs randomly selected from the short- and long-term challenge
experiments (+), 6 uninfected pigs randomly selected fiom both challenge experiments
that received milk only(-), and 2 newborn piglets < 6 hours old from the MSU swine farm
(control). The data represent the mean :1: SEM. “Images in this dissertation are
presented in color.”

138

Table 2.5. Short term challenge experiment 16S RNA PCR analysis results for rapid
identification of Campylobacter and Helicobacter DNA in pig feces.

Helicobacter
' # Pre-infection Post-infection Pre-infection Post-infection

1
2
3
4
5
6
7
8
9
10
ll
12
l3
14
15
l6
l7
18
19
20
21
22
23
24
25
26
27
28
29

 

b)
O

 

a. + tested positive
b. - tested negative
c. x represents missing sample

139

Table 2.6. Long term challenge experiment I6S RNA PCR analysis results for rapid
identification of Campylobacter and Helicobacter DNA in pig feces.

Helicobacter

' # Pre-infection Post-infection Pre-infection Post-infection
6 -
7
8
13
14
15
l6
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39

 

 

a. + tested positive
b. - tested negative
c. x represents missing sample

140

 

Figure 2.4. H&E stained sections from the colons of pigs were examined for T. suis
larvae. Panel A: Control; B: C. jejuni only; C: T. suis only; D: Dual infection, 10X
magnification. Larvae (arrows) were detected in 5 of 6 pigs from each of the T. suis only
and dual infection groups. Samples were taken from the pigs in the 2nd repetition of the
long term challenge experiments only (Jones, dissertation, 2005, reprinted with
permission). “Images in this dissertation are presented in color.”

141

 

Figure 2.5. H&E section from a C. jejuni infected pig (panel A) showing sloughed
epithelial cells in the crypts (black arrow), and a T. suis infected pig (panel B) showing
the mixed inflammatory infiltrate in the lamina propria associated with the larvae (green
arrow). Inflammatory cells included eosinophils (black arrow), plasma cells (blue arrow),
lymphocytes (red arrow), and neutrophils (yellow arrow) (40X magnification). Samples
were taken from the pigs in the 2nd repetition of the long term challenge experiments only
(Jones, dissertation, 2005, reprinted with permission). “Images in this dissertation are
presented in color.”

142

 

Figure 2.6. H&E sections of LGCs fi‘om infected pigs. Crypt distension was present in
the pigs that received C. jejuni only (panel A) and in the dually-infected pigs (panel C),
but crypts were not distended in the pigs that received T. suis only (Panel B). (10X
magnification). Samples were taken from the pigs in the 2nd repetition of the long term
challenge experiments only (Jones, dissertation, 2005, modified and reprinted with
permission). “Images in this dissertation are presented in color.”

143

 

 

 

 

 

 

 

Je'unum Proximal colon Distal Colon
was 1:1 .3 1:1 :3 1: 1:
m1:11::11:1::n1:
MC.-. m::11:1::1:c:
m-a m::11:1::11:11:
mm EIEZIIIIEZIDE
acnmmju:
ms m::1r:11::11:1:
m 13::11:H::1:i:
W m1::n1::1u1:
1:1 11:1 BID 1: 1:
mi 1:11::1:ii:m 1:1
i... a1:1:11:11::11:11:1
:11::11:::11:1m
H... DEJDEEDE
n... I :JJLIichLi:

 

Figure 2.7. Expression of mRN A from a panel of cytokines was measured by Real Time
PCR. The data represent the average fold change in expression fiom infected animals
when compared to the milk control animals. Letters indicate statistically significant
changes: A: p5 0.05; B: p: 0.01. Tissue samples were taken fiom the pigs in the 2nd
repetition of the long term challenge experiments only (Jones, dissertation, 2005,
reprinted with permission). “Images in this dissertation are presented in color.”

144

Tables 2.7. Short term challenge: clinical assessment of diarrhea in weaned,
conventionally-reared swine infected with T. suis and/or C. jejuni.

 

 

Experiment Treatment # of pigs with diarrhea
group
Day 0 Day 1 Day 2
Short (2 days)“ Group 1
2nd repetition c. jejuni 0/6 2/6 0/6
of experiment
only
Group 2
C. jejuni
and T. suis 0/6 1/6 4/6
Group 3
T. suis 06 0/6 0/6
Group 4
E. coli DHS-a 06 0/6 0/6
Group 5
Uninfected 06 0/6 0/6

 

All animals were assessed for diarrhea during fecal sampling on days 0, 1, and 2 of the
short term challenge experiment.

a The four pigs from each treatment group in the first repetition of the short term
challenge experiment were not assessed for diarrhea.

145

Tables 2.8. Long term challenge: clinical assessment of diarrhea in weaned,
conventionally-reared swine infected with T. suis and/or C. jejuni.

 

 

Experiment Treatment # of pigs with diarrhea
group
Day 0 Day 21 Day 22 Day 23
Long (23 days) Group 1
Both C. jejuni 0/11 1/11 2/11 3/11
repetitions
combined Group 2
C. jejuni
andeuis 0/11 6/11 6/11 7/11
Group 3
Tsuis 0/11 5/11 5/11 10/11
Group 4
E. coIiDHS-a 0/11 0/11 0/11 0/11
Group 5
Uninfected 0/11 0/11 0/ 11 0/11

 

All animals were assessed for diarrhea during fecal sampling on days 0, 21, 22, and 23 of
the long term challenge experiments.

146

Table 2.9. Fecal cytokine recovery rate.

 

 

 

 

 

 

 

Cytokine Fecal cytokine recovery rate (%)
lL-8 10
TNF-a 20
IL- 1 -[3 10
IL-4 10
IL-6 20
IL- 1 0 10

 

 

 

 

147

4500‘

 

 

 

 

 

1500

4000

H

o 3500-

3 IDay 0
I 3000-

H IDay 1
E 2500< .

m EJDa 2
Q 2000 y

a

vi

0

I

.3

H

1000

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control and T. suis DHS-a

Treatment groups

Figure 2.8. Changes in IL-8 levels were quantified in the feces of weaned piglets fi'om
the short term challenge experiments. The pigs were inoculated via oral gavage with
various treatments. Fecal samples were collected on days 0, 1, and 2 for further
processing to be used in ELISA. Values shown are means of two identical short term

(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean 1 standard error of the mean (SEM); P=0.56.

148

 

 

 

 

 

H
o
3
a lDay 0
E
\ IDay 1
tn
9. ElDay 2
1:
vi
a
I
E

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control and T. suis DHs-a

Treatment groups

Figure 2.9. Changes in TNF-u levels were quantified in the feces of weaned piglets from
the short term challenge experiments. The pigs were inoculated via oral gavage with
various treatments. Fecal samples were collected on days 0, 1, and 2 for firrther
processing to be used in ELISA. Values shown are means of two identical short term

(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean i SEM; P=0.15.

149

8000‘

 

7000

 

 

6000
lDa 0
5000 y
lDa 1
4000 - Y
DDay 2

 

3000

 

 

2000

IL-l-p in pg/ml stool

1000

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control and T. suis DHS-a

Treatment groups

Figure 2.10. Changes in IL-l-B levels were quantified in the feces of weaned piglets
from the short term challenge experiments. The pigs were inoculated via oral gavage
with various treatments. Fecal samples were collected on days 0, 1, and 2 for further
processing to be used in ELISA. Values shown are means of two identical short term
(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean i SEM; P=0.27.

150

 

 

 

 

. IDay O

 

IDay 1
DDay 2

 

 

 

IL—4 in pg/ml stool

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis DH5-alpha

Treatment groups

Figure 2.11. Changes in IL-4 levels were quantified in the feces of weaned piglets fi'om
the short term challenge experiments. The pigs were inoculated via oral gavage with
various treatments. Fecal samples were collected on days 0, l, and 2 for firrther
processing to be used in ELISA. Values shown are means of two identical short term

(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean i SEM; P=0.26.

151

4500

 

 

 

 

1000
500

'6' 4000 ,

O

n 3500»

m

a 3000 IDay 0
F1 2500 IDa 1
n- 2000 y
3 1500 DDay 2
\D

I

q

H

 

Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis DH5—alpha

Treatment groups

Figure 2.12. Changes in IL-6 levels were quantified in the feces of weaned piglets fi'om
the short term challenge experiments. The pigs were inoculated via oral gavage with
various treatments. Fecal samples were collected on days 0, 1, and 2 for firrther
processing to be used in ELISA. Values shown are means of two identical short term

(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean i SEM; P=0.45.

152

 

 

IDay 0
IDay 1

 

DDay 2

 

 

IL-10 in pg/ml stool

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis DES—alpha

Treatment groups

Figure 2.13. Changes in IL-10 levels were quantified in the feces of weaned piglets
from the short term challenge experiments. The pigs were inoculated via oral gavage
with various treatments. Fecal samples were collected on days 0, l, and 2 for further
processing to be used in ELISA. Values shown are means of two identical short term
(2 days) challenge experiments with a total of 10 pigs in each group. The data represent
the mean i SEM; P=0.79.

153

3500.00 ‘

 

 

 

 

1000.

H

8 3000.

ii 2500. IDay 0
a IDay 21
E: 2000. DDay 22
fl 1500' ElDay 23
‘I'.

(D

I

.:1

H

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control and T. suis DHs-a

Treatment groups

Figure 2.14. Changes in fecal IL-8 levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected from infected, weaned piglets.
The values shown are means of two identical long term (23 days) challenge experiments
with a total of 1 1 pigs in each group (n=8 in the E. coli DHS-a group). The data
represent the mean 1 SEM; P 2 0.05.

154

2500- , 7 . _- __ , ”a

 

2000

 

‘IDay 0
’IDay 21
_ DDay22
»ElDay23

 

 

1500 ‘

 

1000 "

 

 

 

 

TNF-a in pg/ml stool

   

 

 

 

 

 

 

 

 

 

 

 

,3".

 

 

 

 

Milk only C. jejuni C. jejuni T.suis E.col.i DHS—a
and T.suis

Treatment groups

Figure 2.15. Changes in fecal TNF-a levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected fiom infected, weaned piglets.
The values shown are means of two identical long term (23 days) challenge experiments
with a total of 11 pigs in each group (n=8 in the E. coli DHS-a group). The data
represent the mean i SEM; P 2 0.05.

155

 

 

 

 

 

§
U 50004 if
n l
a 4000 4
\ l
a .
3000
n l
a
a, 2000 1
I l
H
,_'-. 1000 *7 7
H
0 r
Milk Control C. jejuni C. jejuni and T. suis E. coli DES—a

T. suis

Treamnt groups

Figure 2.16. Changes in fecal IL-l-B levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected from infected, weaned piglets.
The values shown are means of two identical long term (23 days) challenge experiments
with a total of 11 pigs in each group (n=8 in the E. coli DH5-a group). The data
represent the mean i SEM. * Represents the significant difference of the values for the
T. suis-infected pigs on day 23 in comparison to the values for the milk only control pigs;
P S 0.05.

156

 

450

 

 

 

 

 

H
g 400
f; 350 IDay o
E 300 IDay 21
B 250 ElDay 22
°' 20° IDay 23
g 150
‘1 100
l
A 50
H
O .
Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis HHS-alpha

Treatment groups

Figure 2.17. Changes in fecal IL-4 levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected from infected, weaned piglets.
The values shown are means of two identical long term (23 days) challenge experiments
with a total of 1 1 pigs in each group (n=8 in the E. coli DHS-a group). The data
represent the mean i SEM; P 2 0.05.

157

 

 

IDay 0
IDay 21
EIDay 22
IDay 23

 

 

 

IL-6 in pg/ml stool

 

 

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis DES-alpha

Treatment groups

Figure 2.18. Changes in fecal IL-6 levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected from infected, weaned piglets.
The values shown are means of two identical long term (23 days) challenge experiments
with a total of 11 pigs in each group (n=8 in the E. coli DHS-a group). The data
represent the mean 1 SEM; P 2 0.05.

158

 

 

IDay O
IDay 21
DDay 22
away 23

 

 

 

IL-10 in pg/ml stool

 

 

Milk C. jejuni C. jejuni T. suis E. coli
Control only and T. suis DES—alpha

Treatment groups

Figure 2.19. Changes in fecal IL-lO levels were measured using ELISA at various time
points over a 23-day period. Fecal samples were collected from infected, weaned
piglets. The values shown are means of two identical long term (23 days) challenge
experiments with a total of 11 pigs in each group (n=8 in the E. coli DHS-a group). The
data represent the mean :t SEM; P 2 0.05.

159

Chapter 3: “Evaluation of proinflammatory cytokine production in intestinal pig

epithelial cells infected with Campylobacterjejzmi”

Abstract

C. jejuni is one of the leading causes of gastroenteritis throughout the world.
Enterocytes serve as the first line of defense during bacterial infection, and C. jejuni—
enterocyte interaction may play a significant role in the production of an acute
inflammatory response. It has been shown in vitro that C. jejuni interacts with intestinal
epithelial cells and stimulates the production of proinflammatory cytokines and
chemokines. Therefore, we hypothesized that cultured undifferentiated, crypt-like
intestinal pig epithelial cells (IPEC-l cells) secrete TNF-a, IL-l-B, and IL-8 following C.
jejuni infection. To test this hypothesis, we infected cultured IPEC-1 cells with C. jejuni
and measured IL-8, TNF-a, and lL-l-B production. Supernatants were collected from
IPEC-l cells at O, 8, 24, and 48 hours post-C. jejuni infection, and cytokine expression
was measured using an enzyme-linked immunoabsorbant assay (ELISA). C. jejuni
infection of IPEC-1 cells significantly induced TNF-a and IL-l-B production. In
addition, we found that IPEC-1 cells constitutively secrete IL-8, and IL-8 production was
not induced by C. jejuni infection. Our data suggest that induced TNF-a and IL-l-B
production and constitutively secreted IL-8 may play a role in an innate inflammatory
response that generates or modulates immune responses during C. jejuni infection in vivo.
We also pretreated undifferentiated IPEC-l monolayers with recombinant swine lL-8,
TNF-a, or IL-l-B to determine the direct effects of these cytokines on C. jejuni

adherence, invasion, and the transepithelial electrical resistance of IPEC-l cells. Our

160

findings suggested that neither lL-8 nor lL-l-B pretreatments had a significant effect on
C. jejuni invasion, adherence, or the transepithelial electrical resistance of IPEC-1 cells.
In addition, we found that TNF-a did not significantly affect C. jejuni adherence to IPEC-
] cells or the transepithelial electrical resistance of IPEC-l cells. Taken together, the data
presented here suggest that the induced production of TNF-a and IL-l-B and the
constitutive production of IL-8 from intestinal epithelial cells are indicative of an acute
innate host immune response that may be responsible in part for eliminating these

bacteria from the host in vivo.

16]

Introduction

Camp}'lobacterjejzmi is a Gram negative, microaerophilic, rod-shaped bacterium
which is known throughout the world as one of the leading causes of acute gastroenteritis
(34). Documented clinical symptoms related to C. jejuni infection are abdominal cramps,
diarrhea, fever, general malaise, backache, headache, and arthralgias (5, 6, ll, 12, 34). In
human clinical infection studies, flesh blood, mucus, and polymorphonuclear leukocytes
are found in diarrheic stool samples (34). In most cases, illness is self-limiting, and the
duration of the ilhiess is usually 1 week or less (34). In addition to the immediate effects
of acute Campylobacter infection, there are long-term sequelae associated with
campylobacteriosis such as reactive arthritis (ReA), Reiter’s syndrome, Fisher syndrome,
and Guillain-Barré Syndrome (41). These post-infectious sequelae result in serious
health problems and have a significant economic impact (19, 28, 35, 41, 49, 54). The
causative mechanisms of disease by Campylobacter infections are not well understood.
However, it is believed that host immune responses, such as cytokine production, may
play an important role in the pathogenesis of the disease.

Presently, there is limited knowledge about immune responses elicited during C.
jejuni infection. In vitro, human intestinal epithelial cells (INT 407), monocytic cells,
avian primary chick kidney cells, and an avian macrophage cell line secrete pro-
inflammatory cytokines (TNF-a, IFN-y, IL-I-B and IL-6) and chemokines (IL-8, MCP-l,
and MIP-l) following infection with viable C. jejuni (4, 25, 27, 31, 43). In these systems,

C. jejuni invade and are found within vacuoles. C. jejuni also invades intestinal pig

I62

epithelial cells (IPEC-1) with a differentiated phenotype and stimulates IL-6, IL-10, and
IL-18 production (Parthasarathy, dissertation, 2004 and Jones, dissertation, 2005).

In a Campylobacter swine disease model, weaned piglets infected orally with C.
jejuni show MCP-1, TNF-a, IL-12p40, IFNy, IL-4, and IL-10 mRNA expression in the
jejunum at 48 hours post-infection, and C. jejuni infection causes lesions in the jejunum
and mild crypt distension with sloughing epithelial cells and mucus production. (Jones,
dissertation, 2005). These reported findings suggest that cytokines may play a significant
role in C. jejuni-induced intestinal inflammation and disease pathology. Further
evaluation of C. jejuni-host cell interaction and cytokine expression is necessary to
elucidate the immune response elicited during infection.

There has been limited data reported which suggest that C. jejuni interaction with
or invasion of epithelial cells play a role in stimulating cytokine production (4, 9, 25-27,
43). C. jejuni invasion of intestinal epithelial cells has been observed in vitro and in vivo
and has been suggested as playing an important role in the pathogenesis of
campylobacteriosis (56, 59). However, to date, a direct correlation between C. jejuni
invasion of intestinal epithelial cells and subsequent cytokine production resulting in
disease and pathology has not been established. These types of studies cannot be done in
human patients. However, in C. jejuni-infected pigs, the bacterial cells have been found,
via irnmunohistochemical staining, to be localized in the crypts of the distal colon, the
lymphoglandular complexes (LGCs, and within the follicle-associated epithelium of the
LGCs (10, 39)(Jones, dissertation, 2005). Also, haemotoxylin and eosin (H&E) staining
of intestinal tissue samples from these C. jejuni-infected pigs showed mild crypt

distension with sloughed epithelial cells and mucus. Likewise, in these experiments,

163

proinflammatory cytokine production was associated with C. jejuni infection of tissues
(Jones, dissertation, 2005). Because it has been shown that C. jejuni-host-cell interaction
can trigger an immune response (such as stimulating intestinal epithelial cells to produce
pro-inflammatory cytokines and chemokines) and these events are associated with
disease and pathology, we hypothesize that pro-inflammatory cytokine production of
IECs may also affect epithelial cell integrity and play a role in affecting C. jejuni
adherence, invasion, and/or the transepithelial resistance of intestinal epithelial cells.
Others have shown that specific cytokines (e. g. TNF-a, IFN-y, and IL-l-B) can alter
intestinal epithelial cell function or integrity of the epithelial barrier, thereby facilitating
bacterial-enterocyte interactions and causing serious pathological consequences (24, 48,
51).

We designed this study to better understand the role played by undifferentiated,
crypt-like intestinal epithelial cells in a natural host in immunity and immunomodulation
following C. jejuni infection. We used undifferentiated, crypt-like IPEC-1 cells derived
from an unsuckled newborn piglet to evaluate pro-inflammatory cytokine production
following C. jejuni infection and some of the possible roles these cytokines play in C.
jejuni infection. Because C. jejuni-enterocyte interaction has been shown to trigger an
acute immune response in vitro and in vivo, we hypothesized that undifferentiated, crypt-
like IPEC-I cells secrete TNF-a, IL-l-B, and IL-8 in response to C. jejuni infection. Our
findings show that C. jejuni induces these swine intestinal epithelial cells to produce
TNF-o. and IL-l-B and that IL-8 is not induced by C. jejuni exposure but is constitutively
secreted by these cells. These findings suggest that this early, innate response may

participate in stimulating adaptive ThI-type adaptive immune responses which play an

164

important role in bacterial elimination in other hosts. In addition, to elucidate the roles of
pro-inflammatory cytokines in C. jejuni-intestinal epithelial cell interaction, we also
evaluated how the treatment of IPEC-l cells with recombinant swine TNF-a, IL-l-B, or
IL-8 directly affects C. jejuni adherence, invasion and/or the transepithelial electrical
resistance. We found that these pro-inflammatory cytokines do not influence C. jejuni
adherence and/or invasion of intestinal epithelial cells, nor do they stimulate changes in

the monolayer integrity to facilitate C. jejuni infection.

165

Materials and Methods

Bacteria and growth conditions

C. jejuni strain ATCC 33292 was grown at 37°C, 5% C02 on Bolton agar plates.
This strain was originally isolated from a human with enteritis and was rederived by a
single passage in a gnotobiotic pig. Escherichia coli DH5-0t was a noninvasive negative
control for the invasion assay, and E. coli 0157:H43 was a positive control for the
adherence assay. E. coli 0157:H43 (DEC7A) was kindly provided by Dr. Thomas
Whittman of Michigan State University. The E. coli strains were grown on Bolton agar
at 37°C. All bacterial culture media were obtained fiom Oxoid (Oxoid Inc, Basingstoke,
Hampshire, England) unless otherwise stated. All bacteria were taken from frozen
stocks, streaked for isolation onto agar and incubated for 24-48 hours. For inoculation,
the C. jejuni and E. coli isolated colonies were subcultured for 20 hours for confluent
growth, harvested with sterile swabs, and suspended in RPMI 1640 medium (lnvitrogen,
lnvitrogen, Frederick, MD) without phenol red, fetal bovine serum (lnvitrogen,
Frederick, MD) or insulin-transferrin selenium (lnvitrogen, Frederick, MD). The optical
density (OD560) was adjusted to 0.1 to achieve 5 X 108 colony forming units per milliliter.
Dilutions of the bacteria were made in RPMI 1640 medium (lnvitrogen, Frederick, MD)
without phenol red, fetal bovine serum (F BS; lnvitrogen, Frederick, MD) and insulin-
transferrin selenium (ITS; lnvitrogen, Frederick, MD) or Bolton broth (C. jejuni and E.

coli strains) as required to achieve a particular bacterial dose.

166

Cultured epithelial cells

IPEC-1 (intestinal pig epithelial cells) is a non-immortalized cell line originally
derived from the small intestine of an unsuckled neonatal piglet. In this study, these cells
were used in an undifferentiated state to represent the rapidly dividing basilar intestinal
epithelial crypt cells. Two-to-three days after seeding, they form confluent monolayers
but do not have tight junctions or distinct apical or baso-lateral surfaces, which is
characteristic of the morphology of intestinal epithelial crypt cells.

All culture media and growth supplements were obtained from Invitrogen
(Frederick, MD) unless otherwise stated. Growth flasks, flat bottom polystyrene plates,
and transwells were obtained from Coming (Costar, Corning, NY). IPEC-1 cells were
routinely maintained in RPMI 1640 medium containing 5% fetal bovine serum (FBS) and
supplemented with 1% insulin-transferrin selenium (ITS) and grown to a confluent
monolayer in T75 flasks at 37°C in a humidified atmosphere with 5% C02. For infection
studies, the cells were gently washed with versene and released from the flask with
0.05% Trypsin-EDTA. The cells were resuspended in RPMI 1640 supplemented with
5% FBS and 1% ITS. A subsample (50 ul) of the cells was mixed 1:1 with volume of
trypan blue (Sigma, St. Louis, MO), then loaded onto a hemocytometer (Hausser
Scientific, Horsham, PA), and viable cells were counted on a Nikon inverted microscope
(Mager Scientific, Dexter, MI). Cells were seeded onto fibronectin coated (20 ug/ml)
(Sigma, Inc., St. Louis, MO) 12-well Costar tissue culture plates (Costar, Corning, NY) at
the appropriate density for each experimental design. The cells were incubated at 37°C
in humidified atmosphere with 5% C02 and allowed to grow 2-3 days to form confluent,

undifferentiated monolayers.

167

Experimental Design

C. jejuni infection of IPEC-l cells for lL-8, IL-l-B, and TN F -a analysis

To determine the effect of C. jejuni on IL-8, IL-l-B, and TNF-a secretion from
undifferentiated crypt-like cells, confluent IPEC-l monolayers were seeded at a density
of 4 x 106 per well and were infected with C. jejuni at multiplicity of infection (MOI) of
40:1 and 200:1 and 400:1. The C. jejuni inocula were prepared by diluting the stock
(5 x 108 CF U/ml, OD560 0.1) to the desired multiplicity of infection. The negative control
treatment was RPMI 1640 medium without phenol red, PBS or ITS. The positive control
treatment for IL-8 and TNF-a induction was phorbol myristate acetate (PMA) and
calcium ionophore (CI), each at a final concentration of 100 ng/ml. The positive control
treatments for IL-l-fl induction were recombinant swine TNF-a (500 pg/ml per well) or
phytohemagglutinin (FHA; 50 11ng per well) (Table 3.1). All treatments were prepared
and diluted in RPMI 1640 medium without phenol red, FBS or ITS to eliminate any false
positive results. Each treatment was applied in a final volume of one milliliter, and three
replicates of each treatment were analyzed for cytokine production. The IPEC-1 cells
were exposed to these treatments for O, 8, 24 and 48 hours. The supernatants were
collected from the wells at the time intervals described. In order to measure cytokine
expression at 0 hours, the growth medium containing RPMI 1640 supplemented with 5%
FBS and 1% ITS was removed, and the treatments were immediately applied to the
confluent cell monolayer, and the supernatants were immediately collected. All
supernatants from each treatment group were centrifuged at 4°C, 10,000 rpm for 5

minutes to remove cell debris, transferred to fresh 1.5 ml tubes, and stored at -80°C.

168

 

Levels of secreted lL—8, IL-l-B, and TNF-a were measured from the supernatants by

enzyme-linked immunoabsorbant assay (ELISA).

Assay for IL-8, IL-l-B, and TNF-a secretion

IL-8, IL-l-B, and TNF-a were measured using swine sandwich ELISA kits
obtained from Biosource International (Camarillo, CA); assays were performed according
to the manufacturer’s instructions. Each assay contained a IL-8, IL-l-B, or TNF-a
standard protein control. A microplate reader (EL800 Universal Microplate Reader, Bio-
Tek Instruments, Winooski, Vermont) was used to measure the absorbance at 450 nm,
and the concentration of IL-8, IL-l-B, or TNF-a in each sample was quantified using

KCjunior® software (Bio-Tek Instruments, Winooski, VT).

Invasion assay

To determine the direct effect of exogenous IL-8, IL-l-B, or TNF-a on C. jejuni
invasion of IPEC-l cells, confluent monolayers were pretreated with recombinant swine
IL-8 (0, 100, 250, 500, 750, or 1000 pg/ml); IL-I-B (0, 3000, 30,000, or 300,0000 pg/ml);
or TNF-a (0, 50, 500, or 5000 pg/ml) (Biosource International, Camarillo, CA) in a dose-
response design (Table 3.2). The cytokine concentrations used in this experiment were
determined by reports from the literature and preliminary data from previously conducted
do se-response experiments in our laboratory. Each cytokine was diluted to the
appropriate concentration in RPMI 1640 medium without phenol red, FBS or ITS and
applied to the cells in a final volume of 1 ml. The monolayers were incubated at 37°C in

a humidified atmosphere with 5% CD; for 5 hours. The cytokine pretreatment medium

169

 

was removed; the monolayers were not washed in order to avoid cell damage or
detachment. C. jejuni ATCC 33292 or E. coli DHS-a (noninvasive positive control)
inocula were added to the confluent IPEC-1 cells at a MOI of 100:1 (approximately

2 x 108 CFU/ml) and incubated at 37°C in a humidified atmosphere with 5% CO2 for 3
hours to allow bacterial invasion of the cells. The medium was removed and gentamicin
(lnvitrogen, Frederick, MD), diluted in RPMI 1640 without phenol red, at a final
concentration of 100 ug/ml, was added to the monolayers. The monolayers were
incubated for 1 hour to kill the extracellular bacteria, and then the gentamicin was
removed. The monolayers were lysed with 0.1% sodium deoxycholate (Sigma, Inc., St.
Louis, MO) in 1X Phosphate Buffered Saline (0.01 M). The internalized bacteria were
enumerated by serial dilution and spreading on Bolton agar. After 48 hours incubation,
the colonies were counted to determine the number of internalized bacteria. All

treatments were performed in triplicate.

Adherence Assay

To determine the direct effect of exogenous IL—8, IL-l-B, or TNF-a on C. jejuni
adherence to IPEC-1 cells, confluent monolayers were pretreated with each recombinant
swine cytokine as previously described for the invasion assay (Table 3.2). After cytokine
pretreatment, C. jejuni ATCC 33292 or E. coli 0157:H43 (DEC7A) (adherence positive
control strain) inocula were added to the confluent IPEC-l cells at a MOI of 100:1
(approximately 2 x 108 CF U/ml) and incubated at 37°C in a humidified atmosphere with
5% CO2 for 1 hour to allow bacterial adherence to the cells. The medium was then

removed; the monolayers were not washed in order to avoid cell damage or detachment.

I70

The monolayers were lysed with 0.1% sodium deoxycholate (Sigma, Inc., St. Louis, MO)
in 1X Phosphate Buffered Saline (0.01 M). The cell-associated (adherent) bacteria were
enumerated by serial dilution plating on Bolton agar and counting CFUs at 48 hours after

incubation. All treatments were performed in triplicate.

Transepithelial resistance assay

To determine the effect of exogenous recombinant cytokines on IPEC-1
mono layer integrity, we measured the transepithelial resistance (TER) across the
monolayers before and after treatment with recombinant swine IL-8, IL-l-B, or TNF-a
(Biosource International, Camarillo, CA). IPEC-l cells were seeded at a density of 2 x
106 per well onto 24-Transwell inserts (6.5 mm diameter, 3 pm pore size, Costar,
Corning, NY) coated with 20 ug/ml fibronectin in order to measure the TER. The cells
were incubated at 37°C in a humidified atmosphere with 5% C02 and allowed to grow 2-
3 days to form confluent monolayers. All treatments were prepared and diluted in RPMI
1640 medium without phenol red, FBS or ITS and applied to the cells in a final volume
of 1 mL.

The TER across the IPEC-l monolayers was measured before cytokine treatment
using an electrode (EVOMX, World Precision Instruments, Sarasota, FL) to assess
monolayer integrity. TER values between 150-600 Qcm2 were considered evidence of
confluent monolayers. The monolayers were exposed to recombinant swine IL-8 (250
pg/ml), IL-l-B (3000 pg/ml), TNF-a (500 pg/ml), or 0 pg/ml. They were next incubated
at 37°C in a humidified atmosphere with 5% CO2 for 5 hours, and then the TER was

measured again. All treatments were performed in quadruplicate.

171

Statistical Analysis

ELISA data were analyzed using the mixed model procedure in the Statistical
Analysis System (SAS)(SAS Institute Inc., Cary, NC) program. The fixed effects in this
model were time and treatment. The random effect in this model was the plate. P values

S 0.05 were considered significant.

Invasion and adherence data were analyzed using the mixed model procedure in
the Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC) program. The fixed
effects in this model were experiment and treatment. The random effect in this model

was the plate. P values S 0.05 were considered significant.

Transelectrical resistance (TER) data were analyzed in the Statistical Analysis
System (SAS)(SAS Institute Inc., Cary, NC) program. A generalized linear model with
one factor (treatment) was used to determine significance. P values 5 0.05 were

considered significant.

172

Results
Sensitivity of TNF-a, IL-l-B, and lL-8 ELISAs

Figures 3.1 to 3.3 show the standard curves for the TNF-a, IL-l-B and IL-8
ELISAs, respectively. The regression curves for these assays were obtained using
KCjunior® software (Bio-Tek instruments, Winooski, Vermont). The TNF-a, IL-l-fl and
IL-8 regression curves showed significant dose response curves with the concentration
range and antibody titer used, respectively, R = 1.000; R = 0.9999; R = 0.9999. The
lower limit of detection of the TNF-a and IL-I-B assays is 2 6 pg/ml, and the lower

limit of detection of the IL-8 assay is 2 10 pg/ml.

Cytokine secretion from C. jejuni-infected IPEC-1 cells

Different ratios of bacteria to IPEC-I cells were used in these experiments to
determine the time course of induction and optimize the assay (Table 3.1).

We exposed undifferentiated IPEC-1 cells to C. jejuni 33292 for 0, 8, 24, and 48
hours to determine levels of TNF-a, IL-l-B, and lL-8 expression following bacterial
infection. Figure 3.4 shows that IPEC-1 cells significantly secreted TNF-a post-C. jejuni
infection at an optimal MOI of 400:1 at 8 hours (176.97 :h 15.25 pg/ml), 24 hours (354.98
i 15.25 pg/ml), and 48 hours (181.81 :1: 15.25 pg/ml) as compared to the RPMI 1640
medium control at 8 hours (16. 56 :t 18.67 pg/ml), 24 hours (22.83 i 15.25 pg/ml), and
48 hours (28. 07 d: 15.25 pg/ml); all p < 0.0001. We found that C. jejuni-infected IPEC-1
cells showed maximum induction of TNF-a production at a bacterial MOI of 400:1 at 24
hours post-infection. We also note that at 0 hours an elevated level of TNF—a was

detected in our RPMI 1640 medium control, and we speculate that this response was

1.73

likely due to physical stress on the IPEC-I cells induced by the rapid manipulation during
the experimental technique (described in the materials and methods) used to challenge the
cells.

We also found that exposure of IPEC-1 cells to C. jejuni 33292 significantly
induced IL-l-B (Figure 3.5). By 24 hours post-infection, IL-l-B was induced at a C. jejuni
MOI of 400:1(134.27 i 16.12 pg/ml) as compared to the RPMI 1640 medium control
(40.47 i 16.12 pg/ml; p = 0.025). In preliminary trials, IPEC-1 cells were tested for
positive IL-I-B induction using phytohemagglutinin (PHA 50 ug/ml) or recombinant
swine TNF-a (500 pg/ml); over a wide range of time points. Levels of IL-l-B were found
to be beneath the level of detection of the assay (TNF-a stimulation) or were not
significantly different from the RPMI 1640 control (PHA stimulation). Therefore,
induction in this assay was defined as a positive response that was more than 3 fold
greater than the control. Because the possible positive control treatments were weak or
unavailable, this could be an experimental drawback. Follow-up experiments were
conducted to determine if the recombinant TNF- or was active, and results showed that
administered recombinant TNF- a actively stimulated IPEC-1 cells to induce IL-8 (data
not shown). However, in this case, recombinant swine TNF- u did not effectively
stimulate IPEC-1 cells to significantly produce IL-l-B.

IPEC-1 cells were not stimulated by C. jejuni 33292 exposure to secrete
significant levels of IL-8; however, IL-8 protein was constitutively expressed by IPEC-1
cells. The positive control (PMA/CI) significantly induced lL-8 by 8 hours, and IL-8
levels remained significantly elevated through 48 hours as compared to the RPMI 1640

medium control (Figure 3.6A, all p < 0.0001). Figure 368 shows that IL-8 is

174

constitutively expressed by IPEC-1 cells. From the supernatants of the RPMI 1640
(control), C. jejuni M01 40:1, and C. jejuni M01200:1 treatment groups, we detected IL-
8 production at 24 hours post-treatment and continued detecting increased IL-8 levels
through 48 hours. From the supernatants of the C. jejuni 400:1 treatment group, we
detected lL-8 production as early as 8 hours post-treatment and continued detecting a rise
in IL-8 levels through 48 hours. Although increased IL-8 levels were detected in the
supernatants of each treatment group, there were no statistically significant changes in
IL-8 secretion in response to C. jejuni infection of IPEC-1 cells (Figure 3.63, p > 0.05).
In these experiments, we determined that IL-8 secretion and accumulation was apparently

time related and not dependent on the C. jejuni challenge dose given.

Effects of cytokine pretreatment on C. jejuni invasion and adherence of IPEC-1 cells
IPEC-l cells were pretreated with IL-8, TNF-a, or IL-l-B to determine if these
cytokines have a direct effect on the interaction between C. jejuni and crypt-like basal
epithelial cells (IPEC-l cells), specifically, effects on the integrity of the epithelial cell
barrier. Treatment of IPEC-1 cells with recombinant swine IL-I-B or IL-8 did not have a
significant effect on C. jejuni adherence and/or invasion (IL-IB, Figures 3.10 and 3.11;
IL-8, Figures 3.13 and 3.14; all p > 0.05). Therefore, C. jejuni 33292 did adhere to and
invade IPEC-1 cells regardless of recombinant swine IL-l—B or IL-8 pretreatment of
IPEC-1 cells. Also, treatment of IPEC-1 cells with recombinant TNF-a did not have an
effect on C. jejuni adherence to IPEC-1 cells (Figure 3.8, p > 0.05), and we could not
draw a conclusion about its effects on C. jejuni invasion of IPEC-l cells because of the

variability observed in the number of internalized bacteria (Figure 3.7, p > 0.05). E. coli

175

DH5-a (a noninvasive strain) was the control for the invasion assays and neither TNF-a,
IL-l-B, nor lL-8 affected E. coli DH5-a interaction with IPEC-1 cells; E. coli DH5-a did
not invade IPEC-l cells regardless of cytokine pretreatment of the IECs. E. coli
0157:H43 DEC7A was the control for the adherence assays, and neither TNF-a, IL-l-B,
nor IL-8 affected E. coli 0157:H43 DEC7A adherence to IPEC-1 cells (TNF-a, Figure

3.9; IL-IB, Figure 3.12; IL-8, Figure 3.15)(all p > 0.05).

Effects of cytokine pretreatment on IPEC-l transepithelial resistance (TER)

IPEC-l cells were pretreated with IL-8, TNF-a, or IL-l-B to determine whether
these cytokines have a direct effect on the transepithelial electrical resistance of IPEC-l
cells. Treatment of IPEC-1 cells with recombinant swine IL-8, TNF-a, or IL-l-B did not
have a significant effect on the transepithelial electrical resistance of IPEC-1 cells (Figure
3.16; p > 0.05). IPEC-l cells pretreated with RPMI 1640 medium alone had a TER of
~300 Qcmz. Experimental TER values between 150-600 52cm2 were considered as
evidence of confluent monolayers with no evidence of damage to the integrity of the cells

post-treatment with recombinant swine cytokines.

176

Discussion

C. jejuni invasion or stimulation of intestinal epithelial cells (IECs) has been
shown to significantly induce the expression and up-regulation of pro-inflammatory
cytokines and chemokines (4, 25-27, 32). In vivo, a robust pro-inflammatory cytokine
response is detected in crypt-like epithelial cells (Jones, dissertation, 2005), and
Campylobacter organisms are associated with these surface epithelial cells within the
crypts and were found within the crypts of lymphoglandular complexes in the distal colon
(Jones, dissertation, 2005)(40). Pro-inflammatory cytokines such as IL-8, TNF-a and IL-
l-B can act as mediators of inflammation and regulators of immune responses which are
associated with an innate immune response that facilitates bacterial elimination during
infection (37). In this study, we hypothesized that intestinal pig epithelial cells (IPEC-1
cells), which serve as a model of undifferentiated, crypt-like cells, secrete pro-
inflammatory cytokines (specifically IL-8, TNF-a, and IL-l-B) in response to C. jejuni
infection when challenged in vitro. We also examined the treatment of confluent IPEC-l
monolayers with recombinant swine IL-8, TNF-a, or IL-I-B to determine the direct
effects of these cytokines on C. jejuni adherence, invasion, and/or the transepithelial
electrical resistance of the IPEC-l cells.

IPEC-1 cells were exposed to 5 x 108 CFU/ml of C. jejuni 33292 for 0, 8, 24 and
48 hours. At 8, 24, and 48 hours post-infection, we measured significantly increased
levels of TNF-a compared to the uninfected controls. Maximum TNF-a induction was
detected 24 hours following bacterial infection at an MOI of 400: 1. Likewise, we

measured significant levels of IL-l-B at 24 hours post-infection. Based on the data

177

presented here, C. jejuni challenge induces IPEC-l cells to secrete TNF-a and IL-l-B at
significant levels. This finding is comparable to other in vitro reports that demonstrate
the production of pro-inflammatory cytokines by intestinal epithelial cells following C.
jejuni infection (4, 9, 25, 27). Human intestinal epithelial cells, INT 407 cells, secrete an
array of pro-inflammatory chemokines (IL-8, MCP-l, GROalpha, GROgamma, and
gammaIP-IO) when stimulated with C. jejuni (4, 27). C. jejuni has also been shown to
stimulate INT-407 cells to produce intracellular IFN-y, IL-lO, TNF-a, and IL-4 (4, 47).
Induction of pro-inflammatory cytokines to C. jejuni challenge is also observed in viva.
Pigs rectally challenged with C. jejuni for 1 hour show significant up-regulation of
intracellular TNF-a, IL-18, IL-8, GM-CSF, IL-l-B, iNOS, and MCP-1 in the distal colon
and lymphoglandular complexes (LGCs)( Jones, dissertation, 2005). It has also been
demonstrated in Balb/c mice that C. jejuni induces a peritoneal inflammatory cytokine
response and increased peritoneal phagocyte oxidative activity (31, 47). Given that
increased pro-inflammatory cytokine expression elicited by C. jejuni infection of
intestinal cells has been demonstrated in vitro and in viva, it was not surprising that we
detected significant elevated TNF-a and IL-l-B levels secreted by IPEC-l cells following
C. jejuni exposure; the production of these cytokines likely plays a significant role in host
innate immunity to this bacteria.

Furthermore, in this in vitro, C. jejuni-challenge study, the cytokine response of
the undifferentiated IPEC-l cells is consistent with other in viva and in vitro findings (4,
9, 25, 27, 53) (Parthasarathy, dissertation, 2004 and Jones, dissertation, 2005). Because
IPEC-1 is a non-irnmortalzied cell line, the cytokine secretion pattern is likely more

reflective of the physiology in the host than that represented by an immortalized cell line.

178

This is strengthened by the fact that concurrent studies conducted in our laboratory have
shown that C. jejuni also invade differentiated IPEC-1 cells and stimulate these cells to
produce cytokines such as IL-18, IL-6, or IL-10 (Parthasarathy, dissertation, 2004 and
Jones, dissertation, 2005). Likewise, previous in viva data from our laboratory show that
C. jejuni stimulates a proinflammatory cytokine response after invading intestinal tissue
and has been identified in the intestinal crypt cells of infected pigs (Kathryn, dissertation,
2005). Our findings support the hypothesis that primary, undifferentiated intestinal pig
epithelial cells (IPEC-1) can serve as a more suitable model to mimic the enteric immune
response of human undifferentiated intestinal crypt cells as compared to other in vitro
systems that involve the use of immortalized cell lines, such as INT 407, T84, and HEp-2
cells. IPEC-1 cells were derived from a newborn unsuckled piglet, and pigs are
monogastric and have dietary habits and anatomical and physiological characteristics
similar to humans (7). Therefore, the overall similarities between cultured primary
intestinal pig epithelial cells (IPEC-1) and human intestinal cells allow for a better
evaluation of bacterial virulence, host immune mechanisms, and the possible role played
by intestinal epithelial cells as an important source and initiator of enteric immune
responses following assault.

The functions of IL-l-B and TNF-a are similar and play a significant role in
enteric immune responses. TNF-a activates neutrophils, mononuclear phagocytes and
eosinophils which can amplify the mucosal‘inflammatory response to efficiently eradicate
infectious microorganisms (1). In viva, mice exposed to a primary L. manacytagenes or
C. jejuni infection produced significant plasma TNF-a which is believed to enhance

innate immune functions(37). Together or independently, TNF-a or IL-l-B can also

179

stimulate inflammatory cells to produce IL—8 which contributes to neutrophil influx, and
the combined action of TNF-a, IL-l-B and IL-6 contribute to systemic acute-phase effects
which can induce fever (increased body temperature is not conducive for the growth of
some pathogenic bacteria)(57). In addition, it is suggested that IL-l-B plays a pivotal role
in early events leading to inflammation (50, 57). In vitro, nontransformed epithelial cells
rapidly express IL-l-B mRN A, and secreted IL-l-B initiates and enhances the acute
inflammatory response following bacterial infection (16, 29, 57). IL-l-B has also been
shown to be predominantly expressed in undifferentiated cells located in the basal
mucosal crypts following acute experimental colitis . Therefore, in agreement with these
findings, the present data suggest that the significant production of TNF-a and IL-l-B in
response to C.jejuni infection of IPEC-l cells may play a significant role in initiating and
modulating immune responses that facilitate the elimination of C. jejuni infection in viva.

IL-8 is a proinflammatory cytokine that is a chemoattractant which induces
adherence to vascular endothelium and extravasation into tissues (17). We found that
IPEC-1 cells constitutively secrete IL-8 protein, and IL-8 production was not induced by
C. jejuni infection. The constitutive expression of IL-8 by the undifferentiated, crypt-
like IPEC-1 cells is consistent with other reports showing that various intestinal epithelial
cell lines (T84, Caco-2, SW620, and HT29) constitutively secrete IL-8. Also, some of
these cell lines can significantly express IL-8 depending on the stimulant (17, 18, 25, 43).
Even though IPEC-1 cells were not induced to secrete IL-8 following C. jejuni exposure,
the constitutive expression of IL-8 protein constitutes a significant inflammatory response
when one considers the number of epithelial cells lining the colon. In viva, the gut

epithelium is continuously exposed to a variety of harmless microflora, antigenic dietary

180

constituents, and bacterial pathogens that can damage it by directly invading the mucosa
or by toxin production. It is possible that there is a continuous, basal level of
inflammation within the intestinal mucosa which can be up-regulated when necessary in
response to pathogens and noxious antigens thereby providing protective immunity (16,
60). The constitutive production of IL-8 at low levels along the gut mucosal epithelium
could be partially responsible for a consistent low-level. inflammatory cellular infiltration
of neutrophils and monocytes which would be favorable to the host (16, 25, 43).
Therefore, we suggest that intestinal pig epithelial cells (IPEC-l cells) constitutively
secrete IL-8 protein as a molecular inflammatory mediator of innate host defense which
may play a role in limiting microbial exposure at the mucosal surface and maintaining
tissue integrity for low level insults. 0n the other hand, mature differentiated epithelial
cells lining the villus tips of the colon or dendritic cells and macrophages populating the
lamina propria immediately underlying the epithelium may be responsible for the IL-8
expression hypothesized to draw neutrophils to the this site.

Because others have reported that IL-8 is significantly induced via NF KB
activation through lea degradation following Salmonella Dublin, enteroinvasive
Escherichia coli, Yersinia enterocolitica, Helicobacter pylori, or C. jejuni invasion or
stimulation of IECs (15, 20), we believe it is necessary to discuss a few possible reasons
why IL—8 was not significantly induced by C. jejuni infection of IPEC-1 cells in our
study. High concentrations of diverse bacteria and bacterial products continually interact
with the intestinal mucosal epithelium. Because of this intimate contact, it is critical that
the intestinal epithelia facilitate the control and maintenance of a normal healthy state to

avoid persistent, dysregulated inflammation. Gut epithelial cells express toll-like

181

 

receptors (TLRs) which are a family of transmembrane receptors also termed pattem-
recognition receptors (PRRs) (33, 42). TLRs play an important role in host innate
immunity by recognizing pathogen-associated molecular patterns (PAMPs) present on
diverse microbes including Gram-positive and Gram-negative bacteria, fungi, and
mycobacteria (33). In general, infection of IECs can lead to activation of the TLRs
which then initiates intracellular signaling cascades which can lead to the activation of
the NFKB/Rel family transcription factors and cytokine production (2, 3, 38).

NFKB has been described as the central regulator of the intestinal epithelial cell
innate immune response induced by enteroinvasive bacterial infection (20, 38). NFKB is
activated by many extracellular stimuli via TLR interaction, and there are many signaling
pathways that lead to NF KB activation which leads to the expression of many genes,
including those encoding inflammatory cytokines (14, 38). More specifically, IL-8
production has been associated with TLR4 receptor signaling via LPS stimulation and
NFKB activation (2, 3, 13, 21). It has been shown that immune-mediated signals regulate
TLR-4 and MD-2 (a TLR4 coreceptor accessory molecule) expression, and changes in
TLR-4 and MD-2 expression impact host cell responses to bacterial lipopolysaccharide
(LPS) (20, 46, 60). In addition, it has been reported that TLR4 is barely detectable in
isolated primary intestinal epithelial cells (3 8). Therefore, it is possible that decreased
expression of Toll-like receptor-4 (TLR-4) and MD-2 or inhibition of intracellular
signaling via the transcription factor can affect assessment of proinflammatory gene
expression in some in vitro systems (3, 33). Hence, if TLR4 and its accessory molecules
are only present at low levels or absent from the cell surface of IPEC-1 cells (isolated

primary intestinal epithelial cells), then C. jejuni LPS either weakly interacts with TLR4

182

and its accessory molecules or does not bind the receptor complex to induce significant
production of IL-8 thereby causing IPEC-1 cells to be unresponsive to this particularly
bacterial component (C. jejuni LPS). It is also reasonable to think that C. jejuni-infected
IPEC-l cells can significantly produce IL-l-B and TNF-a and do not significantly induce
IL-8 expression because induction of these cytokines is likely regulated via different
Toll-like receptor signaling or different intracellular signaling pathways. It has been
demonstrated that the flagellin of other bacteria, such as Listeria manacytagenes and
Salmonella typhimurium , can bind to TLR5 receptors (TLRs that specifically recognize
bacterial flagellin) to stimulate TNF-a, IL-8, or lL—l—B production (22, 33, 55), whereas,
C. jejuni flagellin has been reported to be a poor stimulator of TLR5 and does not play a
significant role in stimulating IL-8 production nor has it been tested to determine if it
stimulates TNF—a or IL-l-B production (58). Even though not tested in this study, we
suggest that C. jejuni-infected IPEC-1 cells express lL-l-B, TNF-a, or IL-8 via different
TLR ligand signaling recognition or other intracellular signaling pathways. Additionally,
recent multilocus sequence typing studies of C. jejuni strains in our lab suggest that some
strains have LOS variants that may be more stimulating than others. Further studies are
needed to elucidate the regulatory mechanisms involved in the interaction between C.
jejuni bacterial products or components and intestinal epithelial cell TLR activation,
intracellular signaling pathways, and cytokine gene expression and secretion.

Secreted cytokines can exert autocrine (self-stimulation), paracrine (adjacent),
intracrine (within the cell) and some endocrine (distant) actions (23, 33, 42). Cytokines
have been shown to modulate bacterial-enterocyte interactions in vitro. For instance,

cytokines can disrupt intestinal epithelial cell function or barrier integrity (24, 48, 51).

183

Increased bacterial adherence has been associated with IFN-y pretreatment of IECs
suggesting that IFN-y augments bacterial adherence to the intestinal epithelium (24). In
addition, IF N -y and TNF-u act synergistically on the epithelial barrier by causing reduced
TER and altering cell morphology (21). Likewise, concurrent studies in our laboratory
have shown that recombinant swine IL-4 (rIL-4) pretreatment of differentiated IPEC-1
cells significantly reduced the TER of IPEC-1 cells, increased C. jejuni internalization,
and caused differential effects on adherence of two C. jejuni strains (Parthasarathy,
dissertation, 2004). Because of the impact cytokines have on enterocytes and the
possible roles they play in bacterial-enterocyte interaction, in this study, we pretreated the
IPEC-1 cells with recombinant swine IL-8, TNF-a, or IL-l-B to determine if these
cytokines would affect C. jejuni invasion, adherence and/or the transepithelial electrical
resistance (TER) of IPEC-1 cells. Our results showed that C. jejuni 33292 does adhere to
and invade IPEC-l cells, and these data support previous findings from our laboratory
(Parthasarathy, dissertation, 2004). However, neither IL-8 nor IL-l-B has a significant
direct effect on C. jejuni invasion, adherence, and/or changes in transepithelial electrical
resistance of undifferentiated IPEC-1 cells. Also, treatment of IPEC-1 cells with TNF-a
did not significantly affect C. jejuni adherence or changes in the TER of the IECs.
However, we were unable to draw conclusions about the effects of TNF-a on C. jejuni
invasion of IPEC-1 cells because of the variability observed in these experimental results.
Additional experiments could be conducted to address this concern; however, the lack of
a significant effect under the rigorous experimental conditions employed, suggest efforts
might be better placed elsewhere. Even though there was not a direct effect on C. jejuni

adherence to or invasion of IPEC-l. cells (in response to 1L-8 or IL-l-B pretreatment) or a

184

 

direct effect could not be determined following TNF-a pretreatment in vitro, it is still
possible that IL-8, TNF-a, or IL-l-B may affect epithelial function in a paracrine-like or
another autocrine-like manner during C. jejuni infection in viva. For instance, it has been
demonstrated that cytokines can facilitate enterocyte proliferation, maturation, MHC
class II expression, cytokine receptor expression or apoptosis in healthy and diseased
states (21, 48, 51). In addition, TNF-a and TGF-a have been shown to stimulate
immature crypt cell proliferation, and TGF—B and INF-y can inhibit mitotic activity (52).
IFN-y can alter immature crypt epithelial cell turnover and upregulate MHC class II
expression (51). Therefore, alterations of cytokine concentrations as a consequence of
bacterial exposure and intestinal inflammation may contribute to serious pathologic
consequences. Further elucidation of the effects of distinct cytokines on epithelial cell
growth, phenotype, firnction and bacterial-enterocyte interactions would be of importance
in the understanding of cytokine mediated regulation of mucosal immune responses
associated with C. jejuni infection.

In summary, we showed that undifferentiated, crypt-like intestinal pig cells
(IPEC—1) can serve as a suitable in vitro model for studying C. jejuni-enterocyte
interaction and host enteric immune responses elicited by C. jejuni during infection. We
also found that C. jejuni infection of these cells significantly induces TNF-a and lL-l-B
production. In addition, we observed that IPEC-1 cells constitutively secrete IL-8. The
data presented here suggest that TNF-a and IL-l-B production and constitutively secreted
IL-8 are significant inflammatory responses which are associated with an acute innate
immune response and may lead to a healing Thl-type adaptive immune response. It is

likely that this inflammatory response can generate and/or modulate the immune response

185

to benefit the host by contributing to the elimination of the bacteria. In addition, we
found that neither lL-8 nor IL-l-B had a direct effect on C. jejuni adherence, invasion, or
the TER of IPEC-l cells. Likewise, we found that TNF-a did not have a direct effect on
C. jejuni adherence to IPEC-l cells or the TER of IPEC—1 cells. Even though these
findings showed no direct effect of these cytokines on C. jejuni interactions (specifically
adherence, invasion, or changes in IPEC-l TER) with the swine intestinal epithelial cells,
they could possibly have other autocrine- or paracrine-like effects which could play a role
in host inflammatory immune responses associated with C. jejuni infection. For example,
TNF-a and IL-l-B can stimulate intestinal epithelial cells to produce C3, a complement
component which is associated with host acute-phase inflammatory responses (21, 44, 45,
48, 51). So, given that our data support the hypothesis that TNF-a, IL-l-B, and IL-8 are
cytokines that are important components of the inflammatory immune response to enteric
pathogens, we can utilize this information to more specifically determine, characterize,
and elucidate the possible roles played by IL-8, TNF-a, and/or IL-l-B during C. jejuni

infection.

186

10.

11.

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192

 

Table 3.1. IPEC-1 cells treatments for cytokine measurement (ELISA)

 

 

 

 

 

 

 

 

Treatment
Group Treatment
1 Positive controls:
IL-8 and TNF-a phorbol myristate acetate (PMA) (100 ng/ml)
calcium ionophore (CI) (100 ng/ml)
IL- 1 -B phytohemagglutinin
(FHA, 50 ug/ml)
or recombinant swine TNF-a
(rTNF-a, SOOpg/ml)
2
Campylobacterjejuni 33292 (MOI 40:1)
3
Campylobacterjejuni 33292 (MOI 200: I)
4
Campylobacterjejuni 33292 (M01 400: 1)
5 Negative control:
RPMI 1640 without phenol red, fetal bovine serum (FBS), or
insulin-transferrin-selelium (ITS)

 

I93

 

 

Table 3.2 Recombinant swine cytokine pretreatment concentrations for invasion and
adherence assays

All treatments were suspended in RPMI 1640 without phenol red, fetal bovine serum
(F BS) or insulin-transferrin-selenium (ITS). The negative control was identified as

medium only.

 

 

 

 

 

 

 

 

 

 

 

Treatment Recombinant IL-8 Recombinant IL-l-B Recombinant
Group (rIL-8) (rIL-l-B) TNF-a
(rTNF-a)

Negative control: Negative control: Negative control:

1 Medium only Medium only Medium only
0 pg/ml 0 pg/ml 0 pg/ml

2 100 pg/ml 3000 pg/ml 50 pg/ml

3 250 pg/ml 30,000 pg/ml 500 pg/ml

4 500 pg/ml 300,000 pg/ml 5,000 pg/ml

5 750 pg/ml Not applicable Not applicable

6 1000 pg/ml Not applicable Not applicable

 

 

194

 

1.2

 

 

     

 

 

 

 

 

 

 

1 _—
E
= 0.8 at
v
o
2 06
a .
.o
h
,2 0.4 r 0 Standards
4: — Standard
0.2 - curve
I
O T I I I 7
0 500 1000 1500 2000 2500 3000

Concentration (pg/ml)

4 Parameter (y = (A - D) / (1 + (x/C)"B) + D)
A=19.1384 B=-1.0057 C=47262. 147 D=0.0298, R-Square = 1.000

Figure 3.1. Standard curve for swine TNF-a used in the ELISA. The curve is
representative of three identical experiments.

I95

 

1.2

 

 

 

 

 

 

 

 

1 +
P
g 0.8 -—
g .
‘
cu
U 0.6 "r
:
a
a:
E 0.4 -- 0 Standards
E — Standard
< 0 2 fl curve

0 we 1 1 t :

0 500 1000 1500 2000 2500 3000

Concentration (pg/ml)

4 Parameter (y = (A — D) / (1 + (x/C)"B) + D)
A=2.556O B=-I.1 l 16 C=3407.3799 D=0.0077, R-Square = 0.9999

Figure 3.2. Standard curve for swine IL-l-B used in the ELISA. The curve is
representative of three identical experiments.

I96

 

0.9 r
0.8 ‘r
0.7 t
0.6 “r
0.5 at
0.4 2*

 

 

  

0 Standards
— Standard
curve

0.3 *—

Ahsorhance (nm)

0.2 “r
0.1 *-

 

 

 

 

 

 

 

0 , i i l l
O 500 1000 1500 2000 2500

Concentration (pg/ml)

4 Parameter (y = (A — D) / (1 + (x/C)"B) + D)
A=3.3492 B=-1.3873 C=3570.7411 D=0.0087, R-Square = 0.9999

Figure 3.3. Standard curve for swine IL-8 used in the ELISA. The curve is
representative of three identical experiments.

197

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

400
*
350
:4; 300 B0 hours
\
g 250
- * * 138 hours
a 200
'd I
'3' 150 :i': _ .24 hours
ll,
2 ll :l: :l: B48 hours
g I“ ' I'l r't
50 ‘ I'l 'l' I'I Ill _d
m III '|' r't u't
Us I 'I' I'I I't
O _.

 

 

 

PMA/CI RPMI 1640 C. jejuni C. jejuni C. jejuni
MOI 40:1 MOI 200:1 MOI 400:1

Treatment groups

Figure 3.4. TNF-a secretion by IPEC-1 monolayers after exposure to C. jejuni 33292.
Phorbol myristate acetate (PMA 100 ng/ml) and calcium ionophore (CI 100 ng/ml) were
used as the positive control. RPMI 1640 medium was used as the negative control. All
treatments were performed in triplicate and are representative of three identical
experiments. Values shown represent the mean 3: SD. An asterisk (*) indicates the
significant difference between C. jejuni MOI of 400:1 and the RPMI 1640 (uninfected)
control at 8, 24, and 48 hours post-infection (P < 0.0001).

198

180

 

 

 

 

 

: 160
E 140 I0 hours
m
‘9 120
e 100 1118 hours
4.)
g 80 , .
I I I .24 hours
H 60 :l : I
I
g 40 i: i i
3 ': ' I :48 hours
a 20 'I I I

'l : I

l

rTNF- PHA RPMI 1640 C. jejuni C. jejuni C. jejuni
alpha MOI 40:1 MOI 200:1 MOI 400:1

Treatment groups

Figure 3.5. IL-l-B secretion by IPEC-1 monolayers after exposure to C. jejuni 33292.
Recombinant swine TNF-alpha (500 pg/ml) or phytohemagglutinin (PHA 50 ug/ml) was
used as the positive control. RPMI 1640 medium was used as the negative control. All
treatments were performed in triplicate and are representative of three identical
experiments. Values shown represent the mean 3: SD. An asterisk (*)indicates a
significant difference between C. jejuni MOI of 400:1 and the RPMI 1640 (uninfected)
control at 24 hours post-infection (P < 0.001).

199

Figure 3.6 (A and B). The two graphs represent experiments conducted at the same
time. (A) Positive control: IPEC-l monolayer positively induced to secrete IL-8
following phorbol myristate acetate (PMA 100 ng/ml) and calcium ionophore (CI 100
ng/ml) exposure. RPMI 1640 medium is the negative control. Values shown represent
the mean i SD. An asterisk (*)indicates a significant difference between the positive
control values at 8, 24, and 48 hours as compared to the RPMI 1640 control, P value was
< 0.0001. (B) IL-8 secretion by IPEC-l monolayers after exposure to C. jejuni 33292.
RPMI 1640 medium was used as the negative control. All treatments were performed in
triplicate, and the values shown represent three identical experiments. Values shown
represent the mean i SD. For all values, P was > 0.05 compared to the RPMI 1640
(uninfected) control.

200

Figure 3.6A.

(pg/m1)

IL-B

9000

8000

7000

6000

5000

4000

3000

2000

1000

O

0 hours

Figure 3.68.

IL-8 (pg/m)

1200

1000

 

8 hours 24 hours

Time (hours)

48 hours

 

fiPMA/CI
IRPMI 1640

 

 

 

 

 

RPMI 1640

 

C. jejuni C. jejuni
MOI 40:1 MOI 200:1

Treatment groups

201

 

C. jejuni
MOI 400:1

 

IO hours

m8 hours

I24 hours

E48 hours

 

 

 

 

 

 

 

 

 

 

 

 

 

    

 

.51
7“ i; *1

O 50 500

 

 

Internalized bacteria (CPU)
N
O
O

 

 

 

 

 

Swine rTNF-alpha concentrations (pg/m1)

Figure 3.7. IPEC-1 monolayers were pretreated with increasing amounts of swine
rTNF-a for 5 hours to determine the effect of swine rTNF-or on C. jejuni 33292 invasion
of intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were performed in triplicate. Results shown are representative of
three experiments. The values shown represent the mean :1: SD. For all values, P was >
0.05 compared to the RPMI 1640 control.

202

 

 

Cell-associated(adherent)
bacteria x 10*5

 

 

 

0 50 500 5000

Swine rTNF-alpha concentrations (pg/m1)

Figure 3.8. IPEC-1 monolayers were pretreated with increasing amounts of swine
rTNF—a for 5 hours to determine the effect of swine rTNF-a on C. jejuni 33292 adherence
to intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were in triplicate. Results shown are representative of three
experiments. The values shown represent the mean i SD. For all values, P was > 0.05
compared to the RPMI 1640 control.

203

 

 

 

bacteria x 10‘5

 

 

 

Cell-associated (adherent)

0 50 500 5000

Swine rTNF-alpha concentrations (pg/m1)

Figure 3.9. IPEC-I monolayers were pretreated with increasing amounts of swine
rTNF-a for 5 hours to determine the effect of swine rTNF-o. on E. coli 0157:H43
DEC7A adherence to intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used
as the negative control. All treatments were in triplicate. Results shown are
representative of three experiments. The values shown represent the mean i SD. For all
values, P was > 0.05 compared to the RPMI 1640 control.

204

1400

 

 

 

 

 

B
I.
8 1200
.‘l
H 1000
0
U
u 800
a
.n
u 600
0
:1
H 400
I
a
u 200
0
J.)
a
H

 

 

0 3000 30000 300000

Swine rIL-l-beta concentrations (pg/m1)

Figure 3.10. IPEC-1 monolayers were pretreated with increasing amounts of swine
rIL-I-B for 5 hours to determine the effect of swine rIL-I-B on C. jejuni 33292 invasion
of intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were in triplicate. Results shown are representative of three
experiments. The values shown represent the mean i SD. F or all values, P was > 0.05
compared to the RPMI 1640 control.

205

 

Cell-associated (adherent)
bacteria x 10‘5

 

0 3000 30000 300000

Swine rIL-l-beta concentrations (pg/m1)

Figure 3.1 1. IPEC-I monolayers were pretreated with increasing amounts of swine
rIL—I-B for 5 hours to determine the effect of swine rIL-l-B on C. jejuni 33292 adherence
to intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were in triplicate. Results shown are representative of three
experiments. For all values, P was > 0.05 compared to the RPMI 1640 control.

206

 

 

Cell-associated(adherent)
bacteria x 10‘5

 

 

 

O 3000 30000 300000

Swine rIL-l—beta concentrations (pg/m1)

Figure 3.12. IPEC-I monolayers were pretreated with increasing amounts of swine
rIL-I-fi for 5 hours to determine the effect of swine rIL-l-B on E. coli 0157:H43 DEC7A
adherence to intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the
negative control. All treatments were in triplicate. Results shown are representative of
three experiments. The values shown represent the mean i SD. For all values, P was >
0.05 compared to the RPMI 1640 control.

207

 

 

 

 

Internalized bacteria (CFU)

 

 

Swine rIL—8 concentrations (pg/m1)

Figure 3.13. IPEC-1 monolayers were pretreated with increasing amounts of swine rIL-8
for 5 hours to determine the effect of swine rIL—8 on C. jejuni 33292 invasion of
intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were done in triplicate. Results shown are representative of three
experiments. The values shown represent the mean i SD. For all values, P was > 0.05
compared to the RPMI 1640 control.

208

 

 

 

 

 

 

 

Cell-associated (adherent)
bacteria x 10‘5

 

0 100 250 500 750 1000

Swine rIL-8 concentrations (pg/ml)

Figure 3.14. IPEC-1 monolayers were pretreated with increasing amounts of swine rIL-8
for 5 hours to determine the effect of swine rIL-8 on C. jejuni 33292 adherence to
intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the negative
control. All treatments were done in triplicate. Results shown are representative of three
experiments. The values shown represent the mean :1: SD. For all values, P was > 0.05
compared to the RPMI 1640 control

209

450

 

400

 

350

 

300
250
200

150

bacteria x 10‘5

100

50

 

 

Cell-associated (adherent)

 

0 100 250 500 750 1000

Swine rIL-B concentrations (pg/m1)

Figure 3.15. IPEC-1 monolayers were pretreated with increasing amounts of swine rIL-8
for 5 hours to determine the effect of swine rIL-8 on E. coli 0157:H43 DEC7A
adherence to intestinal epithelial cells. RPMI 1640 medium (0 pg/ml) was used as the
negative control. All treatments were in triplicate. Results shown are representative of
three experiments. The values shown represent the mean i SD. For all values, P was >
0.05 compared to the RPMI 1640 control.

210

TER %

 

Pretreatment 0 pg/ml rIL-B rIL—l—beta I‘I'NF—alpha
control 250 pg/ml 3000 pg/ml 500 pg/ml
Treatments

Figure 3.16. Effect of swine rIL-8, rIL-I-B, and TNF-a on transepithelial electrical
resistance (TER) of IPEC-1 cells. The TER was measured before and after treatment.
The pretreatment control represents the TER before any treatments were applied to the
IPEC-1 cells. RPMI 1640 medium (0 pg/ml) was used as the negative control. All
treatments were done in quadruplicate. The values shown represent the mean :1: SD. For
all values, P was > 0.05 compared to the pretreatment control and RPMI 1640.

211

Chapter 4: Summary and Conclusions

The long term goals of this laboratory project were to elucidate the
mechanisms by which concurrent C. jejuni and T. suis infection cause severe disease and
pathology in young swine. The identification and characterization of these mechanisms
will be beneficial fi‘om both animal and human health perspectives. C. jejuni is a food-
bome pathogen that has been identified throughout the world as one of the leading causes
of human gastroenteritis (5, 6, 11). There have been significant advances in
understanding the mechanisms by which C. jejuni causes disease, but few immunological
studies have been pursued. This is especially true of those using a naturally occurring
animal model and associated in vitro systems such as swine intestinal epithelial cells. T.
suis is a nematode parasite found in the cecum and colon of swine. Young infected pigs
can acquire catarrhal enteritis with serious pathology, such as anorexia, diarrhea, anemia,
dehydration, retardation of growth, and death (32, 33). C. jejuni is commonly found in
the colon of conventionally-reared pigs without signs of pathology; however,
simultaneous T. suis infection results in severe disease and pathology (22-24). The
severe pathology observed mimics the clinical disease of Campylobacteriasis in humans.

For this dissertation project, we addressed a two-fold hypothesis. The central
hypothesis is that C. jejuni induces natural host resistance via pro-inflammatory cytokine
responses, which arise as a result of infection. A related hypothesis is that pro-
inflammatory cytokine responses elicited by C. jejuni can be down-regulated in the
presence of T. suis, thereby promoting C. jejuni infection These hypotheses were tested

by evaluating pro- and anti-inflammatory cytokine expression in the feces and intestinal

212

tissues of pigs infected with C. jejuni and T. suis and more specifically by assessing the
secretion of lL-8, TNF-a, and IL-l-B from cultured C. jejuni-infected intestinal pig
epithelial cells (IPEC-1 cells), which served as a model system for undifferentiated,
crypt-like cells. Additionally, we evaluated the effects of these secreted cytokines on
bacterial-host cell interaction and intestinal mucosal integrity.

This dissertation project addressed three specific aims. The first specific aim was
to evaluate secreted IL-8, IL-l-B, TNF-a, IL-4, IL-6, and IL-10 expression in the feces of
pigs after single or dual challenge infections with C. jejuni and T. suis. Two
experimental designs were used to test this specific aim: a short-term challenge
experiment (2 days) and a long-term challenge experiment (23 days). In the short-term
challenge experiments we assessed the acute pro-inflammatory response to C. jejuni and
T. suis infection. In these experiments, conventionally, weaned piglets were assigned to
separate treatment groups and were simultaneously infected with 2 x 108 cfu C. jejuni
and/or 2500 T. suis embryonated eggs or 2 x 108 cfu E. coli DHS-a. Fecal samples were
taken prior to inoculation (day 0), and on days 1 and 2 post-inoculation. The fecal
samples were prepared for analysis, and the supernatants were used in an ELISA to
measure pro- and anti-inflammatory cytokine responses following concurrent infection.
In the long-term challenge we assessed the pro-inflammatory cytokine response to C.
jejuni in the presence of adult stage T. suis larvae. The pigs were assigned to separate
treatment groups. One group of pigs was orally inoculated with 2500 T. suis
embryonated eggs, and 21 days post-T. suis inoculation, these pigs were orally inoculated

with 2 X 108 cfu C. jejuni. The other assigned groups were singly inoculated with 2500

T. suis embryonated eggs, 2 x 108 cfu c. jejuni, or 2 x 108 cfir E. coli DHS-a. Fecal

213

samples were taken prior to inoculation (day 0), and on days 21, 22, and 23 post-
inoculation. The fecal samples were prepared for analysis, and the supernatants were
used in an ELISA to measure pro- and anti-inflammatory cytokine expression during
concurrent infection. Also, on day 23 of the long-term challenge experiments, tissue
samples were taken from the intestine, and cytokine mRN A expression was measured
using real time PCR. The second specific aim was to measure secreted IL-8, TNF-a, and
lL-l-B from cultured swine intestinal epithelial cells (IPEC-l cells) following C. jejuni
infection. In these in vitro experiments, IPEC-l cells were cultured in an undifferentiated
state to mimic intestinal crypt cells and infected with C. jejuni 33292. Culture
supernatants were removed at 0, 8, 24, and 48 hours after infection and analyzed for IL-8,
TNF-a, and IL-l-B protein expression by ELISA. The third specific aim was to
determine whether pro-inflammatory cytokines (specifically, IL-8, TNF-a, and IL-l-B)
play a significant role in C. jejuni adherence, invasion, and/or the transepithelial electrical
resistance (TER) with persistent exposure. In the adherence and invasion assays, IPEC-l
cells were cultured in an undifferentiated state to mimic intestinal crypt cells and treated
with various concentrations of recombinant swine IL-8, TNF-a, and IL-l-B for 5 hours.
Recombinant swine IL-8, TNF-a, and IL—l-B supernatants were removed, and the
pretreated IPEC-1 cells were infected with C. jejuni for 1 hour to allow bacteria to adhere
to the cells or 3 hours to allow bacteria to invade the cells. After 1 hour, the adhered
(cell-associated) bacteria were enumerated by serial dilution plating. After 3 hours, the
gentamycin assay was performed, and intracellular C. jejuni were enumerated by serial
dilution plating. Also, IPEC-1 cells were pretreated with recombinant swine IL-8, TNF-

a, or IL-l-B to determine if cytokine exposure affects the transepithelial electrical

214

resistance (TER) of these cells. Undifferentiated IPEC-1 cells were grown to confluency,
and the TER was measured prior to treating these cells with recombinant swine IL-8,
TNF-a, or IL-l-B. Subsequently the IPEC-l cells were treated with recombinant swine
IL-8, TNF-a, and IL-l-B for 5 hours, and then the TER was measured.

To assess the effects of concurrent T. suis and C. jejuni infection on cytokine
expression, we used our established swine infection model system set up as two
experimental designs (short term; day O—day 2 and long term; day O-day 23) challenge
experiments and measured pro- and anti-inflammatory cytokines in the feces and
intestinal tissues of infected pigs. In viva results from the long-term challenge
experiments (day O-day 23) showed that pigs that received T. suis alone showed a
significant decreased IL-l-B in the feces by 23 days post-inoculation, and the dually-
infected pigs showed moderately decreased IL-I-B expression. Also, on day 23, IL-I-[S
was significantly decreased in the T. suis-infected pigs compared to IL-l-B expression in
the C. jejuni-infected pigs. These and other findings suggest that T. suis likely
predominates in driving the cytokine response by down regulating the expression of this
pro-inflammatory cytokine during infection. This is not surprising because in most
helminth infections a Th2 response is elicited with induced production of IL-4, IL-5, IL-
6, IL-9, IL-10, and IL-13 during infection (13, 38) This pattern of cytokine expression is
characteristic of a Th2 response which has been shown to predominantly limit Th1
cytokine-induced inflammation (Th1 cytokines- Il-12, IFN-y, IL-8, TNF-a, and IL-1-
B)(l3, 39) and result in warm expulsion.

In the short-term challenge experiments, there were no significant changes in

secreted lL-8, IL-l-B, TNF-a, IL-4, IL-6, or IL-10 expression. Generally, in pigs that are

215

naturally infected with T. suis, signs of pathology are not seen until 21 days post-T. suis
infection, it is reasonable to believe that results from the long term challenge (23 days)
would likely reflect the immune response of the natural infections more than the short-
term challenge (2 days). This also suggests that immunity to T. suis ova or first stage
larvae may be limited.

In the swine model experiments, we determined the cytokine recovery rate for
these fecal ELISAs to be 10—20%. This low recovery rate may have been due to protein
degradation associated with freezer storage, the loss of protein during the fecal
preparation due to the cytokines binding to the solid fecal matter, or protein degradation
due to protease activity. Because of these possible drawbacks, the fecal ELISA data was
only moderately useful and we relied instead on additional data obtained directly by
measuring cytokine expression in gastrointestinal tract tissues in areas where the
pathogens reside.

In the long-term challenge experiments, after fecal sampling on day 23, the pigs
were euthanized and tissue samples were taken hour the jejunum, proximal colon, and
distal colon of our pigs. In the proximal colon of the two groups of pigs that received T.
suis, Real Time PCR analysis showed increased expression of IL-13 mRNA (Th2
cytokine) and decreased IL-8 and IL-12 expression (Th1 cytokines). Compared to the
uninfected control pigs, IL-13 expression was upregulated 3 to 4 fold, and IL-8 and IL-12
was decreased 2 to 3 fold and 2 to 4 fold, respectively. Additionally, expression of MCP-
], a chemokine, and IL-5, a Th2 cytokine, were significantly decreased 2 fold in the dual-

infected pigs compared to the uninfected control pigs. Also, there were no significant

216

changes in cytokine mRNA expression in the pigs that received C. jejuni alone as
compared to the uninfected controls.

These data provide evidence that supports the hypothesis that T. suis infection can
stimulate the production of a Th2 cytokine (IL-13) which can down-regulate pro-
inflammatory cytokines (IL-8 and IL-12) in the proximal colon of pigs. In vitro, it has
been shown that IL-13 can decrease IL-8 secretion from human intestinal epithelial
cells(21). It has also been shown that IL-13 protects BALB/c mice from LPS-induced
lethal endotoxemia, and decreased IL-12 production is correlated with this protective
effect (27). In addition, it has been shown in our laboratory that differentiated intestinal
pig epithelial cells stimulated with T. suis excretory-secretary products secrete IL-10
(31). In general, IL-10 production has been associated with helminth infections, and
more specifically, it has been shown to be a critical component in the development of a
polarized Th2-type immune response which has been correlated with host resistance and
survival during Trichuris infection (37). Although IL-IO mRN A was not significantly
upregulated in any of the tissue samples from the pigs that received T. suis, it is possible
that peak expression of IL-10 occurred before tissue sampling on day 23 of the
experiment, and by day 23, IL-IO levels had possibly gone back down to baseline levels.
Altemately, it is possible that low constant levels of IL-10 could play a role in
downregulating pro-inflammtory cytokine expression in order to maintain intestinal
mucosal integrity. More to the point, previous data showed that significant production of
IL-10 from cultured IPEC-1 cells following T. suis ESP exposure occurred at 24 hours
post-infection (31). In vitro and in viva studies have demonstrated IL-10 and IL-13

inhibition of intestinal inflammatory responses such as Th1 -type cytokine production,

217

and this inhibition is believed to diminish the severity of the inflammatory responses
thereby reducing clinical disease activity (8, 20, 21, 37). Taken together, the data
presented here are a supportive extension of previous findings in our laboratory, and
collectively these findings suggest that it is possible that the down regulation of IL-8, IL-
12 and possibly MCP-1 mRN A expression in response to T. suis infection may be due to
IL-13 production and possibly IL-10 in response to helminth infection. Therefore, it is
reasonable to suggest that T. suis plays a role in the down-regulation of pro-inflammatory
cytokine expression, and that this response can alter host conditions which promote
secondary bacterial infection.

In the distal colon, significant changes in cytokine expression were observed only
in the pigs that received T. suis alone. IL-l-B, IL-6, and IFN-y expression were increased
3 to 4 fold, and IL-13 expression was increased 3 fold compared to uninfected controls.
lL-l-B, IL-6, and IFN-y production is characteristic of an inflammatory response. The
pigs showing these responses had diarrhea and lesions in these sites. This observed
inflammatory response distal to the sites of warm attachment (in the cecum and proximal
colon) may be the result of secondary bacterial infection. Previous data have shown that
T. suis infection plays a role in facilitating secondary bacterial invasion of many species
of resident bacteria, including C. jejuni, C. cali, C. lari, Campylobacter spp. (unidentified
species), and E. coli (24).

In this study, in spite of our best efforts to support the organism in samples during
transport, culture alone was not sensitive enough to detect or identify C. jejuni after
infection. We found it necessary to use multiple diagnostic tests (TaqMan Real time

PCR, RFLP analysis of Campylobacter 23S rRNA gene, 16S rRNA PCR analysis and

218

ELISA) to detect the inoculated strain of C. jejuni after infection. When C. jejuni was
detected by TaqMan analysis in the distal colon of l of 6 pigs that received T. suis-only
in screens of pigs post inoculation, we realized the possibility that prior infection with
this organism or with other Campylobacter spp., or other epsilon proteobacteria may have
occurred in these pigs. Suspicions of a contaminating bacterium were firrther aroused
when RFLP analysis also showed positive results for C. coli and a Campylobacter spp.
that gave a unique banding pattern that was not consistent with C. jejuni, C. lari, C. coli,
or C. upsaliensis in jejunum, proximal and distal colon samples from most of the pigs in
the treatment groups. ELISA results for a wide range of these bacteria showed that the
pigs were most reactive to Helicobacter spp. that may have caused problems in both
serological and molecular assays. Helicobacter is a closely related member of the epsilon
proteobacteria group with similar outer membrane proteins. Finally, we tested fecal
supernatants for the presence of other Campylobacter species and Helicobacter species
using 16S rRNA PCR analysis. Here we found that ahnost all pigs in the short- and long-
term challenge experiments tested negative for the presence of Campylobacter species
pre- and post-infection. However, several pigs tested positive for the presence of
Helicobacter species pre- and post-infection. Taken together these data provide the best
evidence to suggest that the pigs were exposed to Helicobacter spp. prior to the
experiment and that the cytokine responses measured may have been affected by this
prior bacterial exposure. We must consider that these results are consistent with the
possibility that the increased inflammatory cytokine response seen in the distal colon of
the pigs infected with T. suis alone may actually represent host resistance to secondary

bacterial infection.

219

It is also possible that the effects of ESP produced by 4th stage larvae that have
traveled to the distal colon could contribute to increased expression of IL-13 and IL-6. It
has been shown that T. suis-infected pigs have serum antibodies as early as 21 days post
inoculation that recognize a 20kDa glycoprotein isolated from ESP collected fi'om adult
worms cultured in vitro (17). It has been demonstrated in vitro that T. suis ESP treatment
of differentiated IPEC-1 cells causes a dose dependent cytotoxic response and
significantly decreased transepithelial electrical resistance (TER)(1, 2). Also, cultured
differentiated and undifferentiated IPEC-1 cells secrete IL-6 and IL-10 following ESP
exposure (31). Therefore, it is reasonable to consider that the cytotoxic effects of ESP on
intestinal epithelial cells can stimulate an inflammatory cytokine response in the distal
colon. We should consider that ESP may have direct effects on intestinal epithelial cells
and could account in part for the increased cytokine production in viva at sites distal to
warm attachment.

Interestingly, we also found that there were no significant changes in cytokine
expression in the distal colon of pigs that received C. jejuni alone or those that were
dually-infected or in the proximal colon of pigs that received C. jejuni alone. In deed, we
recognized through the use of multiple diagnostic tests that the pigs from our in viva
experiments were likely exposed to Helicobacter spp., a wildtype C. jejuni, other
Campylobacter spp., or epsilon proteobacteria prior to the experiments. The timing and
source of acquisition is not known, but pigs were farrowed and reared with their mothers
in a solid floor shower-in shower-out confinement facility and then were moved directly
to the containment colony for these studies. Because our data provides evidence

suggesting that these pigs were likely exposed to Helicobacter spp. (epsilon

220

proteobacteria-campylobacter-like organisms) at birth or shortly after birth on the MSU
swine farm, it is reasonable to believe that the C. jejuni administered during our
experiments served as a secondary bacterial challenge. A possible scenario is that these
pigs acquired immunity after early exposure to epsilon proteobacteria such as
Helicobacter, and the host cytokine and chemokine responses to the experimental C.
jejuni inoculum are more representative of host resistance to secondary bacterial infection
associated with protective immunity. Therefore, this scenario affected the interpretation
of the results, but it did not negate our findings.

Nevertheless, in the jejunum (a division of the small intestine), we found that pigs
receiving C. jejuni alone showed significant changes in cytokine expression. The
expression of MCP-I, TNF-a, IL-12p40, IFN-y, IL-4, and IL-10 was increased 3 to 5 fold
compared to uninfected controls. In vitro, C. jejuni induces INT 407 cells to produce
intracellular MCP-1, TNF-a, IFN-y, IL-4, and IL-10 (18), and Helicobacter pylori
induces gastric epithelial cells to produce IL-8 and MCP-l (15, 28, 29, 34). Thus, the
cytokine changes observed could be due to Campylobacter or Helicobacter interaction
with the intestinal epithelial cells in the jejunum. Additionally, it is likely that the jejunal
samples taken from our pigs contained some Peyers patches along with surrounding
epithelium Peyers patches are lymphoid follicles in the small intestinal mucosa that
serve as important components in local and systemic immune responses. Peyers patches
are functionally similar to LGCs of the colon. It has been demonstrated in oral-
challenged germfiee piglets that C. jejuni targets LGCs and induces robust cytokine
responses (22, 23) (Jones, dissertation, 2005). It is possible that C. jejuni or Helicobacter

stimulated the jejunal Peyers patch to produce a similar cytokine response, which likely

221

 

influenced our results. Clearly, the net effect of C. jejuni exposure on the jejunum was
elevation of proinflammatory cytokines. To distinguish the exact cell types fi‘om which
this response arises, further work should be done to fractionate tissues in the jejunum
prior to measuring cytokine responses. The data here suggest that in the absence of T.
suis, C. jejuni or epsilon proteobacteria stimulate pro-inflammatory cytokine responses in
the jejunum. Furthermore, in the gastrointestinal tract of chickens rechallenged with
Salmonella enterica Serovar Typhimurium, it has been shown that significant changes in
proinflammatory cytokine and chemokine (IL-6 and MIP family chemokine) expression
occur in the ileum and cecal tonsils, which are also segments of the small intestine (40).
Additionally, in the jejunum of dually-infected pigs, GM-CSF was down-regulated 5 fold
compared to uninfected controls. The pattern of reactivity in the jejunum over the five
groups of pigs suggests that T. suis is likely contributing to the down-regulation of pro-
inflammatory cytokine expression in the jejunum which is similar to the
immunomodulatory effects that occurred in the proximal colon.

In addition, we observed that clinical diarrhea was a prominent sign of disease
throughout the experiments. Diarrhea was observed in pigs that received C. jejuni only,
T. suis only or bath. However, more frequent and more severe diarrhea was observed in
the dually infected pigs and the T. suis only-infected pigs compared to the other treatment
groups, thereby implying that T. suis is mainly responsible for causing the diarrhea.
Additionally, diarrhea was mostly observed in the long-term challenge experiment where
T richuris infection had progressed for 21 days. As has been reported frequently in
literature reports on T richuris-induced disease, diarrhea usually begins around 21 days

post-infection and is commonly called the “21 day scours” (9). In both experiments,

222

 

some of the C. jejuni-infected pigs had mild diarrhea which is commonly seen in
campylobacterosis (7). It has also been reported by Mansfield et al. in a previous

C ampylabacter swine disease model experiment that some of the C. jejuni-infected pigs
showed clinical signs of mild diarrhea at a less degree of severity than the dually-infected
or T. suis only-infected pigs (23). The uninfected pigs and E. coli DHS-u infected pigs
showed no signs of diarrhea. The observation of severe diarrhea in pigs infected dually
or with T. suis alone has provided some evidence to suggest that T. suis plays a
significant role in diarrhea] disease.

Furthermore, C. jejuni invasion or stimulation of intestinal epithelial cells has
been shown to induce pro-inflammatory cytokine production (16, 18). In viva, C. jejuni
invade the lymphoglandular complex (LGCs) entrapped crypts and the follicle-associated
epithelium of the LGCs following a primary challenge infection (23)(Jones, dissertation,
2005). A robust pro-inflammatory cytokine response is expressed in the LGCs upon C.
jejuni infection (Jones, dissertation, 2005). In viva and in vitro studies have shown that
C. jejuni interacts with enterocytes which serve as the first line of defense during
bacterial infection (16, 19). My second specific aim was to assess IL-8, IL-l-B and TNF-
a production from undifferentiated, crypt-like intestinal pig epithelial cells (IPEC-1 cells)
following C. jejuni infection. This primary neonatal cell line was chosen because it
complemented our swine model system The cells were grown to form confluent
monolayers and infected with C. jejuni for 0, 8, 24, and 48 hours. The supernatants were
collected at each time point and analyzed using ELISA. TNF-a was secreted from the
IPEC-l cells by 8 hours post-inoculation, and continued production was measured

through 48 hours post-inoculation. Likewise, IL-l-B was secreted by the IPEC-1 cells by

223

24 hours post-inoculation. We found that IL-8 was constitutively secreted by the IPEC-1
cells, and was not induced by C. jejuni infection. The data presented here suggest that
the induced production of TNF-a and IL-l-B and the constitutive production of IL-8 fiom
intestinal epithelial cells are indicative of an acute innate host immune response. It is
likely that this inflammatory response can generate and/or modulate the immune response
to benefit the host by contributing to the elimination of the bacteria.

In the third specific aim, we assessed whether IL-8, TNF-a, or IL-l-B treatment of
IPEC-l cells had an effect on C. jejuni invasion, adherence, and/or the transepithelial
resistance (TER). IPEC-1 cells were grown on transwell inserts for 2 days to form
confluent monolayers. The TER of the confluent monolayer was measured prior to
treatment. Then, either IL-8, TNF-a, or IL-l-B were applied to the confluent monolayers
and incubated for 5 hours, and the TER was measured again. The data presented fi‘om
this work, showed that neither lL-8 nor IL-l-B have a direct effect on C. jejuni invasion,
adherence, and/or the TER of IPEC-l cells. Also, treatment of IPEC-1 cells with TNF-a
did not significantly affect C. jejuni adherence or changes in the TER of the IECs. This
finding suggests that these cytokines do not act in an autocrine-like manner to
specifically affect C. jejuni adherence, invasion, and/or the TER of IPEC-1 cells. Even
though there was not a direct effect on C. jejuni adherence to or invasion of IPEC-l cells
(in response to IL-8 or IL-I-B pretreatment) or a direct effect could not be determined
following TNF-a pretreatment in vitro, it is still possible that IL-8, TNF-a, or IL-l-B may
affect epithelial function in a paracrine-like or another autocrine-like manner during C.
jejuni infection in viva. For instance, it has been demonstrated that cytokines can

facilitate enterocyte proliferation, maturation, MHC class II expression, cytokine receptor

224

 

expression or apoptosis in healthy and diseased states (14, 30, 35). In addition, TNF-a
and TGF-a have been shown to stimulate immature crypt cell proliferation, and TGF—B
and INF-y can inhibit mitotic activity (36). IFN-y can alter immature crypt epithelial cell
turnover and upregulate MHC class II expression (35). Therefore, alterations of cytokine
concentrations as a consequence of bacterial exposure and intestinal inflammation may
contribute to serious pathologic consequences. Further elucidation of the effects of
distinct cytokines on epithelial cell growth, phenotype, firnction and bacterial-enterocyte
interactions would be of importance in the understanding of cytokine mediated regulation

of mucosal immune responses associated with C. jejuni infection.

225

Future Directions

The results fi'om this dissertation project opened other avenues for further
research that would enhance understanding of several aspects of this dual infection
phenomenon. In chapter 2 we orally infected immunocompetent, conventionally reared
pigs that had a complete intestinal microbial population with C. jejuni, T. suis, or both
and analyzed fecal cytokine expression and cytokine expression in the jejunum, proximal
colon, and distal colon. In this study, we found that our pigs were probably exposed to C.
jejuni, other Campylobacter spp. or epsilon proteobacteria prior to the experiments, and
this finding significantly impacted the interpretation of our current results. Therefore, it
would be imperative to use gnotobiotic pigs for firture studies in order to definitively
characterize and gain a better understanding of in viva cytokine profiles and kinetics
during concurrent C. jejuni and T. suis infection. Further analysis of cytokine expression
using in situ hybridization and cytokine antibody arrays would allow us to determine if
there is a correlation between cytokine mRNA expressed in intestinal tissues and secreted
cytokine (protein) expression, respectively. During the concurrent infection, analysis of
cytokine expression at different times throughout the course of a long term challenge
experiment (days 0-45) would allow us to examine acute and long term inflammatory
responses (beginning at 24 hours postinfection and continuing throughout the length of
the experiment) to C. jejuni infection and would also allow us to examine changes in
cytokine expression while T. suis developmental changes were occurring. Extending the
evaluation time out to 41 or 45 days would allow a more thorough assessment of cytokine

responses throughout the entire prepatent period of T. suis luminal development. In

226

 

addition, we would examine pathologic changes to correlate lesion progression with
cytokine changes. This would entail having a large number of infected pigs so that a
subset could be sacrificed at intervals after infection.

In general, T. suis infection triggers a Th2 type response involving the cytokine
production of IL-4, IL-10, and IL-13 which plays a role in warm expulsion fi'om the
host(13, 38). This Th2-type response can down-regulate ThI-type innate host immune
responses (e. g. IL-l-fi, TNF—a, INF-y, IL-8, and IL-12) which generally cause bacteria
elimination from the host (13, 38, 39). In chapter 2 we discussed our results that suggest
that T. suis likely mediates lL-8 and IL-12 down-regulation by producing IL-13 in the
proximal colon of pigs, in viva. Even though we did not measure IL-4 or IL-10 mRN A
expression in any tissues from the pigs in the in viva experiments, it would be useful to
determine the possible roles of these cytokines in vitro. In previously reported data from
our laboratory, recombinant swine IL-4 has been shown to weaken tight junctions
between IPEC-1 cells, and this could allow bacteria to breach the epithelial barrier and
gain access to the basolateral surface of cells. Undifferentiated and differentiated IPEC-1
cells could be treated with IL-4, IL-10 and/or IL-13 in a dose response experiment
followed by infecting these cytokine pretreated IPEC-1 cells with C. jejuni. The
supernatants of these C. jejuni-infected cells could be used in ELISA to measure lL-8,
TNF-a, IL-l-B, IFN-y, and IL-12 production. This experiment could determine if Th2-
type cytokines (IL-4, IL-10, and IL-l3) effect Thl-type cytokine production (IL-I-B,
TNF-a, and IL-8) from C. jejuni-infected swine intestinal epithelial cells, in vitro. It

would also be interesting to challenge these cells with multiple strains of C. jejuni from

227

 

patients to see if particular gene content or gene expression correlates with enhanced host
response.

In chapter 3 we measured secreted IL-8, TNF-a, and IL-l-B from undifferentiated
crypt-like swine intestinal epithelial cells (IPEC-l cells) following C. jejuni infection.
Previous reports from our laboratory have shown that C. jejuni specifically interact with
colonic crypts and are associated with mucus near goblet cells (23). It has also been
demonstrated that intestinal epithelial cells can be stimulated by C. jejuni to produce
proinflammatory cytokines and chemokines (16, 18). We measured secreted IL-l-a and
TNF-a from C. jejuni-infected IPEC-1 cells, but only showed that IPEC-1 cells
constitutively secrete IL-8. This raises the question as to why IL-8 is not induced by C.
jejuni infection of IPEC-1 cells. One way to address this question could be to determine
if primary intestinal pig epithelial cells (IPEC-1 cells) lack or have decreased expression
of Toll-like receptor 4 and MD-2 (accessory molecule) on the cell surface using PCR
analysis or flow cytometry. It has been demonstrated that IL-8 production is associated
with TLR4 receptor signaling via LPS stimulation and NFKB activation (3, 4, 10, 12, 14).
Once it has been determined whether IPEC-l cells lack or have decreased TLR4 and
MD-2 on the cell surface, we could use western blot analysis of intracellular proteins to
study the related intracellular signaling pathways of genes encoding proteins involved in
inflammation. More specifically, we could evaluate the interaction of TLR4 and MD-2
binding to C. jejuni LPS, and determine if this interaction stimulates the NFKB signaling
pathway to induce IL-8 gene expression (3, 4).

Also, in chapter 3, our data showed that IL-8, TNF-u, and IL—l-B had no direct

effect on C. jejuni interactions (specifically adherence, invasion, or changes in IPEC-I

228

 

TER) with the swine intestinal epithelial cells, but they could possibly have other
autocrine- or paracrine-like effects which could play a role in host inflammatory immune
responses associated with C. jejuni infection. For example, it has been shown that TNF-a
and IL-l-B can stimulate intestinal epithelial cells to produce C3, a complement
component which is associated with host acute-phase inflammatory responses (14, 25, 26,
30, 35). Therefore, it would be interesting know if intestinal pig epithelial cells (IPEC-1
cells) could produce C3. It is known that IL-l-B is an initiator of inflammation and a
regulator of immune responses, and it induces acute phase protein responses, such as the
production of the acute phase protein complement C3. Undifferentiated and
differentiated IPEC-1 cells could be pretreated with IL—l-B in a dose response
experiment. Confluent monolayers could be exposed to the different concentrations of
IL-l-B. C3 levels could be measured in the supernatants by ELISA, and C3 mRNA
levels would be determined by Northern blot analysis. This experiment would determine
if IL-l-B acts in an autocrine-like manner to stimulate swine intestinal epithelial cells to
produce an acute phase protein, complement C3. In a secondary experiment, the IPEC-1
cells could be pretreated with anti-IL-l-B antibody or IL-lRa (IL-1 receptor antagonist)
to reverse C3 production, if any.

In summary, we have generated data from this dissertation project that has
provided some evidence and understanding of the research defining host immune
responses to concurrent T. suis and C. jejuni infections. We demonstrated in viva that T.
suis plays a major contributing role in mediating changes in cytokine expression during
concurrent T. suis and C. jejuni infection. Using our swine infectious disease model

system, we began to define host local immune responses expressed during this concurrent

229

 

infection. In vitro, we showed that undifferentiated, crypt-like intestinal pig cells (IPEC-
1) can serve as a suitable in vitro model for studying C. jejuni-enterocyte interactions and
host enteric immune responses elicited by C. jejuni during infection. We discovered that
C. jejuni can stimulate intestinal pig epithelial cells (IPEC-l cells) to produce IL-I-B and
TNF-a, and we found that these intestinal pig epithelial cells constitutively secrete IL-8.
Hence, these cytokine responses are indicative of an innate inflammatory immune
response that may be responsible in some part for eliminating bacteria from the host in
viva. In addition, we found that proinflammatory cytokines, such as IL-8, IL- l-B, or
TNF-a, do not appear to directly effect C. jejuni interactions with host cells (particularly
adherence, invasion, or stimulating changes in the transepithelial electrical resistance
(TER) of IPEC-1 cells). Collectively, these data have provided some evidence for T. suis
contributing significantly to cytokine dysregulation seen during concurrent infection, and
these immunomodulatory effects likely promote C. jejuni infection of the intestinal

mucosal epithelium by limiting the natural host resistance to bacterial infection.

230

10.

11.

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