Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1-1-2001

Evaluation of yogurt cultures as a method to reduce Salmonella Typhimurium in swine

Christian Lee Baum Iowa State University

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This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Evaluation of yogurt cultures as a method to reduce Salmonella Typhimurium in swine

By

Christian Lee Baum

A thesis submitted to the graduate faculty

in parti

MASTER OF SCIENCE

Major: Microbiology

Major Professor: D.L. Harris

Iowa State University

Ames, Iowa

2001 11

Graduate College Iowa State University

This is to certify that the Master's thesis of

Christian Lee Baum has met the thesis requirements of Iowa State University

Signatures have been redacted for privacy 111

TABLE OF CONTENTS

Page

CHAPTER 1. GENERAL INTRODUCTION 1

CHAPTER 2. LITERATURE REVIEW 2

CHAPTER 3. MATERIALS AND METHODS 21 In vivo experiments 21 In vitro experiments 28

CHAPTER4. RESULTS 32 In vivo experiments 32 In vitro experiments 45

CHAPTER 5. DISCUSSION 50 In vivo experiments 50 In vitro experiments 57

CHAPTER 6. CONCLUSIONS 60

REFERENCES CITED 61

ACKNOWLEDGEMENTS 72 1

CHAPTER I.GENERAL INTRODUCTION

Thesis Organization

The following thesis consists of a general introduction, review of literature, materials and methods, results, discussion, conclusions, and acknowledgements.

Introduction

Lactobacilli have been evaluated for their pro biotic properties since the beginning of the

20th century. 62 Many reports have focused on the use of lactobacilli for their beneficial, pro biotic effects upon the health and well being of humans and other animals. 27,45,47,51,53,54,64,65,71, 73,101

The recent rise in concern over the association between the sub-therapeutic use of anti- microbials in the swine industry-and the increased prevalence of anti-microbial resistant human pathogens has intensified the search for anti-microbial alternatives. Lactobacilli are

GRAS (generally regarded as safe) organisms and are believed to have beneficial effects on health. Thus, lactobacilli are candidates for use as anti-microbial alternatives.

One potential application for lactobacilli in swine is a pre-harvest method to reduce

Salmonella. Non-typhoidal Salmonella are associated with 1.4 million cases of foodborne illness in the United States per year. 61 Moreover, pork products are estimated to be the source of 6-9 percent of food borne salmonellosis in the United States. 32

The purposes of the following studies were to 1.) evaluate feeding yogurt cultures of two

Lactobacillus isolates as a method to reduce levels of Salmonella enterica serovar

Typhimurium in swine and 2.) develop strains of bacteria to aid in the study of in vivo pro biotic mechanisms of lactobacilli. 2

CHAPTER 2. LITERATURE REVIEW

History of Fermented Foods

For centuries, humans have produced and consumed fermented foods. Until the late 19th and early 20th centuries, fruits, vegetables and milk were fermented for preservation purposes. Egyptians prepared and ate "leben raib", a fermented product of buffalo, cow or goat milk. According to the Bible (Genesis 18:8), Abraham offered several guests a combination of sweet and soured milk. In Russia, the long winters made necessary the fermentation of summer watermelons, apples, cucumbers, and other vegetables. 62

Around the turn of the 20th century, Elie Metchnikoff made several observations that would change the concepts of fermented foods and microbes. One observation was the association between longevity and the consumption of fermented foods. In particular,

Metchnikoff noted that there were more "old people" in Bulgaria and Eastern Europe, where fermented foods were part of the normal diet, than Western Europe, where fermented foods were consumed less frequently. In one case, Metchnikoff reported that a 180-year-old woman maintained her normal daily activities on a diet consisting of barley bread and sour milk. Metchnikoff also suggested that fermented foods were the result of lactic acid that was produced by microbes.

With these observations in mind, Metchnikoff posed the following question,

"Since the lactic fermentation is such an excellent means of preventing putrefaction in general, why should it not exercise this same power and prevent putrefaction in the digestive tube of man?"

Metchnikoff reported the results of a few studies that demonstrated decreased levels of sulfur ethers in the urine of dogs and humans who had been injected with lactic acid microbe 3 or fed fermented foods, respectively. At the time, high levels of sulfur ethers were a measure of intestinal putrefaction.

In essence, Metchnikoff had introduced the concept of bacteriotherapy, which eventually became known as probiotics. In doing so, he proposed two new ideas to microbiology. One concept was the use of fermented foods for purposes other than food preservation. The second concept was that microbes were not necessarily associated with disease, but some had the capacity to benefit the host. Interestingly, Metchnikoff proposed a method for production of fermented milk that is still used today. He suggested that milk first be sterilized and then inoculated with a strain of bacteria that was known to produce lactic acid.

The efficacy of this method was proven by members of Metchnikoff s lab group who daily consumed the fermented milk and reported "gratifying" results.

In 1907, Metchnikoff declared that lactic-acid producing bacteria should occupy the first place of honor among the microbes that are absolutely essential to our well-being. Since then, the beneficial effects and mechanisms of fermented foods upon humans and animals continue to be investigated worldwide. 62

Definition of probiotics

In 1965, Lilly and Stillwell first coined the term "probiotic", meaning "for life" in Greek.

The concept that Metchnikoff introduced in the early 1900's was formally defined by Fuller as "probiotics" in 1989. According to Fuller, probiotics are "live microbial food supplements which beneficially affect the host by improving the intestinal microbial

33 balance." . This definition was expanded by Guarner and Schaafsma to include non-food preparations. They defined probiotics as "living micro-organisms, which upon ingestion in certain numbers, exert health affects beyond inherent basic nutrition". 40 4

Probiotic preparations containing Lactobacillus species have been developed in many forms. In humans, lactobacilli are most often administered in fermented milk products or as

64 92 99 freeze-dried bacterial cultures. • • Frozen, fermented milk, or freeze-dried preparations of bacteria have been developed for use in food production animals, such as chickens and

45 46 51 108 swine. • • • Gels containing live Lactobacillus species, such as Probios™ (Pioneer

International, Johnston, IA) and FastrackTM (The Conklin Co. Inc., Shakopee, MN), are available for treatment of individual animals.

Lactobacilli in Humans

Although Metchnikoff first proposed the use of fermented milk for health benefits in humans over 100 years ago, the interest in this subject has peaked only in the past 20 years.

Indeed, over half of the human clinical trials with pro biotic lactic acid bacteria (LAB), which include Lactobacillus species, that were conducted since 1961 were performed between 1991 and 1998. 66

In order to streamline and standardize the selection of candidate probiotics, the Lactic

Acid Bacteria Industrial Platform (LABIP) has supported the following criteria for human probiotics:

• Be of human origin • Demonstrate non-pathogenic behavior • Exhibit resistance to technological processes • Prove resistance to gastric acid and bile • Adhere to gut epithelial tissue • Be able to persist, albeit for short periods, in the gastrointestinal tract • Produce anti-microbial substances • Modulate immune responses • Have the ability to influence metabolic activities 27

The pro biotic effects of lactobacilli have been evaluated in many different models in humans. Five strains of Lactobacillus, in particular, have demonstrated efficacy in humans. 5

Those strains are Lactobacfllus -aciaophilus [Cl, L. acidophilus NCFOl 748, Lactobacillus

27 rhamnosus GG, Lactobacillus casei Shirota, and Lactobacillus gasseri • Results have shown that different strains of Lactobacillus have different effects in the same model.

Examples of these results are found in Table 1.

Table 1. Effects of lactobacilli in humans Strain (Dosage) Model Effect Reference 10 11 L. casei GG (10 -10 ) Rotaviral diarrhea J, duration of diarrhea Isolauri et al. ( 1991) 10 11 L. casei GG (10 -10 ) " tISC and sASC Kaila et al. ( 1992) 10 L. casei GG (l 0 ) Rotavirus vaccine t IgA seroconversion Isolauri et al. ( 1995) 10 L. casei GG (l 0 ) Salmonella Typhi No effect on Ab Fang et al. (2000) Ty21 a vaccine L. acidophilus La 1 + " t serum specific IgA Link-Amster et al. Bifidobacteria Bb 12 (1994) L. acidophilus Lactose intolerance No effect Saltzman et al. ( 1999) L acidophilus Lactose intolerance J, symptoms Gilliland et al. ( 1982) ISC: Immunoglobulin-secreting cells sASC: specific antibody-secreting cells Ab: antibody

Characterization of normal and diseased flora in humans

In one study, populations of lactobacilli from 75 individuals with either healthy or diseased mucosa were characterized. A total of 250 isolates were tested for their ability to ferment 49 different carbohydrates using the API 50 CH test kit (API systems, S.A.,

Montalieu Vercieu, France). In addition, the end products from glucose were measured for each isolate using HPLC. Results of the in vitro tests were used to assign each Lactobacillus isolate to one of seventeen "clusters". Most clusters contained isolates from both diseased and healthy mucosa and no qualitative differences in strain composition were observed between different parts of the intestine. These results indicate that there is no one dominant species of Lactobacillus within the intestinal flora. Additionally, there was a large difference in the dominant Lactobacillus species between individuals. 63 6

Lactobacilli vs. gastrointestinal disorders in humans

Results of successful treatment of gastrointestinal disorders with lactobacilli have been

reported. Interestingly, different species of Lactobacillus have been associated with the

treatment of different disorders. For example, results show that L. casei strain GG can be

used for the treatment of rotaviral diarrhea in children. Lactobacillus casei strain GG

10 11 administered twice daily, at the rate of 10 - colony-forming units (CFU), to patients with

rotavirus resulted in diarrhea that lasted, on average, one day less than controls fed sterile

43 47 milk. • Results have also shown that Lactobacillus GG is associated with successful

treatment of recurring Clostridium difficile diarrhea. 11 Administration of Lactobacillus plantarum has been associated with a decrease in the symptoms associated with irritable

bowel syndrome (IBS).69 Lactobacillus acidophilus LB has been associated with antagonistic activity against Helicobacter pylori infection. 17 In these models of gastrointestinal disorders

where certain strains of Lactobacillus have been associated with successful treatment, there

are no reports that evaluate the effects of other strains of Lactobacillus.

Lactobacilli vs. cancer in humans

Reports have demonstrated the use lactobacilli to treat human bladder and uterine cancer.

Uterine cervical cancer patients treated with injected heat-killed L. casei YIT9018 and radiation therapy had prolonged survival and increased relapse-free intervals compared to radiation therapy alone. Treatment with L. casei YIT9018 also reduced the severity of radiation-induced leukopenia compared to untreated subjects.71 Orally administered

Biolactis™ powder (a L. casei preparation) has been shown to decrease recurrence of

superficial bladder cancer.7 The application for different strains of Lactobacillus to treat 7 human cancer appears to depend upon the specific form of cancer. There are no reports that evaluate the use of other strains of Lactobacillus to treat these forms of cancer.

Reduction in food allergy in humans

Infants with atopic eczema associated with food allergy were fed 5 x 108 CFU of L. rhamnosus GG daily in their formula for one month. Treated infants had decreased severity of atopic eczema compared to controls fed normal formula. Decreased intestinal inflammatory responses, measured by fecal TNF-a concentrations, were also observed in treated subjects compared to controls. 59

Lactobacilli vs. metabolic activities in humans

Anti-hypercholesterolemia. In one study, daily consumption of two liters of either whole milk or skim milk fermented with Lactobacillus bulgaricus and Streptococcus thermophilus was associated with a significant reduction in hypercholesterolemia using paired samples. 60

Conversely, human subjects who daily consumed Lactinex (Becton Dickinson Microbiology

Systems, Cockeysvile, MD) tablets containing 2 x 106 CFU of L. acidophilus and L. bulgaricus did not have significantly different cholesterol levels compared to controls that consumed placebo tablets. 54 Yogurt enriched with L. acidophilus (109-10 1°CFU/day) did not lower blood cholesterol levels in adults, compared to controls fed un-enriched yogurt. 22

Lactose intolerance. Pasteurized whole milk containing either 2.5 x 108 CFU/ml or 2.5 x

106 CFU/ml of L. acidophilus significantly reduced lactose malabsorption compared to controls fed sterile milk. In the same study pasteurized whole milk containing 2.5 x 10 7

CFU/ml L. acidophilus did not significantly reduce lactose malabsorption. 39 Lactose intolerance was also reduced in children who were fed milk fermented either with L. 8 acidophilus or L. lactis and S. thermophilus. 64 Adults treated twice daily with 1 x 10 1°CFU of L. acidophilus BG2FO4 failed to reduce symptoms associated with lactose intolerance. 92

Characterization of the ecology of lactobacilli administered orally to humans

Characterization of the ecology of lactobacilli administered orally to humans has largely focused on L. rhamnosus GG and L. rhamnosus DR20. Colonoscopies and colonic biopsies were conducted on humans fed 6 x 10 1°CFU/day of L. rhamnosus GG twice daily for 12 days. Fecal recovery of L. rhamnosus GG decreased over time. However, some subjects with culture-negative fecal samples maintained biopsy samples that were culture-positive.

Two weeks after the termination of L. rhamnosus GG treatment, the administered strain was only recovered from biopsy samples of 2 out of 7 subjects. 3 Similar results have demonstrated a "diluting effect" of orally administered L. rhamnosus DR20 after termination of treatment. 99

Lactobacilli in Swine

Lactobacilli have been evaluated in swine for their probiotic effects upon weight gain and disease associated with Salmonella and Escherichia coli. Examples of the effects of

Lactobacillus probiotics in swine are found in Table 2.

Table 2. Effects of lactobacilli in swine Strain (Dosage) Model Effect Reference L. acidophilus (Probios) Weight gain t Pollman et al. (1980) 7 3 strains (Probios) ( 10 ) t Collington et al. (1988) 8 L. reuteri (10 /ml) Weight gain -1, Cole et al. (1987) Coliforms -1, Stomach and duodenum 9 10 L. acidophilus RP32 (10 -10 ) Weight gain No effect Yuste et al. (1992) 9 5 strains (Ferlac-2) (10 ) Salmonella t colon bacteria Letellier et al. (2000) Typhimurium No effect on shedding or challenge tissue colonization 9 pCFl (10 ) Salmonella -1, shedding Nisbet et al. (1999) ( containing Lactobacillus Typhimurium -1, cecal colonization species)55 challenge 9

Weight gain in swine

In 4-week-old pigs, Probios™, a commercial product containing L. acidophilus, plus virginiamycin increased average daily gain (ADG) and feed conversion (FC) by 9.7% and

4.4%, respectively, compared to controls fed virginiamycin alone. 84 Similar positive effects of Lactobacillus fermentation products, including Probios TM, on weight gain and feed

19 51 conversion have been reported by others • .

Reports that do not demonstrate the efficacy of lactobacilli on weight gain in pigs also exist. In one study, Probios ™ did not affect the growth of grower-finisher pigs.84 Two-day- old pigs fed 100% or 50% yogurt diets had significantly lower ADG and FC compared to

18 animals fed a conventional milk diet • Administration of a nonviable Lactobacillus fermentation product (LFP) at 0.18, 0.36, or 0.72 ml/kg did not significantly effect weight gain in either weaned or growing-finishing pigs.41 L. acidophilus, the same species contained in Probios™, did not affect ADG and FC when administered to three-week-old pigs at rate of

1 x 109 or 1 10 1°CFU/day. 108 These results may indicate significant differences that exist among different strains of Lactobacillus. Others have reported that lactobacilli do not significantly affect ADG and FC. 102

Lactobacilli vs. Salmonella and E.coli in swine

For two primary reasons, the administration of probiotics to pigs has focused on pigs four weeks of age or younger. One reason is the treatment or prevention of diseases such as

Salmonella and E. coli associated with young pigs. A second reason is derived from the hypothesis that the most opportune time for a probiotic to colonize and establish in the gastrointestinal tract is during the first few days of life. Ferlac-2 (Rossell Institute, Montreal, Canada), consisting of L. acidophilus, L. rhamnosus,

L. bulgaricus, Enterococcus faecium, and Streptococcus thermophilus, fed to 12-day-old pigs at 2 x 109 CFU/day resulted in fewer Salmonella culture-positive tissues compared to controls fed a diet without Ferlac-2. In the same animals, Ferlac-2 did not reduce fecal

52 53 shedding of Salmonella. • Competitive exclusion (CE) cultures, containing Lactobacillus

55 9 10 species , administered at rates between 5 x 10 and 5 x 10 cfu/animal have been used to reduce colonization and shedding of Salmonella Typhimurium in pigs two days old or

5 89 younger, compared to untreated controls. • A fermented product consisting of L. lactis in combination with colostrum has been associated with increased survival and decreased

65 scouring in newborn pigs challenged with E. coli .

Characterization of intestinal flora and orally administered lactobacilli in swine

In the colon of normal pigs, L. acidophilus and L. fermentum comprise 8 .1 % and 3. 7%, respectively, of the total flora adherent to the colon wall. Conversely, in dysenteric pigs,

91 there are no lactobacilli attached to the colonic mucosa • Denaturing gradient gel electrophoresis (DOGE) of 16S ribosomal DNA and plasmid profiling of Lactobacillus isolates from nom1al pigs indicate that differences exist in the predominant Lactobacillus populations between individuals of the same litter. DOGE and plasmid profiling have also demonstrated that the predominant Lactobacillus populations change dramatically within an

95 98 individual pig during the first two weeks of life • . The influence of weaning upon

Lactobacillus populations attached to the ileum, pars esophagus and cecum has also been demonstrated by ribotyping. 50 The same study showed no difference in the predominant strains of Lactobacillus in pigs that were either fed a com-soy diet, a com-soy diet with 40% lactose, or remained with the sow during days 28 to 3 8 of age. 50 The ecology of orally 11 administered L. reuteri has been characterized. When pigs were treated with L. reuteri at a rate of 2.5 x 10 1°CFU/anima l, L. reuteri was recovered from fecal samples for no more than 2 days after treatment was terminated. 95

Prebiotics

Prebiotics are "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, that can improve the host health."36 The combination of probiotics and prebiotics is referred to as a symbiotic. 36 Prebiotics are available in a variety of chemical compositions, examples of which are found in Table 3.

Table 3. Examples of Prebiotics General Carbon Structure Exam le Disaccharides Lactulose Lacitol Polysaccharides Inulin Resistant Starch Oligosaccharides Fructo-oligosaccharides (FOS) Galacto-oligosaccharides Soybean Oligosaccharides

Fructooligosaccharide (FOS) is the prebiotic that has been studied most extensively in swine. FOS delivered in drinking water increased the total number of bacteria in the colonic flora compared to controls with normal drinking water. 52 In a Salmonella Typhimurium challenge model of weaned pigs, a combination ofFerlac-2 and FOS did not reduce the number of tissues that were Salmonella Typhimurium culture-positive but did increase the total number of colonic bacteria compared to controls fed a traditional diet. 52 In one study

FOS administered with L. paracasei to pigs improved immunostimulation compared to controls fed L. paracasei alone.42 12

Probiotics in Chickens

In chickens, the administration of Lactobacillus probiotics has been evaluated for effects

on weight gain, intestinal flora and decreased levels of Salmonella. Examples of results from

these studies are found Table 4.

Table 4. Efficacl of LAB in chickens Strain (Dosage} Model Effect Reference Gut contents Salmonella Infantis ,!, crop, small Nurmi et al. (1973) challenge intestine, cecal colonization CF (29 mixed strains) Salmonella Typhimurium ,!, cecal colonization Corrier et al. (1995) challenge 10 11 Lactobacillus spp. (10 - 10 ) Salmonella Infantis No effect Adler et al. ( 1980) challenge L. acidophilus (NR) Salmonella No effect Stavric et al. (1992) Bifidobacterium bifidum Typhimurium challenge 8 L. salivarius CTC2 l 97 ( 10 ) Salmonella Enteritidis ,!, cecal colonization Pascual et al. ( 1999) challenge 8 Lactobacillus spp. ( l 0 ) Weight gain No effect Watkins et al. (1984) L. acidophilus (109/gm) 1' Jin et al. (2000) 12 strains 1' Lactobacillus (109/gm)

Lactobacilli are usually administered to chickens in one of three preparations: single

strains of Lactobacillus, multiple strains of Lactobacillus, or continuous flow cultures.

While single and multiple strain preparations of Lactobacillus are self-explanatory,

continuous flow cultures require some description. Typically, continuous flow cultures, also

referred to as competitive exclusion cultures (CEC), are derived from chicken cecal contents.

These cecal contents are used to inoculate media in a fermenter. After several days of

growth, the fermenter reaches a "steady-state condition" at which point the bacteria are

isolated and identified. A defined continuous flow culture is then produced from the

bacterial isolates obtained from the steady state conditioned fermenter. All defined

20 68 continuous flow cultures contain at least one strain of Lactobacillus. • 13

Weight gain in chickens

Lactobacillus acidophilus 26 or a mixture of 12 strains of Lactobacillus (2 strains of L. acidophilus, 3 strains of L. fermentum, 1 strain of L. crispatus, and 6 strains of L. brevis) administered in dry feed at a rate of lxl09 CFU/g significantly increased the weight of broilers compared to controls that did not receive any lactobacilli. 45 Others have reported increased body weights in broilers fed L. acidophilus compared to controls. 101

Administration of an unidentified strain of Lactobacillus to chicks via drinking water at a rate of 2 x 108 CFU/chick/day did not significantly influence the weight gain in chicks

2 107 compared to controls that were not fed Lactobacilli. • Others have reported that administration of Lactobacilli to chickens did not result in significant increases in weight gain. 106 The effect of continuous flow culture on weight gain in chickens has not been reported.

Lactobacilli vs. Salmonella in chickens

Many researchers have evaluated probiotics as a method to control Salmonella in chickens. While reports of the efficacy are variable, there has been a distinct progression in the formulation of pro biotic preparations since the early 1970' s. Nurmi and Rantala first described the feeding of normal adult chicken intestinal contents to reduced infection by

Salmonella enterica subsp. enterica serovar Infantis. 70 Due to regulatory restrictions, the trend shifted toward defined mixtures of organisms and continuous flow cultures as previously described. Stavric et al. demonstrated that mixtures of several uncharacterized

Lactobacillus isolates did not reduce levels of Salmonella Typhimurium in chicks challenged with 105 CFU/chick. The inclusion rate of the Lactobacillus isolates was not reported.96

Recently, a single strain of L. salivarius was administered in feed (105 CFU/g) or water (107 14

CFU/ml) to one-day-old chicks that were subsequently challenged with Salmonella enterica

subsp. enterica serovar Enteritidis. In both treatment groups, cecal colonization by

Salmonella Enteritidis was reduced to 0% compared to 70% in untreated controls. 73

Probiotics in Other Animals

Horses

Administration of 30 g of Probiocin (1 x 107 CFU/g of L. plantarum, L. casei, L. acidophilus,

and S.faecium) or 15 g of Quick Fix (2.75 x 108 CFU/g of L. acidophilus, S.faecium,

Bifidobacterium thermophilum, and B. longum) to horses following surgery for colic did not

reduce levels of Salmonella shedding. 72 However, penicillin and gentamicin were

administered for 2 to 3 days during probiotic treatment. There are no reports on the probiotic

effect of lactobacilli alone in horses. 72

Cattle

Lactobacillus acidophilus administered orally to calves at a dose of 3 x 109 CFU in milk

was associated with increased weight gain and feed conversion compared to controls fed

milk without L. acidophilus. 1 However, no significant effect on weight gain was observed

when calves were fed either a nonviable L. bulgaricus fermentation product, live L.

38 94 acidophilus NCFM, or live L. acidophilus C-28 compared to controls fed sterile milk. •

Sheep

Lambs treated twice with 2 ml ProbiosrM gel (1 x 107 CFU of L. plantarum, L. casei, L.

acidophilus and Streptococcus faecalis) per treatment showed a trend towards increased

weight gain between 2 and 6 days of age compared to control lambs treated with an inert placebo gel. Differences in weight between groups ranged from 4.36% to 5.67% between 2

and 6 days of age, respectively. 6 15

Mechanisms Involved in the Beneficial Effects of Probiotics

Several mechanisms for the beneficial effects of probiotics have been suggested, based upon in vitro studies. These mechanisms include decreased pH, inhibition of pathogen adhesion to receptor sites, production of anti-microbial compounds, and the stimulation of immune cells. The pro biotic mechanisms of lactobacilli have also been studied extensively in mouse models of immune function and human disease. Examples of the results from these studies are shown in Table 5.

Table 5. Efficacy of Lactobacillus strain in mice Strain (Dosage) Model Effect Reference 9 L. casei (10 ) Salmonella 20% survival/day dependent Perdigon et al. (1990) Typhimurium challenge 9 L. acidophilus ( 10 ) 20% survival Perdigon et al. ( 1991) L. acidophilus + 100% survival 9 L. casei ( 10 ) 8 L. acidophilus (I 0 ) No effect Filho-Lima et al (2000) 8 L. casei LAB-I (10 ) 75% survival/1' serum IgA Paubert-Braquet et al. (1995) 8 L. casei LAB-2 (10 ) 50% survival 8 L. casei Yakult ( 10 ) 50% survival/1' serum IgA " L. brevis ( 10 8 i.p) SRBC 0TH No effect Bloksma et al. (1979) L. p/antarum (108 i.p.) Adjuvant effect SRBC 0TH: Sheep Red Blood Cell Delayed-Typed Hypersensitivity i.p.: intraperitoneal

Stimulation of immune function

Oral administration of lactobacilli has been demonstrated to increase non-specific and specific mechanisms of the immune system. In Salmonella challenge studies with mice, administration of L. casei fermented milk increased anti-Salmonella antibodies in intestinal fluid and in serum. In the same study, L. acidophilus reduced anti-Salmonella serum antibodies by 50% compared to controls that were not treated with L. acidophilus.

Interestingly, treatment with a combination of L. casei and L. acidophilus increased serum anti-Salmonella antibodies 2.5-fold higher than administration of L. casei alone. 81 Similar 16

increases in serum and intestinal antibodies specific for cholera toxin 100 and trinitrophenyl

(TNP)3 5 have been demonstrated in mice. In humans, oral administration of L. acidophilus

Lal and Lactobacillus GG has increased specific IgA and immunoglobulin-secreting cells

35 47 against Salmonella enterica subsp. enterica serovar Infantis and rotavirus , respectively.

Oral administration of L. acidophilus, L. bulgaricus or L. casei to mice at 109 cfu/day

activated macrophages as measured by lysosomal activity, phagocytosis and carbon

77 9 clearance compared to untreated controls. .7 Interestingly, co-administration of L. casei

and L. acidophilus also activated macrophages in mice, although at levels lower than those

80 observed with each strain alone . Supematants from the fermented milk cultures of these

Lactobacillus strains also increased the number of plaque-forming cells, carbon clearance

rates, and levels of serum antibodies in mice, compared to controls fed sterile milk. 78 In

humans, phagocytic activity of peripheral blood leukocytes has been increased by oral

administration of L. acidophilus Lal compared to untreated controls.24

The addition of L. acidophilus K8, L. bulgaricus A Y, L. plantarum 14D, or L. casei

101/37 to human peripheral blood lymphocytes resulted in the increase in interferon-y

(IFN-y) when cells were stimulated with the mitogen, Con A. 23 Treatment of peripheral

blood lymphocytes with heat-killed or filtered yogurt containing L. bulgaricus and S.

thermophilus did not increase production of IFN-y as did unfiltered, live yogurt

preparations.23 A similar study demonstrated increased production ofIL-lP, TNF-a, and

IFN-y by human peripheral blood mononuclear cells treated with L. casei, L. acidophilus, and

L. helveticus. Human subjects fed yogurt containing 10 11 CFU of L. bulgaricus and S. thermophilus did not have detectable plasma levels ofIL-lP, IL-2, IFN-y, IFN-a, or TNF-a. 17

However, 2'-5' synthase activity of blood mononuclear cells was increased by 83% compared to milk-fed controls. 83

In mice, intraperitoneal injection of 5 x 107, 1 x 107 or 1 x 106 CFU of L. bulgaricus increased serum levels ofIFN-y compared to untreated controls. This effect was augmented with irradiated bacteria but was abolished with heat-killed bacteria. 82 Studies with inbred mice demonstrate that different Lactobacillus strains stimulate different cytokine responses.

Examples of these results are found in Table 6.

Table 6. Crtokine ~rofiles of orallr administered Lactohacillus strains 58 Strain ., ., =- > tD > (JQ (JQ tDr,:i '"O r,:i ., I I = '"O ., = C')C') '"O :t. :t. N "zj 0 tD I 0 •• I R ""CO I r,:i .,tD Q r,:i = -- tD= = tD L. murines NS NS 1' NS NS 1' L. casei NS NS NS NS NS 1' L. gasseri NS NS NS NS NS 1' L. plantarum 14917 NS NS NS NS NS 1' L. plantarum N CIB NS NS NS NS NS 1' L. fermentum 1' NS NS NS NS NS L. brevis 1' NS NS NS 1' 1' L. reuteri 1' 1' NS 1' 1' 1' 1' significant (P < 0.05) increase vs. controls NS: No significant difference vs. controls

Bacteriocins

The first bactericidal activity oflactobacilli was described in 1961. 21 Bacteriocins produced by lactobacilli are typically membrane-active proteins that are less than 20 kDa, resistant to heat, and synthesized as precursors. 44 The activity of Lactobacillus bacteriocins is usually restricted to a narrow range of gram-positive organisms. Bacteriocins from many strains of Lactobacillus have been isolated and characterized. Lactocin 27 (12.4 kDa) and helveticin J (37 kDa) are produced by L. helveticus LP27. 48 Lactobacillus acidophilus N2 18

produces lactacin B, a 6.5 kDa bacteriocin. Plantaracin Bis a 8 kDa bacteriocin produced

by L. plantarum. 48 Recently Gassericin KT7, a 5.0 kDa bacteriocin, was isolated from L.

gasseri KT7. 109

Interference of pathogen adh~esion to intestinal cells

Chauviere et al. first demonstrated an inhibitory effect of live and killed L. acidophilus LB

against adhesion of eterotoxigenic E. coli (ETEC) to the human enterocyte-like Caco-2

cells. 13 Lactobacillus acidophilus LB and L. acidophilus LA 1 have also demonstrated, in a

dose-dependent matter, the ability to inhibit the adhesion and invasion of enteropathogenic E.

coli (EPEC), Salmonella Typhimurium, Listeria monocytogenes, Yersinia paratuberculosis

with Caco-2 cells compared to untreated cells. 10 In these experiments, adhesion and

invasion were inhibited only when either the Caco-2 cells or the pathogen were incubated

with the Lactobacillus species prior to the adhesion/invasion assays. 15

Antibacterial effects

In addition, spent culture supernatant (SCS) from L. acidophilus LB alone is capable of

decreasing the viability of Salmonella Typhimurium in Caco-2 cells, decreased F-actin

accumulation and decreased IL-8 secretion in Caco-2 cells. Mice treated with the SCS of L.

acidophilus LB had decreased levels of Salmonella Typhimurium per gram in feces

compared to untreated controls. 16 Growth of Salmonella Enteritidis, enteropathogenic and

uropathogenic E. coli and Vibrio cholera has been inhibited in co-cultures with L. paracasei

B21060 and B21070 and L. acidophilus B21190. The role of decreased pH in these

observations has virtually been refuted. However, coaggregation of lactobacilli with pathogens has been observed in co-culture. 25 Lactobacillus acidophilus and L. casei together, and L. casei alone, have been administered in fermented milk to reduce mortality 19

and tissue colonization in inbred mouse models of human Salmonella Typhimurium

75 76 81 infection. • •

Genetically Engineered Lactobacilli

Recently, there has been great progress in the development of genetically engineered

lactobacilli. The lack of characterized genetic systems and the intrinsic difficulty in working

with Gram-positive bacteria in molecular biology techniques have provided the greatest

hurdles in the development of recombinant lactobacilli. In spite of these difficulties, many

researchers have developed recombinant strains of Lactobacilli for two primary objectives.

One objective is to evaluate the dissemination and localization of specific strains of

Lactobacillus in vitro and in vivo. The second objective for recombinant lactobacilli is the

development of vaccine vectors.

The development of recombinant bacteria expressing the green fluorescent protein (GFP)

from Aequorea victoria88 has recently become an effective method for tagging specific

strains of bacteria. GFP-expressing bacteria have been used to study host-pathogen

14 28 29 104 interactions and bacteria in various environmental samples. • • • Lactobacillus plantarum expressing GFP have recently been developed by Geoffry et al. 34 With GFP

under the control of the inducible nisA promoter, L. plantarum can be enumerated by flow

cytometry. Furthermore, macrophages that have engulfed GFP-expressing L. plantarum can

be sorted by flow cytometry from macrophages that have engulfed wild-type L. plantarum. 34

Several properties of lactobacilli make these bacteria appealing candidates for recombinant vaccine vectors. Lactobacilli are "generally regarded as safe" (GRAS)

organisms, meaning they are not considered pathogens and may be safely ingested by humans and other animals. In addition, many strains of Lactobacillus possess the intrinsic 20 ability to stimulate the immune system of host animals and thereby act as an adjuvant. In particular, the oral administration of recombinant lactobacilli would allow delivery of antigens directly to the mucosal surfaces of the gut, thereby stimulated local immune responses against specific antigens. One of the shortcomings of killed or inactivated oral vaccines is the lack of persistence within the intestine. If able to colonize the intestine, recombinant lactobacilli may provide a vehicle to circumvent this difficulty.

Lactobacilli that either express surface bound heterologous antigens or secrete heterologous antigens have been evaluated for their ability to induce immune responses to pathogens and induce oral tolerance, respectively. Lactobacilli expressing a fusion protein of

~-glucuronidase and an influenza HA epitope evoked a systemic antibody response to HA after subcutaneous priming followed by an oral booster. 86 The immunogenicity of surface bound tetanus toxin fragment C (TTFC) or secreting myelin basic produced by recombinant

L. casei has been demonstrated in mice by specific antibody responses detected by ELISA following parenteral immunization with whole cell extracts. 57 21

CHAPTER 3. MATERIALS AND METHODS

In Vivo Experiments

Animals and animal care

Three-week-old, conventional, mixed-sex pigs were purchased from either Farm A (Trettin

Farms, Inc., Rockford, Iowa), Farm B (Prairie Gold Farms, Hancock, Minnesota), or Farm C

(Lauren Christian Farm, Atlantic, Iowa). All animals were fed conventional diets with no added drugs appropriate for age and weight. A "pre-starter" diet (36.1 % com and 28.6% soy) was fed during the first 2-3 weeks of each trial and a "starter" diet (50.7% com and 33.4% soy) was fed for the duration of the study. All feed was irradiated with electron beams at a dose of ~7 kilogray (kGy) at the Iowa State University Linear Accelerator Facility. All animals were housed in confined facilities at Iowa State University. In trials 1-5, animals were housed on solid concrete floor. In trial 6 animals were housed on raised decks.

Protocols for each trial were approved by the Committee on Animal Care at Iowa State

University. All animals were acclimatized to the experimental housing conditions and diet for 12 days prior to the beginning of each trial. During the acclimatization period, the animals were determined to be Salmonella culture-negative by pen fecal samples.

Sample collection for Salmonella isolation

Pen fecal samples consisted of five 5-gram samples of feces were collected from the floors in each pen. Fecal and tonsil swabs were collected from individual animals with

Culturette TM swabs. At necropsy, samples for qualitative Salmonella isolation were collected either by swabbing tissues with Culturette TM swabs or by collecting tissue samples in sterile

Whirl packTM stomacher bags. Approximately 3 grams were collected from each tissue except for cecum contents, from which 25 grams were collected. Tissue samples were 22 pulverized with a rubber mallet. For qualitative Salmonella isolation, all tissues were diluted

1: 10 (w/v) in buffered peptone water (BPW) for pre-enrichment, homogenized by hand, and incubated at 37°C for 18-24 hours. After pre-enrichment, 100 µl of BPW was transferred to

9.9 ml Rappaport-Vassiliadis (RV) broth and incubated at 42°C for 18-24 hours. After incubation in RV broth, a sterile cotton swab was used to transfer an aliquot of the cultures onto XLD agar plates. The XLD plates were streaked for isolation and incubated at 3 7°C for

24 hours. Colonies suspected to be Salmonella were picked and transferred to Kliger' s iron agar, SIM semi-solid agar, phenylalanine agar, lysine iron agar, and trypticase soy agar slants and incubated at 3 7°C for 24 hours. Presumptive Salmonella colonies were tested for "O" antigens by agglutination with typing sera. Quantitative bacteriology was performed using the most probable number (MPN) method. All samples were pulverized with a rubber mallet

(except for cecum contents) and diluted 1:10 (w/v) in BPW. Cecum contents were homogenized by hand and the remaining samples were homogenized for 60 seconds on normal setting in a Seward Laboratory Blender (Model 400). Homogenized samples were

8 serially diluted 10-fold to 10- . From each dilution, 1 ml was transferred to each of 5 tubes containing 9 ml ofBPW for pre-enrichment and incubated at 37°C for 18-24. After pre- enrichment, 100 µl from each BPW tube was transferred to 9.9 ml RV broth and incubated at

42°C for 18-24 hours. After incubation in RV broth, a sterile cotton swab was used to transfer an aliquot to XLD agar and each plate was streaked for isolation to determine if each tube was Salmonella culture-positive or culture-negative. Populations of Salmonella were determined with an MPN chart.4

ELISA for detection of serum antibody responses

Serum was tested using the Danish Mix-ELISA as described by Nielsen et al. 67 23

Lactobacillus cultures and yogurt preparation

Cultures of Lactobacillus sp. strain GS-1 and Lactobacillus sp. strain GS-2 were isolated from the pars esophagus of a conventional pig. Stock cultures of each Lactobacillus isolate were maintained at -80°C in MRS broth. A high passage isolate of Lactobacillus sp. strain

GS-1, (GS-lH), was obtained by daily sub-culturing a stock culture of Lactobacillus sp. strain GS-1 in MRS broth for 40 days. Yogurt starter cultures were grown overnight in 9 ml of MRS broth at 3 7°C and serially passaged daily during each trial by transferring ~ 100 µl to

9 ml of fresh MRS. For each yogurt culture, 700 ml of milk (2% fat), supplemented with 2%

(w/v) dextrose, were autoclaved for 17 minutes in 1 L Pyrex™ glass media bottles, cooled to room temperature and inoculated with 7 ml of overnight yogurt starter culture. Prior to each daily feeding, pH readings of yogurt cultures were recorded. The daily titer of Lactobacillus in each yogurt culture was determined by serial 10-fold dilutions in phosphate buffered saline

(PBS, pH 7.4) and subsequent plating on MRS agar. Live yogurt cultures of Lactobacillus sp. strain GS-1 and GS-2 contained at least 108 CFU/ml and 107 CFU/ml, respectively. Yogurt cultures were killed by heating for 1 hour at 100°C or by exposure to electron beam irradiation at~7 kGy at the Iowa State University Linear Accelerator Facility. All yogurt cultures were fed in addition to conventional diets.

Preparation of Salmonella inoculum

Salmonella Typhimurium strain B was originally isolated from a sow with clinical salmonellosis. 8 Stock cultures of Salmonella Typhimurium strain B were grown in brain heart infusion (BHI) broth and maintained at -80°C. Seed cultures of Salmonella

Typhimurium strain B were grown overnight in BHI broth at 3 7°C. The seed culture was 24

diluted 1: 100 in fresh BHI broth and incubated at 3 7°C to a final O.D. 600 of 0.350, which corresponded to ~3 x 108 CFU/ml. The titer of the inoculum was determined by serial 10- fold dilution in PBS, pH 7.4, and plated onto blood agar.

Experimental designs

Trial 1. Twenty pigs from Farm A were individually identified with a numbered ear tag and randomly assigned to one of 4 treatment groups (n=5). Each treatment group was assigned to a separate room. The GS-1 group was fed yogurt fermented with Lactobacillus sp. strain GS-1. The GS-2 group was fed yogurt fermented with Lactobacillus sp. strain

GS-2. The sterile milk group was fed un-inoculated, sterile milk. The strict control group received no treatment. All groups were fed the appropriate yogurt cultures on a daily basis for 8 days prior to and 26 days after inoculation with Salmonella Typhimurium strain B. On day 27, samples of inocula for the GS-1 and GS-2 yogurt cultures were frozen at -80°C and were identified as Lactobacillus sp. strain L T-60 and Lactobacillus sp. strain L T-61, respectively. On day 0, animals were inoculated intranasally with 3 x 108 CFU of

Salmonella Typhimurium strain B via a 1.5 inch Teflon catheter. Fecal samples were collected on days -12, 2, 4, 7, 9, 11, 14, 17, 19, 21 and 24. Palatine tonsil swabs were collected on days 15 and 21. Blood was collected on days -1, 4, 14, 21 and 27. The study was terminated on day 27 and qualitative Salmonella isolation was performed using

Culturette TM swabs from tonsil, mandibular lymph node, liver, spleen, ileocecal lymph node, jejunal lymph node, ileum contents, and colon contents. Tonsil and ileocecal lymph node were also collected for quantitative Salmonella isolation.

Trial 2. Twenty-five pigs from Farm A were individually identified with a numbered ear tag and randomly assigned to one of three treatment groups. Each treatment group was 25 assigned to a separate room. The GS-I group (n=I0) and GS-I/unchallenged groups (n=5) were fed yogurt fermented with Lactobacillus sp. strain GS-I. The sterile milk group (n=I0) was fed un-inoculated, sterile milk. All groups were fed the appropriate yogurt cultures on a daily basis for 8 days prior to and 26 days after inoculation with Salmonella Typhimurium strain B. On day 27, a sample of the inoculum for the GS-I yogurt cultures was frozen at-

800C and was identified as Lactobacillus sp. strain L T-62. On day 0, animals in the GS-I and sterile milk group were inoculated intranasally with 108 CFU of Salmonella

Typhimurium strain B via a 1.5 inch Teflon catheter. Fecal samples were collected on days

-1, 2, 4, 7, 9, 11 , 14, 17, 19, 21, 23, 25, and 27. Tonsil samples were collected on days-I, 2,

5, 7, 9, 11, 15, 21, 23, and 27. Blood was collected on days -1, 7, 14, 21, and 27. Three and two animals from the GS-1 and sterile milk group were randomly euthanized and necropsied on day 2 and day 9, respectively. The study was terminated on day 27. At each necropsy samples for qualitative Salmonella isolation were colleced using CulturetteTM swabs of tonsil, mandibular lymph node, liver, spleen, ileocecal lymph node, jejunal lymph node, ileum contents, and colon contents. Tonsil, ileocecal lymph node, and cecum contents were also collected for quantitative Salmonella isolation.

Trial 3. Twenty-five pigs from Farm A were individually identified with a numbered ear tag and randomly assigned to one of 5 treatment groups (n=5). Each treatment group was assigned to a separate room. The GS-1 group was fed yogurt fermented with Lactobacillus sp. strain GS-1. The IRR group was fed an irradiated preparation of yogurt fermented with

Lactobacillus sp. strain GS-1 . The HK group was fed a heat-killed preparation of yogurt fermented with Lactobacillus sp. strain GS-1. The sterile milk group was fed un-inoculated, sterile milk. The strict control group was not treated. All groups were fed the appropriate 26 yogurt cultures on a daily basis for 8 days prior to and 26 days after inoculation with

Salmonella Typhimurium strain B. Animals in the GS-1, IRR, HK, and sterile milk groups were inoculated with Salmonella Typhimurium strain Bas in previous trials. Fecal, tonsil and blood samples were collected on the same days as in trial 2. The study was terminated on day 27. Tissues were collected from the strict control, sterile milk, and IRR groups.

Qualitative Salmonella isolation was performed on~ 3-gram samples of tonsil, mandibular lymph node, liver, spleen, ileocecal lymph node, jejuna! lymph node, and ileum contents and

~ 25-gram samples of cecum contents. Tonsil and cecum contents were also collected for quantitative Salmonella isolation.

Trial 4. Twenty-five pigs from Farm A were individually identified with a numbered ear tag randomly assigned to one of five treatment groups (n=5). Each treatment group was assigned to a separate room. The GS-1 H group was fed yogurt fermented Lactobacillus sp. strain GS-1 H. The GS-1 group was fed yogurt fermented with Lactobacillus sp. strain GS-1.

The GS-2 group was fed yogurt fermented with Lactobacillus sp. strain GS-2. The sterile milk group was fed un-inoculated, sterile milk. The strict control group was not treated. All groups were fed the appropriate yogurt cultures on a daily basis for 8 days prior to and 26 days after inoculation with Salmonella Typhimurium strain B. Animals in the GS-1 H, GS-1,

GS-2, and sterile milk groups were inoculated with Salmonella Typhimurium strain B as in previous trials. Fecal, tonsil and blood samples were collected on the same days as in trial 2.

The study was terminated on day 27. Tissues were collected from the sterile milk and GS-2.

Qualitative Salmonella isolation was performed on~ 3-gram samples of tonsil, mandibular lymph node, liver, spleen, ileocecal lymph node, jejuna! lymph node, and ileum contents and 27

~ 25-gram samples of cecum contents. Tonsil and cecum contents were also collected for quantitative Salmonella isolation.

Trial 5. Twenty-five pigs from Farm B were individually identified with a numbered ear tag and randomly assigned to one of five treatment groups (n=5). Each treatment group was assigned to a separate room. The GS-1 group was fed yogurt fermented with Lactobacillus sp. strain GS-1. The GS-1 + FOS group was fed yogurt fermented with Lactobacillus sp. strain GS-1 containing 5 grams of fructooligosaccharide (FOS). The GS-2 group was fed yogurt fermented with Lactobacillus sp. strain GS-2. The sterile milk group was fed un- inoculated, sterile milk. The strict control group was not treated. All groups were fed the appropriate yogurt cultures on a daily basis for 8 days prior to and 18 days after inoculation with Salmonella Typhimurium strain B. Animals in the GS-1, GS-1 + FOS, GS-2, and sterile milk groups were inoculated with Salmonella Typhimurium strain B as in previous trials.

Fecal, tonsil and blood samples were collected on the same days as in trial 2. The study was terminated on day 27. Tissues were collected from all treatment groups. Qualitative

Salmonella isolation was performed on~ 3-gram samples of tonsil, mandibular lymph node, liver, spleen, ileocecal lymph node, jejunal lymph node, and ileum contents and~ 25-gram samples of cecum contents. Tonsil and cecum contents were also collected for quantitative

Salmonella isolation

Trial 6. Twelve pigs from Farm C were individually identified with a numbered ear tag and randomly assigned to one of three treatment groups (n=4). Each treatment group was assigned to a separate room. The GS-1 group was daily fed 1.4 liters of yogurt culture fermented with Lactobacillus sp. strain GS-1. The sterile milk group was fed 1.4 liters of un- inoculated, sterile milk per day. The strict control group was not treated. All groups were 28 fed the appropriate yogurt cultures for 8 days prior to and 13 days after inoculation with

Salmonella Typhimurium strain B. On day 0, animals were inoculated intranasally with 3 x

106 CFU of Salmonella Typhimurium strain B via a 1.5 inch Teflon catheter. Fecal and tonsil samples were collected on days -12, 2, 4, 7, 9, 11, and 14. Blood was collected on days -10, 7, and 14. The study was terminated on day 14. Tissues were collected from all treatment groups. Quantitative and qualitative Salmonella isolation were performed as in trial 5. Approximately 3 grams of colon contents were also collected for qualitative

Salmonella isolation.

In Vitro Experiments

Strain identification

The ability of Lactobacillus sp. strain GS-1 and Lactobacillus sp. strain GS-2 to ferment

49 different carbohydrates was tested using the API 50 CH test kit (API systems, S.A.,

Montalieu Vercieu, France) according to the manufacturers' instructions. Inocula were prepared in MRS broth incubated overnight at 37°C. Tests were read after incubation at 37°C for 2 days and 7 days. Results were rated from O (violet) to 5 (yellow), and 3-5 were scored positive.

Antibiotic resistant Lactobacillus mutants

Antibiotic resistant strains of Lactobacillus sp. strain GS-1 and GS-2 were developed using a gradient plating method. Initially, each strain was streaked on MRS agar containing a rifampin gradient of 0-10 µg/ml. Plates were incubated anaerobically overnight at 3 7°C.

Resistant colonies were re-streaked on gradient plates containing increasing amounts of rifampin until colonies resistant to 30 µg/ml were obtained. Rifampin resistant isolates were subsequently cultured in a similar manner on MRS agar containing 30 µg/ml of rifampin and 29 increasing concentrations of streptomycin until isolates resistant to 30 µg/ml of rifampin and

400 µg/ml streptomycin were obtained.

Salmonella Typhimurium x4232 expressing GFP

Plasmids and bacterial strains. Salmonella Typhimurium x4232 (NalR) (courtesy of Dr.

Scott Hurd, NADC, Ames, Iowa) was used as the recipient. E. coli DH5a harboring the pVLacGreen plasmid (courtesy of Dr. Gwyn Beattie, Iowa State University), was used as the donor. p VLacGreen contains green fluorescent protein (GFP) under the control of the Lac promoter, a kanamycin resistance gene, and has a broad host range origin of replication. E. coli DH5a harboring the pRK.2073 plasmid (courtesy of Dr. Gwyn Beattie, Iowa State

University) was used as the helper strain. The plasmid pRK.2073 encodes streptomycin and spectinomycin resistance genes.

Tri-parental mating. The donor, helper, and recipient were each grown overnight at 37°C with 225 r.p.m. rotary shaking. One hundred microliterss of each overnight culture was added to one microcentrifuge tube (mating mix) and inverted gently to mix. Ten microliters from the donor, helper, recipient, and mating mix were spotted onto Lauria-Bertanelli (LB) agar without antibiotics and the plate was incubated overnight at 37°C. The next day, each sample was streaked for isolation onto LB agar containing the appropriate antibiotic.

Individual plates were examined under ultraviolet light to detect the expression of GFP. The exconjugate was identified as Salmonella Typhimurium x4232/GFP.

Phagocytosis assay

Salmonella Typhimurium x4232/GFP was grown overnight in LB broth at 37°C. Two milliliters of the overnight culture were centrifuged at 5000 x g for 15 minutes and resuspended in 100 µl PBS, pH 7.4. Twenty microliters of the PBS suspension were added 30 to each of two tubes (control and experimental) containing 100µ1 of fresh, heparinized blood from a conventional pig. As a positive control for the assay, 10µl of FITC-conjugated E. coli

(courtesy of Dr. Joan Cunnick, Iowa State University) were added to each of two tubes

(control and experimental) containing 100 µl of fresh, heparinized blood from a conventional pig. Control and experimental tubes were incubated for 10 minutes on ice and at 3 7°C, respectively. All tubes were then placed in a 0°C water bath and 100 µl ice-cold quenching solution (3 mg/ml trypan blue in PBS, pH 7.4) were added to each tube. Each tube was briefly vortexed and 2 ml of ice-cold washing solution (1.0% paraformaldehyde in PBS, pH

7.4)) was added to each tube. All tubes were centrifuged at 250 x g for 5 minutes at 4°C and the supernatant was discarded. Two milliliters of lysing solution (0.15 M ammonium chloride, 10 mM sodium bicarbonate, 0.1 mM EDTA disodium) were added to each tube and the tubes were incubated at room temperature for 15 minutes. The tubes were centrifuged at

250 x g for 5 minutes at 4°C. Pellets were washed with 2 ml of wash solution, centrifuged at

250 x g, and decanted. One hundred microliters of propidium iodide (50 µg/ml in PBS, pH

7.4) were added to each pellet. Each tube was vortexed briefly and incubated in the dark at

0°C for 10 minutes. Wet mount slides were prepared and observed with epiflourescence microscopy.

Lactobacillus mini-preps

Lac to bacillus spp. strains GS-1, GS-1 H, GS-2, and L T-60 were each grown overnight at

37°C in 500 ml MRS broth and centrifuged for 20 minutes at 5000 x g. The cells were washed twice in 25 ml of20 mM Tris-HCl (pH 8.0). Cells were resuspended in 20 ml of20 mM Tris-HCl (pH 8.0) containing 5 mg/ml hen egg white lysozyme and 20 ml of 24% polyethylene glycol 8000 and incubated for 2 hours at 37°C. Cells were centrifuged for 20 31 minutes at 5000 x g and washed in 20 mM Tris-HCl (pH 8.0). Cells were resuspended in the cell suspension buffer of the Promega Maxi-Prep™ kit. Plasmids were isolated according to the manufacturer's protocol and resuspended in 100 µl ofTris-EDTA (pH 7.4).

Spot test assay

Lactobacillus spp. strains GS-1, GS-lH, GS-2, LT-60, and LT-61 were grown overnight in

MRS broth at 3 7°C. Five micro liters from each overnight culture were spotted onto MRS agar plates. As a control, 5 µl of MRS broth samples, adjusted for the pH after overnight growth (pH 3.8 and 4.2 for GS-1 and GS-2, respectively) were spotted onto MRS agar plates.

Each plate was incubated anaerobically at 3 7°C for 18 hours. Five milliliters of BHI top agar

(0.75% agar) containing 107 CFU of Salmonella Typhimurium strain B were poured onto the

MRS plates and the plates were incubated aerobically at 3 7°C. Inhibitory activity of

Lactobacilli was observed after overnight incubation.

Agar well diffusion assay

Lactobacillus sp. strain GS-1 and Lactobacillus sp. strain GS-2 were grown overnight in

MRS broth at 3 7°C. Overnight cultures were centrifuged at 5000 x g for 15 minutes and the spent culture supernatant (SCS) was collected. Fifty microliters of SCS from Lactobacillus sp. strain GS-1 and Lactobacillus sp. strain GS-2 were added to 4 mm diameter wells in MRS agar. As a control, MRS broth samples, adjusted for the pH after overnight growth (pH 3.8 and 4.2 for GS-1 and GS-2, respectively) were each added to a well. The plates were incubated at room temperature until each sample was absorbed by the agar. Then, 5 ml of

BHI top agar (0.75% agar) containing 107 CFU of Salmonella Typhimurium strain B were poured onto the MRS plates and the plates were incubated aerobically at 37°C. Inhibitory activity of each Lactobacillus strain was observed after overnight incubation. 32

CHAPTER4.RESULTS

In Vivo Experiments

Trial 1

Serum antibody levels. On day -1, anti-Salmonella serum antibodies were not detected in any animal. The group mean antibody levels are shown in Figure 1. After inoculation with

60

50 -

40

0 30 0 ~Sterile Milk :::!:!0 20 ---GS-1 10 -

0 ..... GS-2 -1 7 14 21 27 -10 Day (Challenge = 0) Figure 1. Trial 1 anti-Salmonella serum antibody levels

Salmonella Typhimurium strain B, the mean anti-Salmonella serum antibody levels in all groups increased through day 14. While there was a trend towards higher anti-Salmonella serum antibody levels in the sterile milk group compared to the GS-1 and GS-2 groups, there was no statistically significant (P > 0.05) difference between any groups on any day after challenge.

Salmonella isolation from fecal samples. Fecal samples collected from all animals on day -12 were Salmonella culture-negative (Figure 2). On day -1, pen fecal samples collected from all groups were Salmonella culture-negative. The strict control group remained

Salmonella culture-negative throughout the study. On day 2, there were fewer Salmonella 33

100%

80%

(1) 70% > 60% Milk (n=5) 0 50% C (n=5) 40% aGS-2 * * *P<0.05 20% ~- Fisher's 10% I- - I- - exact test of 0% proportions -12 2 4 7 9 11 14 17 19 21 24 Day (Challenge = 0)

Figure 2. Trial 1 Salmonella culture-positive fecal samples

culture-positive animals in the GS-1 (40%) and GS-2 (60%) groups compared to the sterile milk group ( 100% ). This trend continued throughout the duration of the study. On days 9 and 11 there were significantly (P < 0.05) fewer Salmonella culture-positive fecal samples collected from GS-1 compared to sterile milk. After day 11, Salmonella was never recovered from any fecal sample collected from the GS-1 group. Salmonella was recovered from fecal samples of at least one animal from the GS-2 and sterile milk groups throughout the study.

Salmonella isolation from tonsil samples. On days 15 and 21, the sterile milk group

(100%) had significantly (P > 0.05) more Salmonella culture-positive tonsil samples than the

GS-1 (0%) group (Figure 3). Although there were fewer Salmonella culture-positive tonsil

samples in the GS-2 group (40%) compared to the sterile milk group on days 15 and 21, the

difference was not statistically significant (P > 0.05) on either day.

Quantitative Salmonella isolation from tissues. On day 27, results of quantitative I bacteriology of Salmonella from the tonsils of the GS-2 group and the ileocecal lymph nodes 34

100%

90%

80%

Q) 70% :.:;> "ci) 60% a..0 Sterile Milk (n=5) 50% _____, GS-1 (n=5) c E!:IGS-2 n=5 40% Q) * P<0.05 a.. 30% Fisher's exact test of 20% proportions 10%

0% 15 21 Day (Challenge = 0)

Figure 3. Trial 1 Salmonella culture-positive tonsil samples from all groups were not obtained due to technical error. There was a non-significant (P >

0.05) trend towards fewer Salmonella CFU per gram of tonsil in the GS-1 (2.0 x 103 ± 3.4 x

3 4 4 10 ) compared to the sterile milk group (1.4 x 10 ± 1.2 x 10 ).

Trial 2

Serum antibody levels. On day -1, anti-Salmonella serum antibodies were not detected

ci 30 +------1------, +Sterile Milk ci '#- 20 +-----~ 'I------, +GS-1

+GS-1 (no challenge)

Day (Challenge = 0)

Figure 4. Trial 2 anti-Salmonella serum antibody levels 35 in any animal. The group mean anti-Salmonella serum antibody levels are shown in Figure

4.The GS-lino challenge group remained sero-negative throughout the study while serum antibody levels in the GS-1 and sterile milk group increased through days 14 and 21, respectively. The mean serum antibody levels did not differ significantly (P > 0.05) between the GS-1 and sterile milk groups on any day.

Salmonella isolation from fecal samples. Fecal samples collected from all animals on day -12 were Salmonella culture-negative (Figure 5). On day -1, pen fecal samples collected

717 100%

90% 4/5 4/5 80% 5n 70% :;:::; 3/5 3/5 -~ 60% - Sterile Milk :: 50% . C 3n 2/5 2/5 l::IGS-1 40% - (V DGS-1 (no a. 30% - ' challenae) - 1/5 1/5 20% i 1~ * P<0.05 by / Ii.I 10% - I Fisher's ·1 exact test of 0% I I proportions -1 2 4 7 9 11 14 17 19 21 23 25 27 Day (Challenge= 0)

Figure 5. Trial 2 Salmonella culture-positive fecal samples

from all groups were Salmonella culture-negative. All animals in the GS-1/unchallenged group remained Salmonella culture-negative throughout the study. On day 2, there were fewer Salmonella culture-positive animals in the GS-1 (43%) group compared to the sterile milk group (100%). This trend continued throughout the duration of the study. After day 11 there were no Salmonella culture-positive fecal samples collected from the GS-1 group while

Salmonella culture-positive fecal samples were collected from the sterile milk group through day 27. 36

100%

90% 8/10 4/5 80% sn Milk Q) 70% 6/10 ,in 3/5 ·en 60% DGS-1 0 a. 50% 'E " 3/7 'o' 215 215 DGS-1 (no 40% . challenge) Q) 1?7 a. 30% ' * P<0.05 20% lk - "' 1/7 t. 1/7 Fisher's r., 10% - 11 exact test of

1 ·1 proportions 0% ,I -1 2 5 7 9 11 15 21 23 Day (Challenge= 0)

Figure 6. Trial 2 Salmonella culture-positive tonsil samples

Salmonella isolation from tonsil samples. On day -1, tonsil samples collected from all animals were Salmonella culture-negative (Figure 6). All animals in the GS-1/unchallenged group remained Salmonella culture-negative throughout the study. On day 2, there were fewer Salmonella culture-positive tonsil samples were collected from animals in the GS-1

( 60%) compared to the sterile milk group (80% ). This trend continued throughout the duration of the study. On day 11, the difference between the GS-1 (20%) and sterile milk group (80%) was statistically significant (P < 0.05). There were no Salmonella culture- positive tonsil samples collected from the GS-1 group after day 9 while culture-positive samples were collected from the sterile milk group through day 23.

Quantitative Salmonella isolation from tissues. On day 2, there was non-significant

(P > 0.05) trend towards reduced mean Salmonella CFU per gram in the tonsils (19.0 ± 26.4

4 4 vs. 2.3 x 10 ± 4.0 x 10 ) , ileocecal lymph nodes (1.36 ± 2.4 vs. 22 ± 19.1), and cecum

4 4 contents (1.8 ± 2.4 vs. 1.6 x 10 ± 2.8 x 10 ) of the GS-1 group vs. sterile milk group, respectively. On day 9 a similar, non-significant (P > 0.05), trend towards decreased 37

Salmonella populations was observed in tonsil, ileocecal lymph node and cecum for the GS-1 group compared to the sterile milk group. On day 27, there was a non-significant (P > 0.05) trend towards reduced mean Salmonella CFU per gram in the tonsils of the GS-1 group (9.8

3 4 4 4 x 10 ± 2.2 x 10 ) compared to sterile milk group (1.4 x 10 ± 1.2 x 10 ). The mean

Salmonella CFU per gram of cecum contents on day 27 was significantly (P < 0.05) lower in the GS-1 group (17 ± 35) compared to the sterile milk group (304 ± 338) (Figure 7).

0 O> 0 ::::::,

C 1.00E+02 +--- 8 Sterile Milk E::, () DGS-1

0 -E 1.00E+01 +--- ::, LL (.) C ro (I)

Figure 7. Trial 2 Salmonella populations in cecum contents on day 27

Cumulative results from trials 1 and 2

Salmonella isolation from fecal and tonsil samples. Results from trials 1 and 2 were pooled (Table 7). There was a significant (P > 0.05) difference in the mean number of days during which Salmonella was recovered from individual fecal samples in the GS-1 group (3 .3

± 1.96) compared to the sterile milk group (17.5 ± 1.96). There was a significant (P > 0.05) difference in the mean number of days during which Salmonella was recovered from individual tonsil samples in the GS-1 group ( 4. 0 ± 1. 4) compared to the sterile milk group 38

Table 7. Summary of Salmonella culture-positive fecal and tonsil samples from trials 1 and2 Treatment Group Parameter Number of Animals Mean Number of culture-positive days (S.E.) GS-I Feces 10 3.3 (1.96)3 GS-2 5 12.0 (2.8) Sterile Milk 10 17.5 (I.96t GS-I Tonsil 5 4.0 (1.4)3 Sterile Milk 5 21 (I .St a,b Different letters indicate P<0.05 for difference between means with a parameter

(21 ± 1.5). There was a non-significant (P > 0.05) difference in the mean number of days during which Salmonella was recovered from individual fecal samples in the GS-2 (12.0 ±

2.8) compared to the sterile milk group.

Salmonella isolation from tissues. The qualitative Salmonella culture results from necropsy on day 27 of trials 1 and 2 were pooled (Table 8). The mandibular lymph node,

Table 8. Isolation of Salmonella from tissues of pigs in trials 1 and 2 Sam pie Sterile Milk GS-1 (positive/total) (positive/total) Tonsil* 7/10 2/10 Mandibular Lymph Node 0/10 0/10 Liver 0/10 0/10 Spleen 0/10 0/10 Jejunal Lymph Node 0/10 0/10 Ileocecal Lymph Node 3/10 1/10 Ileum Contents 2/10 1/10 Cecum Contents 4/10 2/10 Total* 16/80 (20%) 6/80 (7.5%) * P<0.05 for difference in proportions within rows liver, spleen, and jejunal lymph node in all animals were Salmonella culture-negative.

Salmonella was recovered from the tonsil, ileocecal lymph node, ileum contents and cecum contents from the GS-1 group and sterile milk groups. In all culture-positive tissues, there was a trend towards decreased numbers of culture-positive tissues in the GS-1 group 39 compared to the sterile milk group. There were significantly fewer culture-positive tonsils in the GS-1 group (2/10) compared to the sterile milk group (7 /10). Salmonella was recovered from a significantly (P < 0.05) greater proportion of tissues in the sterile milk group (20%) than the GS-I group (7.5%).

Trial 3

Salmonella isolation. On day -12, fecal samples collected from all animals were

Salmonella culture negative (Figure 8). All animals in the strict control group remained

100%

90% f------

80% ~- --

~- -- Q) 70%

f------I- ·w 60% Sterile Milk 0 Q. 50% -- DGS-1 c -- 1, Q) Irradiated f------I\ - I- I- I- I- 40% ,, Q) Q. ;: IIIIHeat Killed 30% f------I- I- I- I- I- 1-- I-

20% -- - I- I- ,, I- I- I- I- I- 1, 10% - ,- -- 1, ! ; 1 0% -12 2 4 7 9 11 14 18 21 25 Days Post-challenge

Figure 8. Trial 3 Salmonella culture-positive fecal samples

Salmonella culture-negative throughout the study. Salmonella culture-positive fecal samples were collected from all animals in the GS-1, HK, and sterile milk groups on day 2 after inoculation (Figure 8). At least one animal in the sterile milk group had a culture-positive fecal sample on each day of sampling. There was no significant (P > 0.05) difference in the number of culture-positive samples collect from any treatment group compared to the sterile milk group on any day. A similar, non-significant trend was observed in Salmonella 40

Table 9. Trial 3 Salmonella isolation from tissues Sample Sterile Milk Irradiated GS-1 (positive/total) (positive/total) Tonsil 4/4 1/5 Mandibular Lymph Node 1/4 1/5 Liver 1/4 5/5 Spleen 1/4 0/5 Jejuna} Lymph Node 1/4 0/5 Ileocecal Lymph Node 2/4 0/5 Ileum Contents 3/4 1/5 Cecum Contents 2/4 0/5 Total 15/32 (47%) 8/40 (20%) isolation from tonsil samples. On day 14, one animal from the sterile milk group while blood was being collected. At the end of the trial, there was no significant (P > 0.05) difference in

Salmonella from the tonsil, mandibular lymph node, lung, liver, spleen, ileum contents, cecum contents, jejunal lymph node, or ileocecal lymph node (Table 9.)

Trial 4

Salmonella isolation. All animals were Salmonella culture-negative prior to inoculation with Salmonella Typhimurium strain B (Figure 9). The strict control group remained

100%

90% --

80% -

Q) 70% - > ·en 60% - 0 Milk a.. -- c 50% - - DGS-1 40% - II LT-63 Q) a.. 30% -

20% -

10% --

0% -12 2 4 7 9 11 14 17 21 23 Day (O=Challenge)

Figure 9. Trial 4 Salmonella culture-positive tonsil samples 41

Salmonella culture-negative throughout the study. On day 2, Salmonella culture-positive fecal samples were collected from all the animals in GS-I and sterile milk groups. At least one animal in the sterile milk group had a culture-positive fecal sample on each day of sampling. There was no significant (P > 0.05) difference in the number of culture-positive samples collected from any treatment group compared to the sterile milk group on any day.

A similar, non-significant trend was observed in Salmonella isolation from tonsil samples.

On day 6, one animal in the GS-I group died. Results of a post-mortem examination indicated that the animal probably died from salmonellosis. On day 18, one animal in the

GS-I group was euthanized due to lameness. At the end of the trial, no significant (P > 0.05) difference was detected from qualitative or quantitative bacteriology of tonsil, mandibular lymph node, lung, liver, spleen,

Table 10. Trial 4 Salmonella isolation from tissues Sample Sterile Milk GS-2 (positive/total) (positive/total) Tonsil 5/5 5/5 Mandibular Lymph Node 3/5 5/5 Lung 1/5 1/5 Liver 1/5 1/5 Spleen 0/5 0/5 Jejuna! Lymph Node 3/5 2/5 Ileocecal Lymph Node 2/5 3/5 Ileum Contents 4/5 5/5 Cecum Contents 5/5 4/5 Total 25/46 (56%) 28/45 (62%) ileum contents, cecum contents, jejunal lymph node, or ileocecal lymph node (Table 10).

Feed Analysis. Oxytetracycline was detected at ~25 ppm in the starter feed but not pre- starter feed from trial 4 (Table 11 ). No detectable amounts of chlortetracycline, penicillin G and sulfamethazine were detected in either the pre-starter or starter feeds in trial 4. 42

Table 11. Antibiotic analysis of feed from trial 4 Antibiotic Pre-Starter Starter Oxytetracycline No detectable amount ~25 ppm Chlortetracylcline No detectable amount No detectable amount Penicillin G No detectable amount No detectable amount Sulfamethazine No detectable amount No detectable amount

Trial 5

Salmonella isolation. All animals were Salmonella culture-negative prior to inoculation with Salmonella Typhimurium strain B. Animals in the strict control group remained

Salmonella culture-negative throughout the study. On days 2 and 4 Salmonella culture- positive fecal samples were collected from all animals in all groups (Figure 10). Salmonella culture-positive fecal samples were collected from at least 3 animals each day after inoculation. There was no significant (P > 0.05) difference in the number of culture-positive samples collected from any treatment group compared to the sterile milk group on any day. A similar, non-significant trend was observed in Salmonella isolation from tonsil samples. At

100% - - -

90% - - -

80% - - f- -

- Q.) 70% - - - > -- - 60% - - f- f- f- - - ·en ,; 0 a.. Milk c 50% ------DGS-1 40% - - - - Q.) -- - a.. + FOS 30% - - f- f- f- - - eGS-2 20% ------11 10% - I- f- - -

0% --,-- '-,-- --,-- --,-- "'- --,-- -12 2 4 7 11 14 17 19 Day (Challenge= 0)

Figure 10. Trial 5 Salmonella culture-positive fecal samples 43

Table 12. Trial 5 Salmonella isolation from tissues Sample Sterile Milk GS-1 GS-1 + FOS GS-2 (eositive/total) (eositive/total) (eositive/total) (eositive/total) Tonsil 5/5 3/3 5/5 4/5 Mandibular Lymph Node 2/5 2/3 2/5 3/5 Lung 1/5 0/3 2/5 0/5 Liver 0/5 2/3 3/5 0/5 Spleen 0/5 1/3 3/5 0/5 Jejunal Lymph Node 1/5 1/3 1/5 2/5 Ileocecal Lymph Node 4/5 1/3 2/5 4/5 Ileum Contents 3/5 1/3 2/5 2/5 Cecum Contents 4/5 3/3 4/5 5/5 Total 20/45 (44%) 14/27 (52%) 24/45 (53%~ 20/45 (44%) the end of the trial, no significant (P > 0.05) difference was detected from qualitative or quantitative bacteriology of tonsil, mandibular lymph node, lung, liver, spleen, ileum contents, cecum contents, jejunal lymph node, or ileocecal lymph node (Table 12).

Feed analysis. No detectable amounts of oxytetracycline, chlortetracycline, penicillin G or sulfamethazine were in either the pre-starter or starter feeds in Trial 5.

Trial 6

Salmonella isolation from fecal samples. All animals were Salmonella culture-negative prior to inoculation with Salmonella Typhimurium strain B. Animals in the strict control group remained Salmonella culture-negative throughout the study. On day 2 there were fewer Salmonella culture-positive samples in the GS-1 group (50%) than the sterile milk group ( 100%) (Figure 11 ). This trend did not continue through the study. On day 14, there were more Salmonella culture-positive samples in the GS-1 group (100%) than the sterile milk group (25% ).

Salmonella isolation from tonsil samples. On day 2, there were no Salmonella culture- positive tonsil samples collected from any animal (Figure 12). On day 4, there was a higher but non-significant (P >0.05) number of Salmonella culture-positive tonsil samples in the 44

100%

90%

80%

Cl) 70% > :,.:; ·w 60% 0 a.. Sterile Milk c 50% - 40% -- DGS-1 Cl) a.. 30%

20%

10%

0% 2 4 7 9 11 14 Day (Challenge= 0)

Figure 11. Trial 6 Salmonella culture-positive fecal samples sterile milk group ( I 00%) compared to the GS- I group (25% ). After day 9, Salmonella was never recovered from any tonsil sample collected from the GS- I group. Salmonella was recovered from tonsil samples in the sterile milk group through the study. There was a significant difference in the mean number of days during which Salmonella was recovered

100%

90%

80%

Cl) 70% :,.:;> ·w 60% - a..0 c 50% Sterile Milk 40% DGS-1 Cl) a.. 30%

20%

10% -

0% - 2 4 7 9 11 14 Day (Challenge = 0)

Figure 12. Trial 6 Salmonella culture-positive tonsil samples 45 from individual tonsil samples in the GS-1 (2.25 ± 4.5) compared to the sterile milk group

(12.75 ± 2.5).

Salmonella isolation from tissues. The mandibular lymph nodes, lung, liver, and spleen were Salmonella culture-negative in all animals (Table 13). There was no significant (P >

0.05) difference in the proportion of tissues that were Salmonella culture-positive in the GS-1 group (35%) compared to the sterile milk group (50%). There was no significant (P > 0.05)

Table 13. Trial 6 Salmonella isolation from tissues Sam pie Sterile Milk GS-1 (positive/total) (positive/total) Tonsil 3/4 0/4 Mandibular Lymph Node 2/4 0/4 Lung 0/4 0/4 Liver 0/4 0/4 Spleen 0/4 0/4 Jejunal Lymph Node 1/4 0/4 Ileocecal Lymph Node 3/4 3/4 Ileum Contents 3/4 3/4 Cecum Contents 4/4 4/4 Colon Contents 4/4 4/4 Total 20/40 (50%) 14/40 (35%)

difference in the mean Salmonella CFU per gram of cecum contents in the GS-1 group (268

± 195) and the sterile milk group (192 ± 221 ). Although the mean Salmonella CFU per gram of tonsil in the GS-1 group was zero (0 ± 0) this was not significantly (P > 0.05) different than sterile milk group (1318 ± 2390).

In Vitro Experiments

Strain identification

Lactobacillus sp. strains GS-1 and GS-2 were tested for their ability to ferment 49 carbohydrates using the API CHL 50 system (Table 14). Based upon carbohydrate 46

Table 14. Carboyhydrate fermentation patterns of Lactobacillus sp. GS-1 and Lactobacillus se. GS-2 Carbohl'.drate GS-1 GS-2 Carbohl'.drate GS-1 GS-2 Glycerol Salicine + Erythritol Cellobiose + D-Arabinose Maltose + + L-Arabinose Lactose + Ribose + Melibiose + D-Xylose Saccharose + + L-Xylose Trehalose + Adonitol Inuline p Methyl-xyloside Melezitose Galactose + + D-Raffinose + D-Glucose + + Amidon D-Fructose + Glycogene D-Mannose + Xylitol L-Sorbose p Gentiobiose + Rhamnose D-Turanose Dulcitol D-Lyxose Inositol D-Tagatose Mannitol + D-Fucose Sorbitol + L-Fucose a Methyl-D-mannoside - D-Arabitol a Methyl-D-glucoside L-Arabitol N Acetyl glucosamine + Gluconate + Amygdaline + 2 Keto-gluconate - Arbutine + 5 Keto-gluconate - Esculine + + (+)Positive(-) Negative fermentation profiles, Lactobacillus sp. strain GS- I and Lactobacillus sp. strain GS-2 were identified as Lactobacillus paracasei subsp. paracasei and Lactobacillus fermentum, respectively. These strains were assigned the following names by our laboratory: L. paracasei subsp. paracasei GS-I and L. fermentum GS-2.

Antibiotic resistant Lactobacillus mutants

Colonies of Lactobacillus paracasei subsp. paracasei GS-I and Lactobacillus fermentum

GS-2 were isolated on MRS agar containing 30 µ1/ml of rifampin and 400 µ1/ml of streptomycin. The isolates were identified as Lactobacillus paracasei subsp. paracasei GS-I

(Rif/Stl) and Lactobacillus fermentum GS-2 (Rif/StrR), respectively. 47

Figure 13. Salmonella Typhimurium x4232 expressing GFP

Salmonella Typhimurium x4232 expressing GFP

The ability of Salmonella Typhimurium x4232 containing the p VLac plasmid to express

GFP was confirmed by exciting colonies grown on an agar plate with a handheld darkreader lamp. Individual bacteria expressing GFP was confirmed by epiflourescent microscopy of a

Figure 14. Porcine phagocytes containing Salmonella Typhimurium x4232/GFP 48

wet mount overnight culture of Salmonella Typhimurium x4232/GFP (Figure 13).

Salmonella Typhimurium x4232/GFP in porcine phagocytes

The ability of Salmonella Typhimurium x4232/GFP to express GFP in porcine phagocytes

was confirmed by epiflourescent microscopy (Figure 14). Porcine phagocytes were

identified by red-colored nuclei. Individual bacteria could be visualized within porcine

phagocytes.

Plasmid profiles

Plasmid profiles of L. paracasei subsp. paracasei GS-1 and L. fermentum GS-2 are shown

in Figure 15A. Three distinct bands (~1.2 kb, 7 kb, and ~20 kb) were resolved in the strain

GS-1 profile while the strainGS-2 profile was not resolved (Figure 15A). Comparison of

plasmid profiles of strains GS-1, GS-1 H, and L T-60 (Figure 15B) revealed no difference in

A B 1 2 3 1 2 3 4 kb 12 2 10 8 7 6 5 4 .6 3

.5

Figure 14. A. 1 kb ladder (lane 1) GS-1 (lane 2) GS-2 (lane 3) B. GS-lH (lane 1) LT-60 (lane 2) GS-1 (lane 3) 1 kb ladder (lane 4). 49 the plasmid profiles of high and low passage isolates of strain GS-1.

In vitro inhibition of Salmonella Typhimurium strain B by Lactobacillus species

Lactobacillus spp. strains GS-1, GS-1 H, GS-2, L T-60, and L T-61 inhibited growth of

Salmonella Typhimurium strain B (Figure 16). Spent media from overnight cultures of strain

GS-1 and strain GS-2 did not inhibit Salmonella Typhimurium strain B grown in the well diffusion assay. Salmonella Typhimurium strain B was not inhibited by the well diffusion assay using 0-25%, 25-50%, 50-75% or 75-100% ammonium sulfate cuts. A pH gradient was associated with each zone of inhibition in Salmonella Typhimurium strain B growth. The pH at the edge of the zone of inhibition was ~4.4 while the pH immediately within the agar underlying the Salmonella Typhimurium strain B confluent growth was ~5.2. MRS broth adjusted to pH 3.8 and 4.2 for GS-1 and GS-2, respectively did not inhibit Salmonella

Typhimurium strain Bin the agar well diffusion assay.

Figure 16. Spot test of Lactobacillus species and Salmonella Typhimurium strain B 50

CHAPTER 5. DISCUSSION

In Vivo Experiments

The initial goal of these experiments was to evaluate feeding yogurt cultures as a method to reduce levels of Salmonella Typhimurium in pigs. Pigs were daily fed one of two

Lactobacillus yogurt cultures prior to and after inoculation with Salmonella Typhimurium

strain B . After the pigs were inoculated intranasally with Salmonella Typhimurium strain B, the presence of Salmonella was monitored from fecal samples, tonsil samples, and internal organs of the pigs. Also, anti-Salmonella serum antibody levels were monitored on a weekly basis after inoculation with Salmonella Typhimurium strain B. It was hypothesized that feeding yogurt cultures would be associated with increased anti-Salmonella serum antibody levels and decreased levels of Salmonella in the feces and tissues of pigs compare to controls.

Trial I was designed to evaluate the anti-Salmonella serum antibody levels in pigs fed L. paracasei subsp. paracasei GS-I yogurt or L. fermentum GS-2 yogurt and inoculated with

Salmonella Typhimurium strain B. On day 7, the mean anti-Salmonella serum antibody

levels in both yogurt groups were lower than those in the sterile milk group. Interestingly, these serum antibody results corresponded to an unexpected decrease in the number of

Salmonella culture-positive fecal samples that were collected from the GS-I and GS-2 groups compared to the sterile milk group. By the end of the trial, the mean antibody levels

in both yogurt groups remained lower than those in the sterile milk group. The trend towards reduced Salmonella culture-positive fecal samples in the yogurt groups also continued throughout the study. In addition, the mean Salmonella CFU per gram of tonsil in the GS-1 and GS-2 groups was lower than the sterile milk group on day 27. This difference, however, 51

___ was not significant due to high variation between pigs and few degrees of freedom. While

both yogurt-fed groups had decreased levels of Salmonella, the effect was greatest in the GS-

1 group. Consequently, the L. paracasei subsp. paracasei GS-1 yogurt culture was selected

for further evaluation.

Trial 2 was designed to repeat trial 1 and to examine the effect of feeding L. paracasei

subsp. paracasei GS-1 yogurt on the Salmonella levels in tissues of pigs shortly (within 9

days) after the pigs were inoculated with Salmonella Typhimurium strain B. Therefore,

internal organs from pigs were collected for Salmonella isolation on days 2, 9 and 27. In trial

1, no Salmonella culture-positive fecal samples were collected from any animal in the GS-1

group after day 9. Thus, it was hypothesized that decreased fecal shedding would correlate

with decreased levels of Salmonella within tissues of pigs by day 9.

In trial 2, the results of Salmonella isolation from fecal and tonsil samples were similar to

those in trial 1. The mean Salmonella CFU per gram of tonsil, ileocecal lymph node, and

cecum contents were lower in the GS-1 group than sterile milk group on days 2 and 9.

However, the difference was not significant due, in part, to the high variation between pigs

and few degrees of freedom.

Cumulative results from trials 1 and 2 demonstrated a model that detected differences in

the probiotic qualities between two species of Lactobacillus. Trials 1 and 2 also showed that

feeding L. paracasei subsp. paracasei GS-1 yogurt repeatedly reduced levels of Salmonella

in tonsil and fecal samples of pigs

Results from trials 1 and 2 only allow the speculation of the in vivo pro biotic mechanisms

of L. paracasei subsp. paracasei GS-1 yogurt. In these trials, there was a dramatic decrease

in fecal shedding of Salmonella in the GS-1 group compared to the sterile milk controls by 52

day 2. However, there was-no significant difference in the mean anti-Salmonella antibody

levels in either group on any day. Since primary adaptive immune responses take between 4

and 7 days to develop, these results indicate that a mechanism( s) other than the stimulation of

an adaptive immune response may account for the reduction in Salmonella levels between

days O and 7. Decreased pH in the gut lumen, competition for adhesion sites and nutrients,

production of anti-microbial substances, and stimulation of non-specific or cellular immune

33 66 responses may also be involved as mechanisms. •

Trial 3 was designed to investigate the mechanism(s) for the reduction of Salmonella in

pigs fed GS-1 yogurt. To evaluate the role of live L. paracasei subsp. paracasei GS-1 and

heat-labile constituents in the yogurt cultures, live L. paracasei subsp. paracasei GS-1 yogurt

cultures were killed either by irradiation or heat, respectively. The results of Salmonella

isolation from fecal and tonsil samples in the GS-1 group and sterile milk group were unlike the results from trials 1 and 2. Furthermore, there was no significant difference between the

Salmonella levels in the tonsil and fecal samples from pigs in either the irradiated or heat-

killed L. paracasei subsp. paracasei GS-1 yogurt groups compared to pigs in the live L. paracasei subsp. paracasei GS-1 yogurt group.

Examination of culture records indicated that the L. paracasei subsp. paracasei GS-1 stock

cultures used to inoculate all of the yogurt cultures for trial 3 had been sub-cultured daily in

MRS broth for 40 days prior to the beginning of trial 3. Experimental evidence has shown that bacteriocins produced by lactobacilli may be plasmid-bome.49 Furthermore, plasmids from some Lactobacillus species are quickly cured as a result of in vitro sub-culturing. 85

Thus, it was hypothesized that plasmid-borne bacteriocin of L. paracasei subsp. paracasei

GS-1 may have been cured during sub-culturing. 53

Trial 4 was designed to evaluate differences between yogurt cultures of high and low

passage isolates of L. paracasei subsp. paracasei GS-1 to reduce levels of Salmonella in

swine. From day O through day 27, there was no significant difference in Salmonella culture-

positive fecal and tonsil samples collected from pigs in the GS-1, GS-1 H, GS-2 and sterile

milk groups. In trials 1 through 4, antibiotic-free feed was ordered from the same feed

source. Analyses of the feed from trials 1-3 for antibiotics were not conducted. However,

analysis of feed from trial 4 revealed that oxytetracycline was present in the starter diet at

~25 ppm. In vitro sensitivity assays showed that both L. paracasei subsp. paracasei GS-1

and L. fermentum GS-2 were sensitive to oxytetracycline. Consequently, it was hypothesized

that oxytetracycline in the trial 4 feed may have inhibited the probiotic effects of L. paracasei

subsp. paracasei GS-1 yogurt that were observed in trials 1 and 2.

Trial 5 was designed to repeat trial 1 using antibiotic-free feed from a different feed

source. In addition, the effects of Lactobacillus paracasei subsp. paracasei GS-1 yogurt

cultures supplemented with fructooligosaccharide (FOS) were evaluated. Herich et al.

showed that the addition of FOS to a Lactobacillus paracasei pro biotic improved the

immunostimulative effects and survival rate of the pro biotic in pigs.42 Thus it was

hypothesized that the addition of FOS would improve the pro biotic effects of L. paracasei

subsp. paracasei GS-1 yogurt. From day O through day 27, there was no significant

difference in Salmonella culture-positive fecal and tonsil samples collected from pigs in the

GS-1, GS-1 + FOS, GS-2 and sterile milk groups.

Argus SC TM (Intervet Inc., Millsboro, DE) is an attenuated Salmonella enterica subsp. enterica serovar Cholerasuis vaccine that has been used to reduce shedding and tissue

12 37 103 colonization of Salmonella Typhimurium in pigs. • • In order to detect differences in 54 tissue colonization and fecal shedding between Argus SC™ vaccinates and non-vaccinated controls, the challenge dose of Salmonella Typhimurium had to be reduced from 108 CFU to

106 CFU per animal. 103 The mechanism of protection by Argus sc™ probably involves an adaptive immune response to Salmonella Cholerasuis that provides cross-protection to

Salmonella Typhimurium. Since yogurt treatment alone cannot induce an adaptive immune response to Salmonella Typhimurium, the challenge dose (108 CFU per animal) used in trials

1-5 may have overwhelmed any protective effect of the yogurt cultures. Thus, it was hypothesized that a lower challenge dose, 106 CFU per animal, would be more appropriate to evaluate feeding L. paracasei subsp. paracasei GS-1 yogurt as a method to reduce

Salmonella Typhimurium in pigs.

Trial 6 was designed to evaluate feeding of L. paracasei subsp. paracasei GS-1 yogurt to reduce Salmonella levels in pigs challenged with 106 CFU per animal. Throughout the trial, results of Salmonella isolation from tonsil samples were similar to those collected in trials 1 and 2. Until day 11, the results from fecal samples were also similar to those collected in trials 1 and 2. On day 14, more Salmonella culture-positive fecal samples were collected from the pigs in the GS-1 group than the sterile milk group. The mean number of days during which Salmonella was recovered from individual animals in the GS-1 group was lower than the sterile milk group. These results were similar to those in trials 1 and 2. Furthermore,

Salmonella was not isolated from any of the tonsils of the pigs in the GS-1 group on day 14.

This difference was not significant due to high variation between pigs and few degrees of freedom.

Several sources of variability may have contributed to the inconsistent reduction of

Salmonella in pigs fed Lactobacillus yogurt cultures. First, different strains of Lactobacillus 55

------may have inherently different probiotic effects. Such strain-dependent variation has been

reported in models of disease, weight gain, and immune function in chickens, mice and

31 46 51 58 73 79 81 84 90 96 106 108 pigs. , , • , , , • • , , - The results from trial 1 showed differences between L.

paracasei subsp. paracasei GS-1 and L. fermentum GS-2 in their ability to reduce the levels

of Salmonella Typhimurium in pigs. Both yogurt cultures were associated with a reduction

in Salmonella levels. However, a more dramatic effect was associated with the L. paracasei

subsp. paracasei GS-1 yogurt.

A second possible source of variability is animal genetics. In the present studies, feeding

GS- I yogurt cultures significantly reduced Salmonella levels in the tonsil samples in pigs

from Farms A and C (Trials 1,2, and 6). Significant effects of feeding L. paracasei subsp.

paracasei GS-I yogurt on Salmonella levels in fecal samples, however, were limited to the

pigs from Farm A (Trials I and 2). Among the pigs from Farm A, the effect of feeding L.

paracasei subsp. paracasei GS-I yogurt on Salmonella levels in fecal samples, tonsil

samples, and internal organs was variable (Trials I through 4 ). Thus variability was

observed between and among farms. As a result, conclusions regarding the influence of

animal genetics in these studies are not possible.

A third source of variation may be related to the origins of L. paracasei subsp. paracasei

GS-I and L. fermentum GS-2. Both were isolated from the pars esophagus of normal pigs. In

some strains of Lactobacillus, the peptidoglycan layer is associated with intrinsic adjuvant

57 76 86 activity. • • Furthermore, the S-layer proteins of some Lactobacillus strains are believed

to be involved in adhesion to epithelial cells and adjuvant activity. 87 In the pig gut,

immunocompetent cells are present in the Peyer' s patches as a part of the GALT (gut-

associated lymphoid tissue). 74 It is possible that L. paracasei subsp. paracasei GS- I and L. 56 fermentum GS-2 are only capable of adhering to the pars esophagus but not the epithelial

cells of the Peyer' s patches. If this is true, immune stimulation by these strains may be

precluded by their inability to associate with Peyer's patches.

A fourth source of variation may be related to the ecology of the gut. According to

DuBois et al., the indigenous micro biota is made up of three parts: the autochthonous flora,

the normal flora and pathogens. 26 Autochthonous bacteria have existed throughout the

evolution of the host and are found in every individual within a species. Constituents of the

normal flora may vary geographically but are present in all members of a specific

community. Allochthonous bacteria, on the other hand, are strains that do not normally

93 97 colonize an individual or a specific niche within an individual. • Consequently, they are

not autochthonous. However, allochthonous bacteria may be normal flora in one community

but not another.

If allochthonous bacteria enter the gut, they probably encounter a very harsh environment

and are not likely to colonize or persist for long periods of time. Consequently, if

administered orally, they may only be recovered in fecal samples for a short period of time

after administration has ceased. Furthermore, allochthonous bacteria are "foreign" to the

host. As a result, they may be capable of stimulating the immune response unlike

autochthonous and normal flora to which the host has been tolerized. In mice, the immune

system was stimulated to a greater extent in animals that were fed human isolates of

Lactobacillus (an allochthonous strain) than in mice fed mouse isolates of Lactobacillus

(autochthonous or normal strains).9

In trials 1 and 2, L. paracasei subsp. paracasei GS-1 and L. fermentum GS-2 may have

been allochthonous, relative to the pigs to which they were fed. As a result, both strains may 57 have stimulated an innate or non-humoral immune response that may have led to resistance to Salmonella infection. In subsequent trials, both strains may have been constituents of the pigs' normal flora. Consequently, the immune system may have been tolerized to these particular strains and was therefore not stimulated by increased numbers of these

Lactobacillus speceis in the gut. In the future, yogurt cultures containing non-swine isolate(s) of Lactobacillus or other LAB would facilitate the evaluation of allochthonous vs. normal flora and their ability to reduce the shedding of Salmonella Typhimurium in swine.

Furthermore, such experiments may lead to the identification of bacteria that exhibit consistent probiotic effects in pigs.

In Vitro Experiments

The goals of these experiments were to 1.) identify Lactobacillus sp. strains GS-1 and GS-

2 to the species level, 2.) characterize Lactobacillus sp. strain GS-1 and GS-2 based upon plasmid profiles and in vitro bactericidal activity against Salmonella Typhimurium, and 3.) develop markers for Salmonella and Lactobacillus species for use in in vivo studies.

Lactobacillus sp. strain GS-1 and Lactobacillus sp. strain GS-2 were identified by the API

50 CHL test as Lactobacillus paracasei subsp. paracasei and Lactobacillus fermentum, respectively. Plasmid profiles of both isolates demonstrated another method to differentiate both strains. The plasmid profile of L. paracasei subsp. paracasei GS-1 resolved three distinct bands while the profile of L. fermentum GS-2 appeared as a smear on the gel. Others have reported poor quality mini-preps from some strains of Lactobacillus.56 Some strains have outer S-layers and peptidoglycan layers that are difficult to lyse. Consequently, mini- preps appear as a smear due to high levels of protein and carbohydrate impurities. 58

The similarity among plasmid profiles of high and low passage isolates of L. paracasei subsp. paracasei GS-1 indicates that the three predominant plasmids found in these isolates are stable during sub-culturing in MRS broth. The inherent stability of these plasmids suggests that they may be candidates for cloning vectors in L. paracasei subsp. paracasei

GS-1.

The bactericidal effect of the Lactobacillus strains against Salmonella Typhimurium strain

B may be due to several factors. In these assays, pH 4.2 or lower in the agar was associated with the zone of inhibition. However, un-inoculated MRS broth controls (pH 3.7 and 4.2 for

GS-1 and GS-2, respectively) did not inhibit the growth of Salmonella Typhimurium strain B in either the spot test assay or the agar well diffusion assay.

9 Bacteriocins, which are biologically active peptides produced by a number of bacteria4 , may also contribute to the bactericidal effect observed. However, concentrated SCS of L. paracasei subsp. paracasei GS-1 and L. fermentum GS-2, obtained by ammonium sulfate precipitation, did not inhibit the growth of Salmonella Typhimurium strain B in either the spot test or agar well diffusion assay. Thus, the mechanisms of in vitro bactericidal effects of

L. paracasei subsp. paracasei GS-1 and L. fermentum GS-2 remain unknown.

Since many species of Lactobacillus are present in high numbers within the gut of animals, it can be difficult to differentiate an administered Lactobacillus isolate from a Lactobacillus isolate that is part of the animals' indigenous flora. L. paracasei subsp. paracasei GS-1

(RifSt) and L. fermentum GS-2 (RifSt) will facilitate detection, isolation and enumeration of these particular strains after they have been fed to pigs. Similar strains have been developed and successfully used to study Lactobacillus colonization in pigs.95 59

The mechanisms of Salmonella Typhimurium dissemination through the body of pigs are

not clearly understood. Fedorka-Cray et al. have shown that Salmonella Typhimurium can

invade and disseminate from the nostrils to the gut and internal tissues of pigs through a

mechanism other than the fecal-oral route.30 In mice, a similar mechanism has been reported

that involves dissemination through CD 18-expressing phagocytes. 105

The GFP-expressing strains of Salmonella Typhimurium x4232 developed in these studies

will be useful in flow cytometric assays. Such assays may provide insight to the mechanisms

of invasion of Salmonella Typhimurium in pigs. Together, the strains of Lactobacillus and

Salmonella developed in these studies may be used to evaluate microbe-microbe interactions

and host-microbe interactions in vivo. Results of such studies may provide a better

understanding of the pro biotic mechanisms of lactobacilli in swine. 60

CHAPTER 6. CONCLUSIONS

In the experiments described herein, various preparations of yogurt cultures from two isolates of Lactobacillus were evaluated as a method to reduce levels of Salmonella

Typhimurium in swine. In two of six trials, feeding live Lactobacillus paracasei subsp. paracasei GS-1 yogurt cultures significantly reduced levels of Salmonella in tonsil and fecal samples from swine. These studies show species-dependent effects of orally administered

Lactobacillus yogurt cultures on the levels of Salmonella Typhimurium in swine. The main conclusion of the present study is that Lactobacillus yogurt cultures can reduce levels of

Salmonella Typhimurium in swine. While the inconsistent results currently preclude the use of this method in swine production, they encourage further research.

Several factors that may influence the pro biotic effects of lactobacilli should be investigated. These factors include animal genetics, composition of the diet, indigenous flora, and the age of the animals. The bacterial strains described in these experiments will aid in the study of these sources of variation and will facilitate the delineation of the in vivo pro biotic mechanisms of lactobacilli. 61

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ACKNOWLEDGEMENTS

I would like to acknowledge several people for their role in my graduate education. I am deeply grateful to my major professor, Dr. Hank Harris, for his patience, encouragement, and

"coaching". I appreciate the opportunity to study under his mentorship. I would also like to thank the other members of my POS committee, Dr. James Dickson and Dr. Aubrey

Mendonca, for their encouragement. I thank all the members of my POS committee for helping me to "step back" and look at this work from a objective, scientific perspective.

I would like to acknowledge the National Pork Producers Council, the Food Safety

Consortium (Iowa State University) and the USDA for funding these projects.

The completion of animal studies would not have been possible without the assistance of several members of the Harris Lab group: Stephen Gaul, Dr. Dachrit Nilubol, Al Loynachan,

Matt Erdman, Jeanne Nugent, Stephanie Wedel, Jason Slater, Carrie Basak, Dr. Peter Akoto,

Dr. Nahkyung Lee, Lisa Miller, Adam Baumann, Brad Chriswell, Tim Tardiff, and Nick

Pryor.

I would also like to thank Rohrk Cutchlow for his help in the early animal studies. Thanks a lot for your friendship, encouragement, and camaraderie.

A special thanks goes to my parents, David and Sandi, my sister, Heather, and my brother,

Ryan. I can't tell how much the last couple years together have meant to me. Dad, thanks for your encouragement, perspective, humor and 24 years of discipleship. Mom, I appreciate all the laundry you did and countless other examples of grace. Most of all, I appreciate your love and support. Heather, thanks for your encouragement, your example, and for being a wonderful source of discernment. Ryan, thank you for two more great years together as brothers and best friends. 73

A special thanks, also, to Deb. I'm grateful for your encouragement, friendship and love.

Thanks for understanding the many late nights. I can't wait to see what the "next chapter" brings!

Finally, I am grateful to God for His grace, strength, love, and salvation that made the completion of this work possible.