Investigation of Swine Dysentery Associated with "Brachyspira

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2014 Investigation of swine dysentery associated with "Brachyspira hampsonii" strain EB107 and comparison of diagnostic methods Bailey Lauren Wilberts Iowa State University

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Investigation of swine dysentery associated with “Brachyspira hampsonii” strain EB107 and comparison of diagnostic methods

Bailey L. Wilberts

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Veterinary Pathology

Program of Study Committee: Eric R. Burrough, Co-Major Professor Michael J. Yaeger, Co-Major Professor Darin M. Madson Jesse M. Hostetter Paul J. Plummer

Iowa State University

Ames, Iowa

2014

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

ABSTRACT………………………………...... vi

CHAPTER 1 GENERAL INTRODUCTION ...... 1

Statement of Problem ...... 1 Specific Aims ...... 1 Dissertation Organization ...... 2 Literature Review...... 3 Historical background ...... 3 Diagnostic assays ...... 8 Identification of a novel etiologic agent of swine dysentery ...... 10 Fluorescent in situ hybridization for bacterial identification ...... 12 Background ...... 12 Technique ...... 13 Challenges ...... 16 Selected applications ...... 17 Pathogenesis of swine dysentery ...... 18 Differential mucin expression of the colonic mucus bilayer in health and disease ...... 21 References ...... 27

CHAPTER 2 COMPARISON OF LESION SEVERITY, DISTRIBUTION, AND COLONIC MUCIN EXPRESSION IN PIGS WITH ACUTE SWINE DYSENTERY FOLLOWING ORAL INOCULATION WITH “BRACHYSPIRA HAMPSONII” OR BRACHYSPIRA HYODYSENTERIAE ... 44

Abstract ...... 44 Introduction ...... 45 Materials and Methods ...... 47 Animals ...... 47 Bacterial strains, growth conditions, and preparation of inocula ...... 47 Animal inoculation...... 48 Molecular identification ...... 49 Animal observations and necropsy ...... 49 Histopathology ...... 50 Immunohistochemistry ...... 51 Histochemistry ...... 52 Image analysis ...... 52

iii

Fluorescent in situ hybridization ...... 53 Statistical analyses ...... 53 Results ...... 53 Animal observations ...... 53 Microbial culture ...... 54 Gross pathology ...... 54 Histopathology ...... 57 Immunohistochemistry ...... 58 Histochemistry ...... 60 Fluorescent in situ hybridization ...... 62 Molecular identification ...... 62 Discussion ...... 62 Acknowledgements ...... 67 References ...... 68

CHAPTER 3 COMPARISON OF CULTURE, PCR, AND FLUORESCENT IN SITU HYBRIDIZATION FOR DETECTION OF BRACHYSPIRA HYODYSENTERIAE AND “BRACHYSPIRA HAMPSONII” IN PIG FECES . 72

Abstract ...... 72 Introduction ...... 73 Materials and Methods ...... 74 Bacterial strains and growth conditions ...... 74 Preparation of fecal dilutions and standard plate counts ...... 75 Fluorescent in situ hybridization ...... 75 Real-time polymerase chain reaction primers, probes, and protocol ...... 77 Preparation of samples from experimentally infected pigs ...... 77 Results ...... 78 Culture ...... 78 Fluorescent in situ hybridization ...... 79 Real-time polymerase chain reaction ...... 80 Fluorescent in situ hybridization: Samples from experimentally infected pigs ...... 80 Discussion ...... 82 Sources and Manufacturers ...... 84 References ...... 85

CHAPTER 4 INVESTIGATION OF THE IMPACT OF INCREASED DIETARY INSOLUBLE FIBER THROUGH THE FEEDING OF DRIED DISTILLERS GRAINS WITH SOLUBLES ON THE INCIDENCE AND SEVERITY OF BRACHYSPIRA-ASSOCIATED COLITIS IN PIGS ...... 87

Abstract ...... 87 Introduction ...... 88 Materials and Methods ...... 90 Animals ...... 90

Diet ...... 91 Inoculum ...... 91 Brachyspira spp. isolation and growth ...... 93 Animal inoculation...... 93 Molecular identification ...... 94 Oral fluids ...... 94 Animal observations and necropsy ...... 95 Histopathology ...... 96 Immunohistochemistry ...... 96 Histochemistry ...... 97 Image analysis ...... 98 Statistical analyses ...... 98 Results ...... 98 Animal observations ...... 98 Microbial culture ...... 99 Average daily gain ...... 101 Large intestine pH ...... 101 Gross pathology ...... 104 Histopathology ...... 104 Immunohistochemistry ...... 106 Histochemistry ...... 109 Discussion ...... 111 Acknowledgements ...... 117 References ...... 118

CHAPTER 5 GENERAL CONCLUSIONS ...... 122

Conclusions ...... 122 Recommendations for future research ...... 124 References ...... 125

APPENDIX PERMISSIONS...... 128

ACKNOWLEDGEMENTS

I would like to thank my committee co-chairs, Eric R. Burrough and Michael J.

Yaeger, and my committee members, Darin M. Madson, Jesse M. Hostetter, and Paul J.

Plummer for their guidance and support throughout the course of this research. I would like to extend a special thank you to Dr. Burrough for his patience with my endless questions, openness to discuss ideas, and valuable insight that was promptly and efficiently provided despite a very demanding schedule.

In addition, I would also like to thank the Veterinary Pathology Department faculty and staff for making my time at Iowa State University College of Veteirnary Medicine a wonderful experience. The department is filled with great individuals that make a fantastic team.

Finally, thanks to my family for their encouragement, patience, respect and love.

ABSTRACT

Swine dysentery (SD) is an important cause of mucohemorrhagic diarrhea in pigs.

Swine dysentery is associated with infection by Brachyspira hyodysenteriae which has historically been the only recognized strongly beta-hemolytic Brachyspira sp. However, in recent years, not all strongly beta-hemolytic isolates have been identified as B. hyodysenteriae using PCR assays specific for this species. Several reports have described putatively novel strongly beta-hemolytic Brachyspira spp. including “Brachyspira hampsonii” associated with SD. A pig inoculation study was used to compare lesions and colonic mucin expression associated with infection by B. hyodysenteriae or “B. hampsonii.”

Diagnosis of SD commonly includes culture which while sensitive is time-consuming and

PCR which while rapid can be limited by fecal inhibition. Due to the limitations of these assays, a same-day fluorescent in situ hybridization (FISH) assay was developed to detect B. hyodysenteriae and "B. hampsonii" in pig feces and the threshold of detection was compared to PCR and culture. Little is published about the pathogenesis of SD; yet the interaction between the colonic microbiota and diet seems to be important. Recently, distillers dried grains with solubles (DDGS), a source of insoluble fiber, has been increasingly included in swine diets. A randomized complete block experiment was used to examine the effect of

DDGS on the incidence of Brachyspira-associated colitis in pigs.

Gross and microscopic lesions are similar following infection with “B. hampsonii” or

B. hyodysenteriae. Histochemical and immunohistochemical evaluation of the colon revealed decreased expression of sulphomucins and mucin 4 and increased expression of mucin 5AC in diseased pigs compared to controls.

vii

The FISH assay effectively detected both Brachyspira spp. in formalin-fixed feces from pigs with SD. Spirochetes were also readily detectable in pen level samples from clinical pigs. Although culture remains the diagnostic assay of choice for surveillance, clinically affected pigs may be identified in a timelier manner using either qPCR or FISH.

Pigs receiving 30% DDGS shed on average one day prior to and developed SD nearly twice as fast as pigs receiving 0% DDGS. These data suggest a reduction in insoluble fiber should be considered a part of any effective disease elimination strategy for SD.

CHAPTER I

GENERAL INTRODUCTION

Statement of Problem

Swine dysentery has been recognized as an economically important cause of colitis in growing and finishing pigs since it was first described in 1921.192 The original etiologic agent, most recently designated B. hyodysenteriae, was first identified in the early 1970s.67,172

More recent investigations have examined the impact of diet on SD;62,141,176 however, the exact mechanisms by which diet effects the pathophysiology of SD remain ill-defined.

Historically, B. hyodysenteriae was thought to be the sole strongly beta-hemolytic

Brachyspira spp. to cause SD. However, there has been an increase in the identification of atypical, strongly beta-hemolytic isolates not identified as B. hyodysenteriae using nox-based

PCR assays 15,189 from an increasing number of SD cases in North America.31,135 One of these atypical, strongly beta-hemolytic spirochetes has been provisionally designated “B. hampsonii.”30 Coincident with the identification of potentially novel etiologic agents associated with SD, distillers dried grains with solubles, a source of insoluble dietary fiber, has been increasingly included in diets of swine. The colon pathology, mucin expression, and impact of insoluble dietary fiber following infection with “B. hampsonii” have not been thoroughly described.

Specific Aims

The overall objective of the studies described herein was to characterize swine dysentery (SD) associated with “Brachyspira hampsonii” strain EB107 and compare a novel diagnostic assay for this pathogen against standard methods. The central hypothesis of this

2 dissertation is that there are minimal differences between clinical disease, pathology, impact of diet, and targeted diagnostics associated with infection by “B. hampsonii” strain EB107 or

B. hyodysenteriae strain B204. The following are specific aims based on the central hypothesis: 1) describe the time to onset of clinical SD and the duration of viable spirochete shedding in pigs inoculated with either “B. hampsonii” strain EB107 or B. hyodysenteriae strain B204 (Chapter 2); 2) describe gross lesions and compare histologic lesion severity and distribution within the colons of pigs that develop SD following infection with EB107 versus

B204 (Chapter 2); 3) characterize the biosynthetic responses in mucin-secreting colonic goblet cells in pigs with histologic lesions consistent with SD following infection with either

EB107 or B204 (Chapter 2 and 4); 4) develop a more timely, antemortem diagnostic assay for SD associated with either “B. hampsonii” or B. hyodysenteriae and compare this assay to qPCR and culture (Chapter 3); and 5) investigate the impact of feeding distillers dried grains with solubles on the development of SD associated with either EB107 or B204 infection

(Chapter 4).

Dissertation Organization

This dissertation is organized in the journal publication format that includes manuscripts previously published, accepted for publication, or submitted for publication. The first chapter (Chapter 1) consists of a general introduction and literature review. The following three chapters (Chapters 2-4) consist of three individual manuscripts that address the specific aims of the research project and the final chapter (Chapter 5) consists of general conclusions and suggestions for future research. References are at the end of individual chapters. One manuscript has been published in Veterinary Pathology (Chapter 2). The second manuscript has been accepted for publication in the Journal of Veterinary Diagnostic

Investigation (Chapter 3). The final manuscript has been submitted to PLoS ONE (Chapter

4). Dr. Bailey Wilberts is the principal author of the manuscripts in this thesis; Dr. Eric

Burrough is the corresponding author of each manuscript; Darin Madson, Joann Kinyon,

Paulo Arruda, Tim Frana, Chong Wang, Drew Magstadt, John Patience, Hallie Warneke, and

Leslie Bower were involved in the experimental design, execution, data analysis and critical review of selected manuscripts.

Literature Review

Historical Background

Swine dysentery (SD) was first noted in September, 1917, in the United States and first described in the literature in 1921 as a “bloody diarrhea” that affected three farms with feeder hogs.192 Following extensive gross and histologic evaluation of field cases as well as pigs following experimental exposure, Whiting et al. (1924) concluded that SD was distinctive from hog cholera describing numerous clinical, gross, and histologic characteristics that remain consistent with the disease currently seen in the North American swine industry and by diagnosticians. Despite the identification of conspicuous spirochetes from the colon of an affected pig in the original description of SD, the etiology was not confirmed until the early 1970s.67, 172 Originally being named Treponema hyodysenteriae,67 the causative agent of SD has gone through numerous reclassifications, most recently being placed in the genus Brachyspira.131

Brachyspira spp. are a diverse group of Gram-negative, oxygen-tolerant, anaerobic spirochetes that resemble short slender snakes with several loose coils (Figure 1.1). Those capable of colonizing the large intestine of swine vary from 5 to 11 µm in length and from

0.2 to 0.4 µm in width having two sets of periplasmic flagella anchored at opposite poles of

4 the spirochete conferring a pronounced corkscrew-like motility allowing for penetration of the colonic mucus bilayer.61

Figure 1.1. Fluorescent in situ hybridization of B. hyodysenteriae demonstrating snake-like morphology with several loose coils.

Swine dysentery associated with infection by B. hyodysenteriae has a worldwide distribution being recognized in multiple countries and regions including the United States,

Canada, Central and South America, the Caribbean, Europe, Africa, Australia, New Zealand, and Asia.152 The incidence of SD has varied within these countries or regions over time.

Until recently the incidence of SD in the United States had diminished due to the development of high health herds, multisite production, early weaning systems, and routine medication.61 In infected herds SD has a considerable negative economic impact due to mortality, diminished average daily gain, and antibiotic costs.61 Transmission is primarily through exposure to feces from clinically infected pigs that have large numbers (105 to 109 organisms/gram) of spirochetes in the colonic mucosa.86 Following clinical recovery, the

5 infectious period remains highly variable between studies ranging from 28175 to 70 days164 indicating that clinically normal recovered carrier pigs are likely an important aspect of disease transmission. B. hyodysenteriae has also been isolated from rheas, rodents, and mallards25,76,77,79,81 providing additional potential reservoirs of disease.

Clinically, swine dysentery is characterized by a mucohemorrhagic diarrhea commonly affecting grow-finish pigs, and less frequently weaners (Figure 1.3). 3,67,113,192,193

The passage of loose or soft, yellow to gray feces commonly precedes the onset of the

1.3 1.4 Figure 1.3. Diarrhea characteristic of swine dysentery with hemorrhage and mucus. Figure 1.4. Loss of body condition of pig with swine dysentery (upper right) compared to unaffected pen mates. characteristic mucohemorrhagic diarrhea which is accompanied by a marked loss of body condition (Figure 1.4).132 Most pigs recover over several weeks;138 however, the duration of clinical disease is age-dependent with the course and severity being less pronounced in older pigs.132,192 Death due to SD is uncommon with effective treatment but may occur due to dehydration, acidosis, or hyperkalemia or a combination of these factors and may reach

90% in non-immune pigs61 or as high as 25% mortality when effective treatment is delayed.138 The incubation period for SD is typically 10 to 14 days in naturally exposed pigs

138 although it may range from 2 days to 3 months61 with an average of 11days132 in

6 experimental disease. Diarrhea in one investigation averaged 6.4 days and ranged from 2 to

19 days. 132

Gross lesions specific for SD are confined to the large intestine including the cecum, colon, and to a lesser extent the rectum although distribution can vary with duration and severity.192 The exudate on the mucosal surface of the large intestine ranges from catarrhal to fibrinonecrotic admixed with variable amounts of hemorrhage (Figure 1.5).

Figure 1.5. Fibrinonecrotic and catarrhal colitis in a pig infected with B. hyodysenteriae. Histologically, crypt lumens are elongated and frequently distended with mucus, the lamina propria commonly contains a moderate to severe neutrophilic infiltration, there is variable necrosis and loss of superficial colonocytes, and spirochetes can often be visualized within crypt lumens, mucus producing goblet cells, and less frequently within disrupted epithelial cells by silver staining (Figure 1.6 and Figure 1.7). 61,67,113,173,174,192

Figure 1.6. Characteristic histologic lesions of swine dysentery with crypt elongation (inset; left brace), superficial colonocytes necrosis, hemorrhage, and neutrophils in the lamina propria.

Figure 1.7. Silver stain with numerous spirochetes present in the crypt lumens (arrow).

Ultrastructural examination reveals cellular degeneration with loss or distortion of microvilli, dilation of the cytocavitary network, and numerous spirochetes within crypt lumens, goblet cells, and colonocytes (Figure 1.8). 55,67,173

Figure 1.8. Electron micrograph with condensation of a colonocyte nucleus and cytoplasm, loss of microvilli, dilation of the nuclear envelope (star), swelling and apical displacement of the mitochondria (arrowhead), numerous cytoplasmic vacuoles (four-point star), and spirochetes present in the lumen (arrow).

Diagnostic Assays

Definitive diagnosis of SD is commonly based upon a combination of 1) microscopic lesions, 2) isolation of a characteristic strongly beta-hemolytic, ring phenomenon–positive spirochete from mucohemorrhagic feces or colonic tissue via microbial culture, 3) speciation using molecular methods such as PCR run on extracts from the same samples, and more recently, 4) fluorescent in situ hybridization using formalin-fixed tissue.

Historically, B. hyodysenteriae has been differentiated from other Brachyspira spp. by strength of beta-hemolysis, indole production, and hippurate hydrolyzation.73

B. hyodysenteriae typically imparts a characteristic strong beta-hemolysis that is enhanced around holes in the agar, producing what is known as the “ring phenomenon” when cultivated anaerobically using selective blood agar (Figure 1.9).73 Although indole-negative isolates have been described, isolates of B. hyodysenteriae typically have positive indole and negative hippurate reactions.31,73 Strength of beta-hemolysis has historically been sufficient to differentiate B. hyodysenteriae from the weakly beta-hemolytic Brachyspira spp. that may also reside in the porcine colon including B. innocens, B. intermedia, B. murdochii, and B. pilosicoli.

Figure 1.9. Blood agar plate with strong beta-hemolysis and “ring phenomenon” (circle) typical of B. hyodysenteriae. While Brachyspira culture using selective agars, such as CVS78 and BJ,92 is highly sensitive, false negative results may occur as a result of inappropriate handling or storage of samples including extreme temperatures, drying, or delays during transport.61 Additionally, it can be technically challenging, typically requires speciation using PCR following isolation, and often takes 6 days to complete which results in a delay in disease diagnosis.

Conversely, PCR results can be provided in 1 to 2 days and this assay is widely used for the detection and identification of B. hyodysenteriae.69 Traditionally, PCR targeted portions of the 23S rRNA gene,101 the nox gene,15 or the tlyA gene42 and was run using the primary isolation plate from microbial culture. Recently, however, multiple techniques have been established to run PCR on DNA extracted directly from feces93,94, 127,163,170,197,202 and intestinal tissue.127,170 Such diagnostic assays commonly allow for quantification of bacterial load with simultaneous identification of other enteric pathogens. The detection limit of such assays varied and included 1.5x103 CFU/ml,202 102 to 103 cells per 0.2 grams of sample,163

102 to 103 cells per gram feces,94 and 104 copies.127 When compared to traditional culture and biochemical tests, a duplex PCR system applied to DNA extracted directly from feces detecting the tlyA gene of B. hyodysenteriae lowered the sensitivity by a factor of a 1,000 to

10,000.146 A more recent investigation using routine diagnostic sampling of 103 herds and comprising 239 fecal samples, estimated diagnostic sensitivity of PCR and culture were

73.2% (95% CI: 62.3; 82.9) and 88.6% (74.9; 99.3), respectively.69

Fluorescent in situ hybridization (FISH) is an additional molecular method that allows for the visualization and speciation of bacteria and has been described for the identification of B. hyodysenteriae in formalin-fixed tissues.19 Much like other diagnostic assays, there are limitations associated with FISH as the assay requires approximately two days or more to complete following fresh tissue collection and is limited to a postmortem diagnosis.

Identification of a Novel Etiologic Agent of Swine Dysentery

There has been an increase in SD diagnoses in growing and finishing pigs in the

United States since 200631 with similar reports from Canada since 2009.135 However, not all

11 strongly beta-hemolytic isolates were identified as B. hyodysenteriae using nox-based PCR assays for speciation.15,189 Several recent reports have described potentially novel, strongly beta-hemolytic Brachyspira spp. including atypical isolates of B. intermedia which were isolated from field cases in the United States,31 “Brachyspira suanatina”147 which was present in pigs and wild mallards in Sweden and Denmark, and “Brachyspira sp.

SASK30446” 64 which was first identified in Canada.

“B. hampsonii” is a recently proposed species that is a strongly beta-hemolytic spirochete which is commonly indole negative. It can be best differentiated from B. hyodysenteriae by its nox gene sequencing.30 Comparison of nox gene sequences revealed that clade II isolates of “B. hampsonii” are nearly identical (≥99% identity) to “B. sp. SASK

30446.”28 Experimental infection with “B. hampsonii” strains following oral inoculation has consistently resulted in clinical disease and lesions similar to, if not indistinguishable from,

B. hyodysenteriae infection in mice26 and pigs.27,153,194

Despite the recent characterization and naming of “B. hampsonii,” an atypical isolate of a strongly beta-hemolytic, indole negative spirochete was first identified in 1991 in the

United Kingdom being isolated from the large intestine of a pig with histologic evidence of colitis.128 Identified as Serpulina innocens by multilocus enzyme electrophoresis, the isolate identified as strain P280/1 induced clinical signs and histologic lesions consistent with SD.128

Later analysis of a nox-based phylogenetic tree, strain P280/1 maintained a separate distinct branch between the B. innocens/B. murdochii branch and the B. hyodysenteriae branch.15

Evaluation of the nucleotide sequences of the nox gene available in GenBank corresponding to various isolates demonstrates that strain P280/1 is more similar to “B. hampsonii” clade I than “B. hampsonii” clade II (Figure 1.10); however, this interpretation is not consistent with

a recent manuscript in which it was concluded that strain P280/1 is more similar to “B.

hampsonii” clade II.30 Additional investigation including whole genome analysis of these

strains is needed to provide more complete information concerning species classification and

potentially pathogenicity.

Figure 1.10. Comparison of nox gene sequences from multiple Brachyspira spp. representing 5 official species and 2 putative species. Fluorescent in situ Hybridization for Bacterial Identification

Background. In situ hybridization (ISH) was developed by two separate groups84,134

using cytologic preparations of Xenopus oocytes that were hybridized with radiolabeled

DNA or 28S RNA and detected by microautoradiography. This development allowed for the

examination of nucleic acid sequences inside cells without altering the morphology of the

cell. Nearly two decades later ISH was utilized by Giovannoni et al. (1988) for the

microscopic detection of bacteria using radioactively labeled rRNA-directed oligonucleotide

13 probes. A year later DeLong et al. (1989) was the first to use fluorescent labeled oligonucleotides for the detection of single microbial cells providing not only a safer and shorter technique with better resolution, but also the ability to detect several target sequences within a single hybridization step.125 Additional advances have increased both the sensitivity and speed of fluorescent in situ hybridization (FISH).9

Technique. Fluorescent in situ hybridization detects nucleic acid sequences through a fluorescent labeled probe that hybridizes to a complementary target sequence in an intact cell. The technique commonly involves five simple steps: 1) fixation of the specimen, 2) preparation of the sample including deparaffinization and/or pretreatment steps, 3) hybridization, 4) washing to remove unbound probe, and 5) mounting and visualization.

Probe design is based upon sensitivity, specificity, and ease of tissue penetration. Probe lengths can vary although typical oligonucleotide probes are between 15 and 30 base pairs in length and are created on an automated synthesizer. 125 Fluorescent labeled oligonucleotides are commercially available and can be stored at -20ºC in the dark for many months.125

Probes can be labeled for direct or indirect detection (Figure 1.11). Direct techniques include one or more fluorescent dye molecules directly bound to the oligonucleotide during synthesis through an aminolinker at the 5’-end or bound enzymatically using terminal transferase to attach fluorescently labeled nucleotides at the 3’-end.126

Common FISH fluorochromes are fluorescein-derivatives (fluorescein isothiocyanate or FITC), rhodamine-derivative (Tetramethyl-Rhodamine-Isotheiocyanate (TRITC), Texas

Red) or cyanine dyes including Cy3 and Cy5 which typically produce brighter staining.125

Indirect detection includes linking the probe to a reporter molecule like digoxygenin that is then detected by a fluorescent antibody,201 labeling the probe with horseradish peroxidase

14 that uses fluorescein-tyramide as a substrate,156 or using polyribonucleotide probes, internally labeled with digoxygenin, with the tyramide signal amplification system.187

When used for the visualization of microbial cells, FISH most commonly targets 16S rRNA due to its genetic stability, combination of conserved and variable regions, and high copy number.198 Other targets include 23S rRNA,6,100,118, 130,177 18S rRNA,108,109,166 and

Figure 1.11. Direct, (A) and (B), and indirect, (C) - (E), labeling of probes with digoxygenin (DIG), horseradish peroxidase (HRP) or Tyramide Signal Amplification (TSA). mRNA.187 The use of16S rRNA which contains approximately 1,600 nucleotides serves as a suitable and diagnostic target; however, in some circumstances 16S rRNA fails to provide enough variability to accomplish inter-species and intra-species differentiation 48 as is the case for Brachyspira spp. In such cases, 23S rRNA, which contains about 3,000 nucleotides, can provide enough variability to make such discriminations.8 Regardless of the target, oligonucleotide probes can be designed using sequence data entries in publically available

15 databases to identify microorganisms at the taxonomic to species level according to the region of rRNA targeted.7,57

Prior to hybridization bacteria are fixed using ethanol, methanol, aldehydes, or a mixture of these reagents. Optimal fixation results in good probe penetration, retention of target RNA through inhibition of endogenous RNAses, and maintenance of cell detail.125

Due to the distinctive character of the cell membrane, fixation of Gram-negative and Gram- positive bacteria differs. For Gram-negative bacteria 3-4% (v/v) formaldehyde or paraformaldehyde solution is sufficient. For Gram-positive organisms, ethanol (50%), ethanol/formalin (9:1 v/v) or heat treatment is suggested22,85,151 and additional pretreatment may be required to increase permeability including lysozyme,156,187 lysostaphin,125 or an enzyme mixture.90

Sample preparation includes placement of specimens onto glass slides that have been treated with a coating agent such as pol-L-lysine99 or silanating agents.126 Additional preparation is required for tissue sections. Specifically, paraffin sections require the removal of paraffin using xylene and dehydration in ethanol.19 The use of proteinase K may also be needed for paraffin or cryo-sections to increase the accessibility of the probe to the target.112

Hybridization occurs in a humidity chamber protected from light usually at temperatures between 37 ºC and 50 ºC for 30 minutes to several hours.125 Hybridization stringency (degree of homology between probe and target sequence) can be adjusted by varying the formamide or salt concentration in the hybridization buffer or hybridization temperature. Maintaining the hybridization temperature below the melting temperature of the probe is required for successful hybridization as such conditions allow for annealing of the probes to target sequences. Formamide weakens the hydrogen bonds between the target

16 sequence and its complementary strand decreasing the melting temperature and allowing the lower temperature to be used with high stringency.125 Formamide also destabilizes the secondary structures of rRNA improving the accessibility of the target sequence. Other reagents can be added to the hybridization buffer to improve permeability of the target cell and include sodium dodecyl sulfate (SDS) or ethylenediaminetetraacetic acid (EDTA).

Following hybridization, slides are washed with pre-warmed distilled water or wash buffer with varying salt concentrations as required for stringency.97 Finally, slides are rinsed with water, dried, and mounted typically with commercially available antifading agents.

Challenges. As with any molecular technique, there are various pitfalls and problems associated with FISH; however, viable solutions are commonly available with appropriate trouble-shooting. Autofluorescence of the microorganisms, material surrounding the bacteria185 and tissue components can occur.124,182 Although some background autofluorescence may be helpful for tissue orientation, it typically masks the specific fluorescent signal. This can be alleviated through subtracting it pixel by pixel during digital image processing or changing the growth media, fixation method, or mounting media which seem to dramatically impact signal intensity.165 Additional remedies include the use of narrow-band filter sets, single amplification systems, or multilabelled fluorescent probes.157,165,166 A lack of specificity can also occur making careful design and critical evaluation of new probes essential. Positive controls in addition to closely related strains or species harboring the target sequences with limited mismatches to the probe as negative controls should be included in every FISH experiment.125

False negative results can be due to insufficient penetration (as discussed above), higher order structure of the target or probe, low rRNA content, or photobleaching. Limited

17 accessibility of the probe due to higher order structure of the target can be addressed through the use of the established accessibility map of E. coli 16S rRNA50 or focusing on areas targeted successfully by previous publications. The potential formation of higher order structures of the probe such as dimers and hairpins can easily be analyzed using the appropriate software during probe design. While commonly abundant, the rRNA content of bacterial cells may vary considerably between species and physiologic state which is directly correlated with growth rate.36,142,186 To increase signal intensity, especially for slow-growing bacteria, the use of bright fluorescent dyes like Cy3 is recommended. Additionally, two or more specific probes labeled with the same dye can be used to increase the number of fluorescent molecules per cell.98 Photobleaching can be diminished through the use of narrow band filter sets, photostable dyes, and antifading mounting media. The best control to address potential false negative results due to probe penetration, rRNA content, or fixation is the concurrent use of a nonspecific bacterial probe such as EUB338.5

Selected applications. Fluorescent in situ hybridization can provide a detailed representation of numerous microenvironments without any selective purification or amplification steps allowing for the investigation of microbial communities in natural environments. This includes aquatic habitats, 4,56,110,145,188, soil,43 and root surfaces.116 Such studies have allowed for a better understanding of the composition, function, and population dynamics of various natural environments in response to natural and manmade pressures.99,125

Fluorescent in situ hybridization has also been utilized in medicine through the analysis of complex microbial communities in the oral cavity,52,53 gastrointestinal tract,49,65,66 and respiratory tract.46,47,71 Investigations of human gut flora have commonly used FISH to

18 detect variations in bacterial populations within fecal samples.49,66 In animals, FISH is used primarily for spatial distribution of bacteria in the intestine and to detect enteropathogens.

19,80,103, 177

Fluorescent in situ hybridization provides a specific and sensitive means by which to visualize whole bacterial cells in a relatively short period of time. This technique can be combined with flow cytometry to process and document a large number of target cells rapidly.

Pathogenesis of Swine Dysentery

Little has been published about the pathogenesis of SD; although the interplay between the resident colonic microbiota and diet appear to impact bacterial colonization and clinical expression of disease. Early investigations demonstrated that the development of SD relied upon the presence of other anaerobic bacteria.68,120,191 Separate investigations demonstrated that pure cultures of B. hyodysenteriae produced SD in conventional susceptible pigs.67,172 Additional work done in cesarean-derived, colostrum-deprived

(CDCD) pigs established that the resulting lesions in CDCD pigs were less severe when compared to those observed in conventional pigs of similar age and genetics.174 The requirement of a normal colonic microbiota for the clinical expression of disease and lesion development was further substantiated by Harris et al. (1978) who found that B. hyodysenteriae could persist in the colon of gnotobiotic pigs but did not result in characteristic lesions or mucosal invasion. Numerous other investigations were undertaken to identify the other microorganisms involved in the pathophysiology of SD. Neither the combination of B. hyodysenteriae and Campylobacter coli nor these agents individually produced lesions in gnotobiotic pigs.20,120 However, additional studies identified gram-

19 negative obligate anaerobic bacteria including Bacteroides vulgatus in combination with B. hyodysenteriae did elicit characteristic lesions of SD in gnotobiotic pigs.68,120 Accordingly, it appears that there is a requirement for additional bacteria as part of the pathogenesis of SD.

More recent studies have investigated the impact of various diets on SD; however, the exact mechanisms by which diet composition influences the pathophysiology of SD remain elusive. Highly digestible diets141 inhibit the colonization of pigs by B. hyodysenteriae. The protective mechanism of a highly digestible diet may be due to reduced fermentation in the large intestine160 and/or involves changes in the microbiota resulting in an increase in species that inhibit the spirochete.88,102,121 However, highly digestible diets are not always associated with protection against SD illustrating the multifactorial and complex nature of the pathophysiology of SD.40,106

Diets rich in inulin have also been shown to inhibit colonization of B. hyodysenteriae62,63, 176 through two possibly distinct or dual mechanisms. First, the fermentation of inulin by the resident microbiota results in the production of volatile fatty acids149 and the lowering of luminal pH which may prevent the colonization by B. hyodysentariae.63 Second, the inclusion of inulin in swine diets decreases the protein:carbohydrate ratio in the hindgut increasing the lactate and butyrate-producing bacteria35,121 and decreasing proteolytic bacteria. Proteolytic bacteria may be synergistic with B. hyodysenteriae in SD pathogenesis especially in the breakdown of glycoproteins which are a major component of the protective mucus bilayer.

The use of highly fermentable carbohydrates,176 which produce the opposite effect than highly digestible diets in the hindgut, protected against SD resulting in a discord in the literature. Yet, the diet composition was based on dried chicory roots and sweet lupins

20 resulting in speculation that the protective effect was due to the presence of inulin in dried chicory roots.62 In the end, it appears that the ingredient digestibility plays a major role in the pathophysiology of disease through the manipulation of the large intestinal microbiota.39,75

Transmission of SD occurs following the ingestion of infected feces. Although the natural infective dose may vary, experimentally, an inoculum of 105 colony-forming units may be sufficient to produce SD.87 Following ingestion, B. hyodysenteriae survives the acidic environment of the stomach eventually reaching, colonizing, and proliferating in the large intestine. Proliferation and colonization of B. hyodysenteriae requires specialized adaptations including the use of numerous metabolic pathways that utilize available substrates and the ability to penetrate the mucus bilayer. Spirochetes appear in the feces one to four days prior to clinical signs87 with diarrhea and lesions being observed when cell numbers reach 106/cm2 of mucosa.74 Spirochetes in close approximation or attached to epithelial cells in the lumen and crypts of the large intestine stimulate an outpouring of mucus;195,196 however, the significance of attachment to epithelial cells is unclear as attachment in cell cultures does not cause cellular damage or invasion. 18,89 Colonic malabsorption due to a lack of active sodium and chloride transport rather than the activity of enterotoxins and/or prostaglandins released from the inflamed mucosa appear to responsible for the development of diarrhea.10,11,155

B. hyodysenteriae strains vary in their virulence,2 but as a definitive virulence gene has yet to be identified the basis of this variation is poorly understood. The role of virulence factors and the immunologic response in the development of clinical disease and lesions has not been fully elucidated. The hemolysin(s) and lipooligosaccharide of B. hyodysenteriae

21 may be responsible for tissue destruction by disrupting the epithelial barrier, resulting in epithelial sloughing. Ensuing invasion by secondary bacteria and the protozoan Balantidium coli may also contribute to lesion development.

Differential Mucin Expression of the Colonic Mucus Bilayer in Health and Disease

The colonic mucus barrier in conjunction with the glycocalyx protects the underlying mucosa from a wide range of potentially damaging insults present in the lumen of the colon.23 The mucus barrier provides an energy source for bacteria114 while partitioning the vast number of microbial cells to the luminal side of the intestinal mucus layer distancing them from the underlying colonocytes.115,183 The barrier is composed of two layers14,168 that are rheologically distinct.171 The outer layer is believed to act as a lubricant and is constantly removed while the inner layer is thought to be a selective physical barrier.24

Mucin is the main functional component of mucus. Mucins are composed primarily, up to 85% by weight, of oligosaccharide side chains136 that protect a protein core from degradation by proteases. Mucins play a crucial role in the adhesion of bacterial cells, 119,143 and bind water conferring most of the gel-like properties of mucus.83 The protein core is rich in proline, threonine, and serine (called PTS sequences).83 PTS sequences are commonly called variable number tandem repeats and can be very long. For example, the largest PTS sequence is located in mucin 2 (MUC2) being about 2,300 amino acids in length.60

Following transcription, mucin gene products enter the rough endoplasmic reticulum where they are N-glycosylated and dimerize due to the cysteine-rich areas at the C- terminal.13,17 Following N-glycosylation, mucins are transferred into the Golgi apparatus12 where they are O-glycosylated96 with subsequent N-terminal oligomerization.58 The resulting mucin granules are then packaged tightly due to numerous calcium ions.83 Upon

22 the release of mucins from the granules, the molecules disassociate from the calcium ions and expand in the presence of the aqueous milieu.136

The apical surface of colonocytes is covered by transmembrane mucins while goblet cells synthesize the secreted gel-forming mucins that form mucus (Figure 1.12).83

Figure 1.12. Simplified representation of a gel-forming mucin produced by goblet cells and transmembrane mucins attached to the apical aspect of colonocytes. Left: MUC2 is packed tightly in goblet cells and when released expands to form flat structures that stack upon one another. The circle highlights the oligomeric structure of MUC2. A MUC2 monomer contains a central core of mucin domains with O- glycans which are bounded at either end of the monomer by numerous disulphide bonds. Right: transmembrane mucins are components of the glycocalyx that cover the apical aspect of colonocytes. Transmembrane mucins are components of the glycogalyx136 and have one large N-terminal domain and a short cytoplasmic C-terminus. 70,72 The luminal aspects of these mucins either have a sea urchin-enterokinase-agrin domain (MUC1, MUC3, MUC12, MUC13, and

MUC17) or NIDO-AMOP-vWD domains (MUC4).83 Neither of these domains are cleaved during synthesis rather they are held together by noncovalent bonds.83

The roles of transmembrane mucins have not been fully elucidated although they are probably involved in cell sensing and signaling;70,161,190 however, some specific interactions have been identified. Binding of enteric pathogens to transmembrane mucins stimulates the inflammatory signaling cascade of epithelial cells.181 MUC1 has a protective affect107 and can modulate growth and apoptosis.51,158,200 MUC3, MUC12, and MUC17 have cytoplasmic tails that associate with PDZ-proteins that regulate proteins such as ion channels.37,95,117

Gel-forming mucins (MUC2, MUC5AC, MUC5B, MUC6, and MUC7) have central mucin domains flanked by an N-terminal portion which is involved in oligomerization and a

C-terminal portion that is involved in forming dimeric structures.83 MUC2 is the predominant mucin present in the large intestine.60 It comprises both the inner dense, attached layer and the outer loose, unattached layer forming large net-like structures.83 Upon release from goblet cells, MUC2 unfolds and expands >1,000-fold in volume forming the inner mucus layer. The inner mucus layer is then converted to the outer mucus layer which allows bacterial penetration. Although the exact mechanisms are not fully understood, the transition is likely due to proteolytic cleavages of MUC2.82

Based upon histochemical properties, mucins have also been broadly classified into neutral and acidic mucins. Acidic mucins can be further classified into sialomucins or sulphomucins based on the presence of terminal sialic acid or sulfate groups on the oligosaccharide chain, respectively.44 Histochemistry using high iron diamine and alcian blue (HID/AB) staining demonstrates the predominance and distribution of sialomucins and sulphomucins in tissue sections. Sulphation of mucins increases both the charge of mucus and resistance of the mucin carbohydrate side-chains to bacterial enzymatic degradation suggesting sulphomucins play an important role inhibiting bacterial invasion.178 Differences

24 in sialomucin and sulphomucin expression have been observed in diseases including inflammatory bowel disease. Specifically, multiple studies have demonstrated a loss of mucin sulphation32-34 in ulcerative colitis resulting in a reduction of negative charge from the secreted mucin. Such difference in sialo- and sulphomucins expression may affect microbial colonization and disease expression.

The mechanisms behind apical exocytosis of granules are not fully understood; however, mucin output in the colon can be amplified due to increased mucin biosynthesis, exocytosis rates, and goblet cell hyperplasia.136 There are two possible exocytosis pathways for secreted mucins: baseline secretion and regulated release due to specific stimuli involving signaling pathways.105 Mucin synthesis and secretion, goblet cell proliferation, and crypt hyperplasia may be mediated by a broad array of factors including neurohumoral, local, and immune signals.16,41,136 Plaiesancie et al. (1997 and 1998) found that mucin output increased following stimulation with prostaglandin E2, peptide YY, serotonin, vasoactive intestinal peptide, interleukin-1β, and NO precursors. Up-regulation of mucin genes have also been found in response to cytokines including IL-9 in the presence of IL-13.167 Lastly, studies have identified ETS transcription factor SAM pointed domain ETS factor to be important in goblet cell proliferation.59,129

The colonic mucus bilayer can act as both an energy source and support media for the growth of the colonic microbiota.111 While a single species may not have the ability to cleave all the linkages of a mucin due to a lack of specific enzymes,154 it has been postulated that the ability of the microbiota to thrive may be a result of the interaction of different species providing the required enzymes for sequential mucin degradation.136 This process

25 produces a carbon and energy source for the microbiota and energy source for the host epithelium through the production of short-chain fatty acids from the degraded material.199

Changes in protein and carbohydrate portions of secreted mucins occur in the diseased state likely diminishing the protective efficiency of the mucus barrier (Figure 1.13).

Figure 1.13. Mechanisms that could affect the inner mucus layer properties allowing bacteria to penetrate a normally impenetrable barrier. Bacterial interaction with the epithelium triggers the mucosal immune response leading to inflammation. Such changes could result in an alteration in the available nutrients and growth conditions potentially changing the bacterial population. Additionally, a reduction in total MUC2 secretions179,184 and increased expression of MUC5AC has been demonstrated in ulcerative colitis.45,159 Increased expression of MUC5AC has also been demonstrated in response to the quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] of gram-negative bacteria.162 Upregulation of MUC5AC can also be induced by LPS inducing a general mechanism by which epithelial cells respond to the presence of bacteria

26 by increasing mucin synthesis thereby preventing bacterial invasion of intestinal cells. In cases of epithelial damage and/or infiltration, it is probable that the contact of bacteria with

Toll-like receptors trigger an immune response which includes increased mucin secretion.1

It appears as though certain bacteria and parasites are capable of targeting human colonic mucins.169,180 Adhesion to mucins is thought to be due to the interaction between external microorganism components and carbohydrate structures of the mucin.136 As the inner mucus layer is generally impervious to bacteria, specialized virulence systems are required for microorganisms to penetrate or interfere with the production of this barrier.

Flagella seem to support bacterial penetration of the colonic mucus bilayer.38,91,133,144

Pathogens can also alter the mucus layer to facilitate their motility by increasing the pH of their surrounding environment to decrease the mucus viscoelasticity29 or disassembling the oligomerized mucin macromolecules by proteolytic cleavage and dissolving the mucus gel.104 Entamoeba histolytica is able to penetrate the colonic epithelium through the use of a lectin-like adhesion to attach to the mucus triggering secretion of a cysteine protease which is capable of cleaving MUC2.104,123,137 Additionally, mucin exocytosis can be stimulated by cholera toxin122 or down-regulated by Clostridium difficile toxin A.21

The pathways of mucin synthesis and secretion may be altered due to numerous factors including direct bacterial degradation, the simple presence of certain bacterial species, or bacterial products.136 It has been hypothesized that the relative ratio of bacterial species comprising the colonic microbiota is of importance to colonic health and disease;150 however, the overall enzymatic spectrum148 or products of the microbiota may be of greater import.

Currently, little has been described regarding mucin expression and mucus composition in

SD.

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CHAPTER 2

COMPARISON OF LESION SEVERITY, DISTRIBUTION, AND COLONIC MUCIN EXPRESSION IN PIGS WITH ACUTE SWINE DYSENTERY FOLLOWING ORAL INOCULATION WITH “BRACHYSPIRA HAMPSONII” OR BRACHYSPIRA HYODYSENTERIAE

Modification of a paper published in Veterinary Pathology

Bailey L. Wilberts, Paulo H. Arruda, Joann M. Kinyon, Darin M. Madson, Tim S. Frana, and Eric R. Burrough

Abstract

Swine dysentery is classically associated with infection by Brachyspira hyodysenteriae, the only officially recognized Brachyspira sp. that consistently imparts strong beta-hemolysis on blood agar. Recently, several strongly beta-hemolytic Brachyspira have been isolated from swine with clinical dysentery that are not identified as B. hyodysenteriae by PCR including the recently proposed species “Brachyspira hampsonii”. In this study, 6-week-old pigs were inoculated with either a clinical isolate of “B. hampsonii”

(EB107; n = 10) clade II or a classic strain of B. hyodysenteriae (B204; n = 10) to compare gross and microscopic lesions and alterations in colonic mucin expression in pigs with clinical disease versus controls (n = 6). Gross lesions were similar between infected groups.

No histologic difference was observed between infected groups with regard to neutrophilic inflammation, colonic crypt depth, mucosal ulceration or hemorrhage. Histochemical and immunohistochemical evaluation of the apex of the spiral colon revealed decreased expression of sulphated mucins, decreased expression of MUC4, and increased expression of

MUC5AC in diseased pigs compared to controls. No difference was observed between diseased pigs in inoculated groups. This study reveals significant alterations in colonic mucin expression in pigs with acute swine dysentery and further reveals that these and other

45 microscopic changes are similar following infection with “B. hampsonii” clade II or B. hyodysenteriae.

Introduction

Brachyspira spp. are Gram-negative spirochetes that have historically been distinguished by their strength of beta-hemolysis on blood agar plates and biochemical reactions including indole cleavage and hippurate hydrolysis.2,9,22 Classically, Brachyspira hyodysenteriae and Brachyspira pilosicoli isolates have been associated with clinical disease in swine14 where B. hyodysenteriae is the causative agent of swine dysentery (SD).17 Clinical isolates of B. hyodysenteriae generally have positive indole and negative hippurate reactions and generate strong beta-hemolysis that is enhanced around slits in blood agar (ring phenomenon).22 Historically, such reactions allowed for differentiation of B. hyodysenteriae from weakly beta-hemolytic Brachyspira spp. that can be present in the porcine colon including B. intermedia, B. murdochii, B. innocens and B. pilosicoli.

Not all strongly beta-hemolytic, ring phenomenon positive isolates are identified as B. hyodysenteriae by PCR. Rather, several strongly beta-hemolytic Brachyspira spp. have been recently reported including atypical isolates of B. intermedia,7 “Brachyspira sp.

SASK30446”,16 and the proposed novel species “Brachyspira suanatina”35 and “Brachyspira hampsonii.”6 Recently, isolates of “B. sp. SASK30446” induced clinical disease and lesions similar to B. hyodysenteriae in mice2 and pigs3,38 following oral inoculation. Comparison of nox gene sequences from isolates with sequences available in GenBank reveals that “B. sp.

SASK 30446” sequences are nearly identical (≥99% identity) over the region compared to clade II isolates of “B. hampsonii”. A representative isolate of this clade, EB107, is the subject of the study reported here.

Clinical SD is characterized by a mucohemorrhagic diarrhea with gross lesions restricted to the large intestine that typically include variable mucosal thickening, hemorrhage, fibrinonecrotic exudate, and variable but often abundant mucus.

Histologically, crypt lumens are frequently distended with mucus, there is moderate to severe neutrophilic infiltration in the lamina propria, and spirochetes can often be visualized within crypt lumens and mucus producing goblet cells by silver staining.

The mucus layer is an important structural feature of the colonic mucosal surface that is responsive to environmental factors in the gut lumen.8,23 The protein component of mucus is composed of large glycoproteins called mucins20 which play an important role in innate mucosal defense.13 Mucins can provide both a barrier and reporting function and are divided into two subgroups: gel-forming mucins and cell surface mucins. 20,34,41 Gel-forming mucins, including mucin 5AC (MUC5AC), are extracellular and secreted by goblet cells while cell surface mucins, including mucin 4 (MUC4), are membrane-tethered with a short, cytoplasmic tail and extensive extracellular domain.20 MUC4 is thought to have a largely anti-adhesive role5 and may provoke a response intended to maintain the integrity of the epithelium.21 MUC5AC is thought to inhibit adherence and invasion of colonic epithelial cells33 and instill resistance to enteric nematode infections.19 Despite the abundant mucus typical of SD, the chemoattractant nature of B. hyodysenteriae to mucus,24,30 and the importance of mucus bilayer in intestinal disease, the histochemical and immunohistochemical characteristics of mucin-secreting colonic goblet cells have not been extensively evaluated in pigs with SD.

The objectives of the study described here were threefold: 1) to describe the time to onset of clinical SD and the duration of viable spirochete shedding in pigs inoculated with

47 either “B. hampsonii” (EB107) or B. hyodysenteriae (B204), 2) to describe gross lesions and compare histologic lesion severity and distribution within the colons of pigs that develop SD following infection with EB107 versus B204, and 3) to characterize the biosynthetic responses in mucin-secreting colonic goblet cells in pigs with acute clinical SD following infection with either EB107 or B204.

Material and Methods

Animals

All procedures were approved by the Institutional Animal Care and Use Committee of Iowa State University. Twenty-six 6-week-old crossbred pigs were obtained from a commercial source with no known previous history of Brachyspira-associated disease and were PCR negative for Lawsonia intracellularis upon arrival. Pigs were separated into three groups with six control pigs and ten pigs per inoculated group. All pigs were ear tagged for individual identification and swabbed rectally to test for the presence of Brachyspira spp. by microbial culture seven days prior to inoculation. Each group of pigs was maintained in a separate room to prevent any contact between groups. Pigs were acclimated to these groups and to the facility for one week. During this period and throughout the study, pigs were fed a non-medicated, corn and soybean diet nutritionally complete for their age.

Bacterial strains, growth conditions, and preparation of inocula

Media, isolates, and challenge inocula were prepared as previously described. 3

Isolates used in this study were obtained from the culture collection at the Iowa State

University Veterinary Diagnostic Laboratory. The “B. hampsonii” clade II isolate (EB107) was previously used in a pig inoculation experiment3 after being recovered from a clinical case of SD in 2011 and was 9 – 12th passage at the time of inoculation. The B.

48 hyodysenteriae B204 isolate was originally recovered from a clinical case of SD in 1972 and was 9 – 10th passage at the time of inoculation.

For isolation of Brachyspira spp., individual rectal swabs were collected at 5, 7, 9, 12,

15, and 19 days post inoculation (DPI) and at necropsy. Swabs were plated onto selective agars within 6 hours of collection and incubated as previously described.3 Specifically, swabs were plated onto trypticase soy agar with 5% defibrinated bovine blood (TSA); CVS selective agar containing colistin, vancomycin, and spectinomycin; and BJ selective agar containing pig feces extract, spiramycin, rifampin, vancomycin, colistin, and spectinomycin.

Plate media used in this study were prepared in-house and passed the quality assurance standards of the Iowa State University Veterinary Diagnostic Laboratory. An anaerobic environment was provided by a commercial system (BD GasPak EZ Anaerobe Container

System, BD Diagnostic Systems, Sparks, MD) and plates were incubated at 41 ± 1°C. Agar plates were observed for growth at 2, 4, and 6 DPI. Mucosal scrapings collected at necropsy were plated and evaluated as described above and were also plated onto MacConkey’s agar and brilliant green agar with novobiocin in addition to tetrathionate broth enrichment subcultured to brilliant green with novobiocin and XLT4 to test for the presence of

Salmonella spp.

Animal inoculation

Pigs were inoculated with an agar slurry as previously described3 with slight modification: pigs received three doses of inoculum (100 ml/dose) administered via gavage

24 hours apart with each administration preceded by a 12-18 hour fast. A 5-g sample of each inoculum was reserved from which 1 gram was vortexed for 45 seconds in tubes with 9 ml of sterile PBS and a few glass beads. A standard plate count procedure was performed by

49 titration of 1 ml of the vortexed sample into 9 ml and carried out to 10-9. The dilution series was plated on trypticase soy agar with 5% defibrinated bovine blood and incubated anaerobically for 6 days with plates being observed on days 2, 4, and 6. Brachyspira spp. grew confluently on the more concentrated dilutions, but discrete colonies were observed from the more dilute plates. Colonies were counted after 6 days incubation to obtain the inoculum titer in colony-forming units (CFU) per ml. Pigs in the B204 group received 5 x

105 CFU/ml, 8 x 105 CFU/ml, and 4 x 105 CFU/ml on DPI 0, 1, and 2, respectively. Pigs in the EB107 group received 7 x 105 CFU/ml, 1 x 106 CFU/ml, and 1 x 106 CFU/ml on DPI 0,

1, and 2, respectively. Pigs in the control group received a sham inoculum consisting of agar material from non-inoculated culture plates prepared in the same manner as for the

Brachyspira inocula.

Molecular identification

The subpassaged isolates used to prepare the inocula were verified to species by PCR assays targeting nox gene sequences as previously described [(B. hyodysenteriae, B. pilosicoli, and B. intermedia)40; (B. murdochii and B. innocens)46; and “B. sp SASK30446”3].

All necropsy isolates were confirmed to species by either direct sample PCR on mucosal scrapings or from primary cultures of mucosal scrapings in cases where mucosal scrapings were negative by PCR.

Animal observations and necropsy

Throughout the study period, investigators were blinded to the molecular identification of the inoculum. Following inoculation, animals were observed at least twice daily for feed consumption, availability of adequate water, and clinical illness. Fecal consistency was determined daily, and each pig received a score based upon the following

50 system: 0 if normal, 1 if soft but formed, 2 if semisolid, and 3 if liquid to watery with an additional 0.5 point added each for the presence of discernible mucus and/or blood.

Animals were euthanized by barbiturate overdose 24-48 hours after the first observation of diarrhea with blood and mucus or at the termination of the study 21 DPI. At necropsy, the entire intestinal tract was observed for gross lesions and the full length of the cecal and colonic lumens were exposed and evaluated for the presence and distribution of luminal mucus, mucosal hemorrhage, and fibrinous exudate. Tissue samples were collected and placed in 10% neutral buffered formalin and included representative sections of jejunum, ileum, cecum, base of the spiral colon (section of the centripetal spiral colon that connects with the cecum), apex of the spiral colon, and a site-independent section of colon representative of overall lesion severity or mid-section of the centrifugal spiral colon if no lesion was present. After 24 hours of fixation, tissue samples were transferred to 70% ethanol and processed routinely for histopathology. Samples were also collected from each pig for microbial culture and direct sample PCR and included a rectal swab and colonic mucosal scrapings.

Histopathology

The investigator was blinded to individual pig numbers and group identification.

Sections were cut to 4 µm and stained routinely with hematoxylin and eosin. Sections of jejunum and ileum were evaluated for the presence of any lesions unrelated to Brachyspira infection. Sections of cecum and spiral colon were evaluated for the presence of ulceration, hemorrhage, and neutrophilic infiltration of the lamina propria. Neutrophils were counted in ten 40X fields and the mean for each section of the cecum, base of the spiral colon, apex of the spiral colon, and lesion or mid-section of the spiral colon was determined. Ulceration was

51 scored as follows: 0 if no ulceration, 1 for focal ulceration spanning 1- 3 crypts, 2 for focal ulceration spanning 3 – 5 crypts, and 3 for focal ulceration spanning more than 5 crypts. An additional 0.5 was added for multifocal ulceration or 1 for multifocal ulceration spanning more than 5 crypts. The lamina propria and lumen were evaluated for hemorrhage and each anatomic location was assigned a score based upon the most severe lesion in that tissue section. Scores were determined as follows: 0 if < 5 red blood cells (RBC), 1 if 6-10 RBC, 2 if 11-20 RBC, 4 if 31-50 RBC, and 5 if > 51 RBC were observed with an additional 1 point added to the score for more than 3 foci of hemorrhage within a single histologic section of a given anatomic location. Hemorrhage scores at all locations were combined to form a composite score. Mucosal thickness was measured in each section using a standard eyepiece micrometer and measurements were taken from an area where the crypts were perpendicular to the mucosal surface with intact epithelium. The mean of three measurements was calculated for each section of the cecum, base of the spiral colon, apex of the spiral colon, and lesion or mid-section of the spiral colon for each pig.

Immunohistochemistry

Primary antibodies for MUC4 and MUC5AC which have previously been shown to have specificity in pig large intestine25 were used for immunohistochemistry (IHC) (Zymed

Laboratories, Invitrogen Corporation, Carlsbad, CA). Sections (5 μm) of the apex were cut onto positively charged slides, placed in an incubator at 57°C for two hours, and deparaffinized through graded alcohol. Endogenous peroxidase was inhibited by application of 3% hydrogen peroxide for 20 minutes followed by three rinses with ultrapure water.

Slides were processed for antigen retrieval by microwaving in Tris/EDTA (pH 9.0) for 1 min followed by a steam chamber for 20 minutes. Slides were then incubated with 1:10 dilution

52 of goat serum and PBS buffer for 20 minutes at 37ºC to saturate non-specific protein binding sites. All primary antibodies were diluted 1:50 in PBS containing 0.1% Tween 20. Slides were incubated with primary antibodies overnight at 4°C in a humidified chamber. After washing two times in PBS, a 5 minute PBS bath, and two more rinses with PBS, sections were incubated with a conjugated secondary antibody (Mouse-on-Farma HRP-Polymer,

Biocare Medical, Concord, CA) for 1 hour at 37ºC and then washed as described for the primary antibody. Slides were subsequently incubated with a commercial chromagen

(NovaRED™, Vector laboratories, Burlingame, CA) for 5 minutes at 37ºC followed by rinsing with ultrapure water. Finally, the sections were counterstained with Shandon’s hematoxylin, placed in Scott’s Tap water for 1 minute, rinsed with ultrapure water, dehydrated through a graded alcohol series, and mounted.

Histochemistry

Sections (4 µm) of the apex were placed onto positively charged glass slides, deparaffinized, and treated with a combination of high-iron-diamine and Alcian blue (pH 2.5)

(HID/AB) staining. Sections were placed in high-iron-diamine solution for 18 hours at 37ºC, rinsed with water, placed in Alcian blue solution for 5 minutes, rinsed with water, dehydrated, and mounted.

Image analysis

Quantitative analysis of histochemical and immunohistochemical assays was performed using a commercial software program (Photoshop CS4, Adobe Systems Inc. San

Jose, CA). Five representative 40X images were captured for each slide where each image had the same number of total pixels. The number of pixels representing positive staining was

53 recorded for each image and the average number of pixels representing positive staining was calculated per slide.

Fluorescent in situ hybridization

An oligonucleotide probe specific for 23S rRNA of Brachyspira spp. (SER1410)1 was used under hybridization conditions as previously described.4 A genus based probe was used to insure the evaluator remained blinded to the molecular identification of the spirochete present in the section. Five 40X fields from each lesioned section of spiral colon were evaluated for localization (within epithelial cells, goblet cells, and crypt lumens) and quantification (few, moderate, many) of spirochetes.

Statistical analyses

A commercial statistical software package (JMP Pro 10, SAS Institute, Cary, NC) was utilized to perform all analyses. A one-way analysis of variance (ANOVA) was used to detect significant differences in mean neutrophilic inflammation, ulceration score, mucosal crypt depth, and composite hemorrhage score with a Tukey-Kramer or Steel-Dwass adjustment for multiple comparisons when a residual plot demonstrated equal or unequal variance, respectively. Following logarithmic transformation, HID/AB staining (sulphated mucins), MUC4 expression, and MUC5AC expression was analyzed using an ANOVA with

Tukey-Kramer adjustment. In all circumstances, values of P ≤ 0.05 were considered significant and means are reported with standard error of the mean.

Results

Animal observations

Loose stools with blood and mucus were first observed 5 DPI in the “B. hampsonii”- inoculated group (EB107) and 8 DPI in the B. hyodysenteriae-inoculated group (B204). The

54 average DPI to the onset of clinical SD for the EB107 group (mean ± SEM = 12.2 ± 5.36) was similar to the B204 group (11.7 ± 2.60). Fecal scores and timing of euthanasia for each pig are summarized by inoculation group in Table 1.

Microbial culture

All preinoculation fecal samples were negative for Brachyspira spp. The days with positive Brachyspira culture for each pig are reported in Table 2.1, and a summary of the percent shedding and PCR results by group are summarized in Table 2.2. Shedding was first detected 5 DPI for both the EB107 and B204 groups. The average period of positive culture to the development of clinical SD was shorter for the EB107 group (2.6 days ± 1.5) than the

B204 group (3.2 days ± 2.5).

Gross pathology

Gross lesions observed in each individual group are summarized in Table 2.3. When present, gross lesions in the intestinal tract were limited to the cecum and large intestines but were most common in the centripetal, apex, and centrifugal portions of the spiral colon.

Lesions consisted of variable degrees of fibrinous exudate on the luminal surface; mucosal thickening, mucosal congestion, and hemorrhage; and excessive luminal mucus.

Representative lesions from each inoculation group are displayed in Figures 2.1 and 2.2.

Cecal lesions were observed only in animals inoculated with B204 and were limited to those pigs in which fibrinous exudate was also present in the majority of the large intestine. Fibrinous exudate was more common in those animals in the B204 group that developed SD (8/9; 89%) than those in the EB107 group that developed SD (1/5; 20%). Of those inoculated with EB107 that developed SD, the frequency of moderate to severe luminal

TABLE 2.1. Summary of fecal scores, Brachyspira culture results, and timing of euthanasiaa

Individual Fecal Scoresb by Days Post-Inoculation Inoculum / Pig 5 7 9 12 14 16 19 21 ID

Sham 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 1 0 3 0 0 0 0 0 0 0 0 4 0 0 1 0 0 0 0 0 5 0 0 0 0 0 1 0 0 6 0 0 0 0 0 0 0 0

B204 13 0+ 1+ 4+e - - - - - 16 0 1.5+ 4+e - - - - - 12 0 0 1+ 4+e - - - - 8 0 0 0+ 4+ 4+e - - - 14 0 0 1+ 3.5+ 4+e - - - 10 0 0.5 0+ 0 4+e - - - 11 0 0 0+ 1+ 4+e - - - 7 0 0 1.5 1+ 4+e - - - 9 1 1.5+ 0+ 1+ 1+ 4+e - - 15 0 0 0+ 0 0 0 0 1e

EB107 21 3+ 4+e ------25 0+ 0+ 4+e - - - - - 17 0 0 0 0+ 2.5+ 4+e - - 18 0 0 0 0+ 1.5+ 4+e - - 23 0 0 1 0 0.5+ 0+ 4+e - 24 0 0 0 0 1.5+ 0 2 2.5e 19 1 1 0+ 2 0 0 0 1e 20 0 0 1+ 0 1 0 0 1e 22 0 1 0+ 0 1 0 0+ 1+e 26 0 1 1 0 1 0 1.5 1e a Control pigs had a maximum daily diarrhea score of 0.167 on days 9, 16, and 19 post-inoculation; never had a positive Brachyspira culture; and were euthanized at 21 days post inoculation. b A fecal score was determined for each pig daily based upon the following system: 0 if normal, 1 if soft but formed, 2 if unformed with semisolid consistency, and 3 if severely liquid to watery with an additional 0.5 point added each for the presence of discernible mucus and/or blood (max score = 4). + Positive Brachyspira culture e Indicates day of euthanasia. Italicized entries represent values from the preceding day for instances where euthanasia occurred between defined sampling days.

mucus was greater (5/5; 100%) than the frequency of those inoculated with B204 that developed SD (6/9; 67%) while the frequency of moderate to severe mucosal hemorrhage. was more common in those inoculated with B204 that developed SD (5/9; 56%) than those inoculated with EB107 that developed SD (1/5; 20%). No gross lesions were observed in the sham-inoculated group.

TABLE 2.2. Positive Brachyspira culture and PCR results in feces and tissue following B. hyodysenteriae (B204) or “B. hampsonii” (E107) challenge Fecal culture results by DPI Necropsy

Positive Positive MS PCR Culture Culture Direct with PCR* Positive Positive Group PCR Positive ID RS MS 5 7 9 12 14 16 19 on MS MS Culture 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% Sham - N/A (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6) (0/6)

90% 90% 90% 90% 100% B204 10% 30% 75% 80% 0% 0% B. hyodysenteriae (100%) (1/10) (3/10) (9/10) (6/8) (4/5) (0/1) (0/1) (9/10) (9/10) (9/10) (9/9)

20% 11% 44% 25% 50% 28% 20% 80% 60% 60% 83% EB107 ”B. hampsonii” clade II (2/10) (1/9) (4/9) (2/8) (4/8) (2/7) (1/5) (8/10) (6/10) (6/10) (5/6) (100%) DPI = Days post inoculation RS = Rectal swab MS = Mucosal scraping * PCR ID is based upon direct mucosal scrape sample with the exception of a single case in which mucosal scrape was negative and PCR was performed on the mucosal scrape culture.

TABLE 2.3. Necropsy lesions in pigs which developed clinical SD following B. hyodysenteriae (B204) or “B. hampsonii” (E107) inoculation

Luminal Mucosal Fibrinous Mucus Hemorrhage Exudate Distribution Inoculum Mild M/S Mild M/S MF D Cecum Base* CP Apex CF DC B204 3/9 6/9 4/9 5/9 5/9 3/9 3/9 6/9 9/9 9/9 8/9 3/9

EB107 0/5 5/5 4/5 1/5 1/5 0/5 0/0 2/5 5/5 5/5 5/5 4/5 SD = Swine dysentery M/S = Moderate or severe MF = Multifocal D = Diffuse CP = Centripetal CF = Centrifugal DC = Descending colon *The base of the spiral colon is the section of spiral colon that connects the cecum to the centripetal portion of the spiral colon

Figure 2.1. Spiral colon; pig, inoculated with B. hyodysenteriae B204. (a) Pig 8: Multifocal fibrinous exudate, mucus, and hemorrhage present in multiple segments of the spiral colon 14 days post inoculation (DPI). (b) Pig 13: Section of spiral colon with diffuse fibrinous exudate, mild hemorrhage, and moderate luminal mucus 13 DPI. (c) Pig 16: Fibrinous exudate, luminal mucus, and hemorrhage in the distal spiral colon 10 DPI. (d) Pig 10: Marked diffuse fibrinous exudate 15 DPI. (e) Pig 11: Severe hemorrhage in the distal spiral colon 15 DPI. Figure 2.2. Spiral colon; pig, inoculated with “B. hampsonii” EB107. (a) Pig 25: Copious luminal mucus 10 days post inoculation (DPI). (b) Pig 21: Multifocal fibrinous exudate, moderate luminal mucus, and mild hemorrhage 7 DPI. (c) Pig 17: Severe luminal mucus 16 DPI. (d) Pig 18: Moderate luminal mucus and mild hemorrhage in the distal spiral colon 17 DPI. (e) Pig 23: Severe luminal mucus 19 DPI.

Histopathology

Site-independent, representative lesion. Microscopic evaluation of site-independent, lesioned sections from diseased pigs or mid-sections of the spiral colon in sham-inoculated pigs is summarized in Figure 2.3. A significant difference was observed in neutrophilic inflammation (P < 0.03), crypt depth (P < 0.03), and ulceration (P = <0.0001) between each infected group and the sham-inoculated group. However, a significant difference in the mean

58 composite hemorrhage score was only identified between the B204-inoculated group that developed disease (7.33 ± 2.73) and the sham-inoculated group (0.50 ± 0.50, P = 0.005) and not between the EB107-inoculated group that developed disease (7.80 ± 4.92) and sham- inoculated group (P = 0.07).

Cecum. Cecal histologic evaluation is summarized in Figure 2.4. Briefly, in the cecum there was a significant difference in cecal ulceration score between the B204- inoculated group that developed disease (2.44 ± 0.52) versus those in the EB107-inoculated group that developed disease (0.20 ± 0.20) and the sham-inoculated group (0.00 ± 0.00) (P =

0.02 and P = 0.008, respectively).

Base of the spiral colon. Histologic evaluation of the base of the spiral colon is summarized in Figure 2.5. At the base of the spiral colon there was a significant difference in mucosal crypt depth between the B204-inoculated and EB107-inoculated groups that developed disease and the sham-inoculated group (P = 0.0003 and P = 0.005, respectively).

A significant difference in the base ulceration score was only observed between the B204- inoculated group that developed disease and the sham-inoculated group (P = 0.016).

Apex of the spiral colon. Histologic evaluation of the apex of the spiral colon is summarized in Figure 2.6. At the apex of the spiral colon there was a significant difference in neutrophilic inflammation (P < 0.03, all analyses), ulceration score (P < 0.04, all analyses), and composite hemorrhage (P < 0.03, all analyses) score between each isolate- inoculated group and the sham-inoculated controls.

Immunohistochemistry Image analysis results of mucin immunohistochemistry are summarized in Figure 2.7.

There was a significant decrease in MUC4 expression between the B204-inoculated and the

Figure 2.3. Means ± SEM associated with histologic lesions at a site-independent, representative lesion in the spiral colon of pigs inoculated with “B. hampsonii” EB107 (n = 5) or B. hyodysenteriae B204 (n = 9) that developed mucohemorrhagic diarrhea or centrifugal spiral colon in sham-inoculated controls (n = 6). Figure 2.4. Means ±SEM associated with histologic lesions in the cecum of pigs inoculated with “B. hampsonii” EB107 (n = 5) or B. hyodysenteriae B204 (n = 9) that developed mucohemorrhagic diarrhea and sham-inoculated controls (n = 6). Figure 2.5. Means ±SEM associated with histologic lesions at the base of the spiral colon of pigs inoculated with “B. hampsonii” EB107 (n = 5) or B. hyodysenteriae B204 (n = 9) that developed mucohemorrhagic diarrhea and sham- inoculated controls (n = 6). Figure 2.6. Means ±SEM associated with histologic lesions at the apex of the spiral colon of pigs inoculated with “B. hampsonii” EB107 (n = 5) or B. hyodysenteriae B204 (n = 9) that developed mucohemorrhagic diarrhea and sham-inoculated controls (n = 6). Neutrophils were counted in ten 40X fields and the mean for each section of the spiral colon was determined. * P < 0.05 and ** P < 0.01.

EB107-inoculated groups that developed SD and controls (P < 0.0001 and P < 0.0001, respectively). The decrease in MUC4 expression was concurrent with a significant increase in MUC5AC expression in pigs that developed SD in the B204-inoculated and the EB107- inoculated groups compared to controls (P = 0.0005 and P = 0.007, respectively.

Representative photomicrographs of tissue sections used for image analysis are displayed in

Figure 2.8 and Figure 2.9.

Figure 2.7. Means ±SEM for image analysis of immunohistochemical and histochemical stains on sections taken from the apex of the spiral colon. * P < 0.01 and ** P < 0.0001. Histochemistry

Image analysis results for mucin histochemistry are summarized in Figure 2.7 with representative photomicrographs of tissue sections used for analysis displayed in Figure 2.10.

There was a decrease in the amount of sulphated mucins at the apex of the spiral colon in pigs that developed SD; however, this difference was only significant between the B204- inoculated group that developed disease (25374.6 ± 3923.0) and the sham-inoculated group

(52401.5 ± 6236.3, P = 0.0023) and not between the EB107-inoculated group that developed disease (31192.0 ± 5162.1) and sham-inoculated group (P = 0.054).

Figure 2.8. Spiral colon, apex; pig; IHC for mucin 5AC (MUC5AC). (a) Pig 16 (B204): Numerous MUC5AC positive goblet cells. (b) Pig 25 (EB107): Many MUC5AC positive goblet cells. (c) Pig 4 (control): Lack of MUC5AC expression in goblet cells. Figure 2.9. Spiral colon, apex; pig; IHC for mucin 4 (MUC4) (a) Pig 16 (B204): Diminished surface and goblet cell MUC4 expression. (b) Pig 25 (EB107): Diminished surface and goblet cell MUC4 expression. (c) Pig 4 (control): Abundant MUC4 expression. Figure 2.10. Spiral colon, apex; pig; high-iron-diamine and Alcian blue (pH 2.5) staining to reveal the presence of sulphated mucins. (a) Pig 16 (B204): Diminished presence of sulphated mucins in goblet cells and crypt lumens. (b) Pig 25 (EB107): Diminished presence of sulphated mucins in goblet cells and crypt lumens. (c) Pig 4 (control): Abundant sulphated mucins in goblet cells and crypt lumens.

Fluorescent in situ hybridization

Both B204 and EB107 were observed within epithelial goblet cells (Fig. 2.11, Fig.

2.12); however, moderate to high numbers of intracellular spirochetes were only present in sections from B204-inoculated pigs. Both B204 and EB107 were observed in sparse to large numbers within multifocal crypt lumens throughout affected sections.

* *

Figure 2.11. Spiral colon; pig, infected with B. hyodysenteriae B204; Fluorescent in situ hybridization for Brachyspira spp. (SER1410). Moderate to high numbers of intracellular (white arrow) and intraluminal (asterisk) spirochetes. Figure 2.12. Spiral colon; pig infected with ”B. hampsonii” EB107; Fluorescent in situ hybridization for Brachyspira spp. (SER1410). Robust numbers of spirochetes in crypt lumens (asterisk) and goblet cells (white arrow).

Molecular identification

All isolated spirochetes and inocula were positive only for the appropriate

Brachyspira sp. in all testing. When positive, direct PCR assays performed on mucosal scrapings revealed the presence of the species inoculated (Table 2) and only a single mucosal scraping was negative by direct PCR assay but positive by microbial culture.

Discussion

Recent work in C3H/HeN mice2 and pigs3 has suggested that lesions associated with infection by clinical isolates representative of clades I and II of “B. hampsonii” are similar to, if not indistinguishable from, B. hyodysenteriae infection. Findings of another recent study

63 using a Canadian isolate of “B. hampsonii” clade II provide further support that lesions in inoculated pigs are similar to those associated with B. hyodysenteriae infection.38 In the present study, multiple gross and microscopic parameters were compared and revealed minimal differences in lesion characteristics, severity, or distribution between pigs infected with either B. hyodysenteriae or “B. hampsonii” and provide unequivocal support for “B. hampsonii” as an agent of SD.

The average DPI to the onset of clinical SD and the average period of shedding prior to the development of clinical SD was similar between the two Brachyspira spp. tested. The duration between inoculation and initial observation of grossly detectable fecal blood and/or mucus in the present study was similar to previous studies.3,26,38 With regard to preclinical spirochete shedding, previous reports have described the appearance of spirochetes in the feces one to four days before observable diarrhea with B. hyodysenteriae,26 and rarely more than two days before the onset of mucoid or hemorrhagic diarrhea with “B. hampsonii” clade

II.38 While this period of preclinical shedding is consistent with a majority of pigs in the present study, a single pig in the B204-inoculated group had four consecutive positive cultures spanning an eight day period prior to the development of diarrhea suggesting the potential for prolonged shedding period and horizontal exposure prior to disease recognition.

No difference in the sensitivity of detection was observed when comparing culture of either rectal or mucosal scraping swabs to direct PCR on colonic mucosal scrapings collected at necropsy in the B204 group with each sample being positive in all nine pigs that developed clinical SD. Similarly, the five pigs that developed clinical SD in the EB107 group had positive rectal and mucosal cultures and appropriate molecular identification via PCR. Two pigs did not develop clinical SD but had either a positive rectal swab or a positive mucosal

64 scraping culture at necropsy. Interestingly, a positive molecular identification via PCR occurred in the pig with the negative mucosal scraping culture while the pig with the positive mucosal scraping culture was PCR negative. Surprisingly, direct PCR assays for

Brachyspira spp. from clinical specimens were no less sensitive than microbial culture in the current study, which is in contrast to a previous report.3 This discordance may be due to differences in the severity of clinical disease and the time between disease onset and euthanasia for necropsy and sample collection. In the current study, pigs were euthanized within forty-eight hours of the onset of clinical SD and all had severe gross and histologic lesions with numerous Brachyspira detected by fluorescent in-situ hybridization. This is suggestive of a large population of infectious organisms that could be easily detected by either selective culture or direct PCR. Accordingly, it seems likely that in acute cases of SD either culture or direct PCR may be equally sensitive; however, in less fulminate cases or in surveillance situations, direct culture of either the colonic mucosa or feces may be of highest diagnostic value.

Given the abundant mucus typical of SD, the chemoattraction of B. hyodysenteriae to mucus,24,30 and the importance of the mucus bilayer in intestinal health and disease, the histochemical and immunohistochemical characteristics of mucin-secreting goblet cells were evaluated in the present study. Evaluation of these cells at the apex of the spiral colon revealed a significant decrease in MUC4 with a significant concurrent increase in MUC5AC in pigs infected with either Brachyspira sp. compared to controls; however, a decrease in sulphated mucins was only statistically significant when comparing B204-inoculated pigs and controls. The lack of a statistically significant difference in sulphated mucins between

EB107-inoculated pigs and controls may be due to the greater variability in sulphated mucin

65 expression in those pigs that developed SD or the low total number of diseased pigs in this inoculation group or both. This is supported by a P-value that approaches significance (P =

0.054); however, further study is warranted to fully evaluate the effect of “B. hampsonii” infection on sulphated mucin expression.

The increased expression of MUC5AC, a gel-forming mucin important in inhibition of bacterial adherence and invasion,33 observed in all infected pigs that developed SD in this study is likely associated with the inflammatory infiltrate present in the lamina propria as induced expression of MUC5AC is caused by proinflammatory cytokines including interleukin-1β (IL-1β),11,33 interleukin-17(IL-17),11 and neutrophil elastase.10 The intense inflammatory infiltrate characterized by neutrophils and probable presence of proinflammatory cytokines pertinent to the differentiation of CD4+ T cells suggests the Th17 cells may be involved in acute SD. Th17 cells play an important role in host defense at mucosal surfaces against extracellular bacteria and have a unique link to neutrophils.45

MUC4 is a membrane-tethered mucin likely important in epithelial cell renewal, differentiation, and signaling.12,34 The decreased MUC4 expression in pigs with acute SD may result from TGF-β repression of the precursor cleavage of MUC4 thereby inhibiting

MUC4 expression.28 MUC4 is also expressed at significantly lower levels on the surface of the crypt epithelium of S. typhimurium-infected pigs compared with non-infected pigs.25

Decreased expression of sulphated mucins constitutes the first step in mucin degradation providing a rich source of nutrients including carbon for bacterial growth37 and improved growth of intestinal Brachyspira spp. has been observed following mucin addition to media.39 Evidence of mucin degradation also suggests a mechanism by which pathogenic

Brachyspira spp. may breach the protective mucus bilayer to access the underlying epithelial

66 cells.39 Mucin sulphatase activity has been documented in bacteria associated with most mucosal surfaces including the enteric genera Bacteroides and Prevotella;36,43,47 However, in a recent comparison of the complete genome sequences of multiple Brachyspira spp., only a limited number of sulphatase genes were identified including three in B. pilosicoli 95/1000, one putative gene in B. murdochii 56-150T, and none in B. hyodysenteriae WA1.44

Sequencing of additional species and strains is needed to better characterize the presence of sulphatase genes in Brachyspira spp. as well as further work to determine the activity of the encoded proteins. To the authors’ knowledge, sulphatase activity has not been described with either B. hyodysenteriae or “B. hampsonii” suggesting the potential necessity of concurrent colonization with another bacterium capable of degrading sulphated mucins in order to induce the decreased expression of these mucins as observed in the present study.

Indeed, the requirement for additional bacteria as part of the pathogenesis of SD has been demonstrated in gnotobiotic pigs.18,29

It is well-recognized that diet has a major impact on SD expression as feeding diets that are either highly digestible32,42 or rich in inulin inhibits colonization by B. hyodysenteriae.15 Sulphatase activity may also be directly impacted by feed composition resulting in a shift of the microbiome of the gut. A recent study revealed that pigs lack the terminal restriction fragments corresponding to the sulphatase-producing Prevotella group when fed cooked rice or cooked rice supplemented with either 10% potato starch or 20% wheat bran.27 Another study found that pigs fed fructan-rich diets, similar to those shown to prevent SD, had a higher proportion of Bifidobacterium thermacidophilum subsp. porcinum and Megasphaera elsdenii possibly resulting in alterations in the intestinal environment.31

Such changes may decrease the sulfphatase activity present in the gut inhibiting penetration of the protective mucus layer and disease initiation.

In summary, the results of the present study reveal that the time to onset of mucohemorrhagic diarrhea and the duration of spirochete shedding were similar for pigs infected with either “B. hampsonii" clade II or B. hyodysenteriae under the conditions of this report. Furthermore, significant alterations in colonic mucin expression were observed in pigs inoculated with either “B. hampsonii” or B. hyodysenteriae within forty-eight hours of the onset of mucohemorrhagic diarrhea and these and other microscopic changes were not significantly different between infected groups thus further supporting a role for either spirochete as a cause of SD. The specific alterations in colonic mucin expression observed suggest the potential importance of proinflammatory cytokines in the induction of differential mucin expression in acute SD as well as the potential need for concurrent colonization with a bacterium possessing sulphatase activity. Further studies exploring these potential relationships are warranted and may shed light on the specific host and microbial factors involved in the pathogenesis of swine dysentery.

Acknowledgements

The authors wish to thank Hallie Warneke for her help with the Brachyspira culture work and maintenance of the isolate collection at the ISU VDL; Diane Gerjets, Jennifer

Groeltz-Thrush, and Toni Christofferson for preparation of the histologic sections; Deb

Moore for her help optimizing the immunohistochemistry procedures; and Kate Sawyer and

Marissa Rotolo, for their assistance with the animal inoculation and necropsy procedures.

REFERENCES

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15. Hansen CF, Phillips ND, La T, et al. Diets containing inulin but not lupins help to prevent swine dysentery in experimentally challenged pigs. J Anim Sci. 2010;88:3327-3336.

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17. Harris DL, Glock RD, Christensen CR, et al. Inoculation of pigs with Treponema hyodysenteriae (new species) and reproduction of the disease. Vet Med Small Anim Clin. 1972;67:61-64.

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19. Hasnain SZ, Evans CM, Roy M, et al. MUC5AC: a critical component mediating the rejection of enteric nematodes. J Exp Med. 2011;208:893-900.

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21. Hinojosa-Kurtzberg AM, Johansson ME, Madsen CS, et al. Novel MUC1 splice variants contribute to mucin overexpression in CFTR-deficient mice. Am J Physiol Gastrointest Liver Physiol. 2003;284:G853-62.

22. Hommez J, Castryck F, Haesebrouck F, et al. Identification of porcine Serpulina strains in routine diagnostic bacteriology. Vet Microbiol. 1998;62:163-169.

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24. Kennedy MJ, Yancey RJ Jr. Motility and chemotaxis in Serpulina hyodysenteriae. Vet Microbiol. 1996;49:21-30.

25. Kim CH, Kim D, Ha Y, et al. Expression of mucins and trefoil factor family protein-1 in the colon of pigs naturally infected with Salmonella typhimurium. J Comp Pathol. 2009;140:38-42.

26. Kinyon JM, Harris DL, Glock RD. Enteropathogenicity of various isolates of Treponema hyodysenteriae. Infect Immun. 1977;15:638–646.

27. Leser TD, Lindecrona RH, Jensen TK, et al. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl Environ Microbiol. 2000;66: 3290–3296.

28. Lomako WM, Lomako J, Soto P, et al. TGFβ regulation of membrane mucin Muc4 via proteasome degradation. J Cell Biochem. 2009;4:797-802.

29. Meyer RC, Simon J, Byerly CS. The etiology of swine dysentery. III. The role of selected gram-negative obligate anaerobes. Vet Pathol. 1975;12:46-54.

30. Milner JA, Sellwood R. Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization. Infect Immun. 1994;62:4095-4099.

31. Mølbak L, Thomsen LE, Jensen TK, et al. Increased amount of Bifidobacterium thermacidophilum and Megasphaera elsdenii in the colonic microbiota of pigs fed a swine dysentery preventive diet containing chicory roots and sweet lupine. J Appl Microbiol. 2007;103:1853-67.

32. Pluske JR, Siba PM, Pethick DW, et al. The frequency of swine dysentery in pigs can be reduced by feeding diets that limit the amount of fermentable substrate entering the large intestine. J Nutr. 1996;126:2920–2933.

33. Raja SB, Murali MR, Devaraj H, et al. Differential expression of gastric MUC5AC in colonic epithelial cells: TFF3-wired IL-1β/Akt crosstalk-induced mucosal immune response against Shigella dysenteriae infection. J Cell Sci. 2012;125:703-713.

34. Ramsauer VP, Pino V, Farooq A, et al. Muc4-ErbB2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand for ErbB2. Mol. Biol. Cell. 2006;17:2931–2941.

35. Råsbäck T, Jansson DS, Johansson KE, Fellström C. A novel enteropathogenic, strongly haemolytic spirochaete isolated from pig and mallard, provisionally designated 'Brachyspira suanatina' sp. nov. Environ Microbiol. 2007;9:983-991.

36. Roberton AM, McKenzie C, Scharfe N, et al. A glycosulphatase that removes sulphate from mucus glycoprotein. Biochem J. 1993;293:683-689.

37. Robinson CV, Elkins MR, Bialkowski KM, et al. Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients. J Med Microbiol. 2012;61:1644-1653.

38. Rubin JE, Costa MO, Hill JE, et al. Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with "Brachyspira hampsonii" strain 30446. PLoS One. 2013;8:e57146.

39. Stephens CP, Hampson DJ. Intestinal spirochete infections of chickens: a review of disease associations, epidemiology and control. Anim Health Res Rev. 2001;2:83- 91.

40. Song Y, Hampson DJ. Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens. Vet Microbiol. 2009;137:129–136.

41. Theodoropoulos G, Hicks SJ, Corfield AP, et al. The role of mucins in host-parasite interactions: Part II - helminth parasites. Trends Parasitol. 2001;17:130-135.

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44. Wanchanthuek P, Bellgard MI, La T, et al. The complete genome sequence of the pathogenic intestinal spirochete Brachyspira pilosicoli and comparison with other Brachyspira genomes. PLoS One. 2010;5:e11455.

45. Weaver CT, Elson CO, Fouser LA, et al. The Th17 pathway and inflammatory diseases of the intestines, lungs, and skin. Annu Rev Pathol. 2013;8:477-512.

46. Weissenböck H, Maderner A, Herzog AM, et al. Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii. Vet Microbiol. 2005;111:67–75.

47. Wilkinson RK, Roberton AM. A novel glycosulphatase isolated form a mucus glycopeptide-degrading Bacteriodes sp. FEMS. Microbiol Lett. 1998;50:195-199.

CHAPTER 3

COMPARISON OF CULTURE, PCR, AND FLUORESCENT IN SITU HYBRIDIZATION FOR DETECTION OF BRACHYSPIRA HYODYSENTERIAE AND “BRACHYSPIRA HAMPSONII” IN PIG FECES

Modification of a paper to be published in Journal of Veterinary Diagnostic Investigation

Bailey L. Wilberts, Hallie Warneke, Leslie Bower, Joann M. Kinyon, and Eric R. Burrough

Abstract

Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or from species-specific polymerase chain reaction (PCR) assays.

While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, while rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization has been described in formalin-fixed tissues; however, completion requires approximately two days.

Due to the time constraints of available assays, a same-day fluorescent in situ hybridization assay was developed to detect B. hyodysenteriae and "B. hampsonii" in pig feces using previously described oligonucleotide probes, Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hours. The PCR assay on fresh feces and fluorescent in situ hybridization on formalin-fixed feces had similar levels of detection.

Culture was the most sensitive method, detecting the target spirochetes at least two log- dilutions less when compared to other assays 48 hours after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from

73 inoculated pigs as part of a previous experiment. Accordingly, fluorescent in situ hybridization on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in

North America.

Introduction

Since 2005 there has been an increase in swine dysentery (SD) diagnoses in growing and finishing pigs in the United States6 with similar reports from Canada since 2009.12

Swine dysentery is characterized by a mucohemorrhagic diarrhea and typhlocolitis and can result in significant economic losses in affected production systems.8 Classically, SD is associated with infection with Brachyspira hyodysenteriae.8 However, a recently proposed novel Brachyspira species, "Brachyspira hampsonii”,5 has been isolated from pigs with mucohemorrhagic diarrhea and experimental infection with “B. hampsonii” strains has consistently resulted in clinical disease and gross lesions that are similar to, if not indistinguishable from, B. hyodysenteriae infection.3,11,14

A definitive diagnosis of SD is commonly based on the isolation of strongly beta- hemolytic, ring phenomenon positive spirochetes from culture of mucohemorrhagic feces or colonic tissue and/or from species-specific polymerase chain reaction (PCR) assays run on extracts of such samples.4 While Brachyspira culture using selective agars is a highly sensitive assay, it can be technically challenging, time-consuming, typically requires speciation using PCR following isolation, and often requires 6 days or longer to complete which can result in a delay in disease diagnosis.4 Polymerase chain reaction assays, while rapid, can be limited by fecal inhibition, particularly in pigs.13 Fluorescent in situ hybridization (FISH) assays have been described for the identification of B. hyodysenteriae

74 and "B. hampsonii" in formalin-fixed tissues.2,4 However, these assays require approximately two days or more to complete following fresh tissue collection.4 Due to the time constraints of currently available assays, the current study reports the development of a same-day FISH assay to detect B. hyodysenteriae and "B. hampsonii" in pig feces using previously described oligonucleotide probes Hyo12102 and Hamp12104 targeting the 23S ribosomal RNA (rRNA) of B. hyodysenteriae and "B. hampsonii", respectively. In the present study, this FISH assay was simultaneously compared to both culture and real-time

PCR (qPCR) on pig feces spiked with progressive dilutions of B. hyodysenteriae and "B. hampsonii" to determine the threshold of detection for each assay at 0 and 48 hours after sample preparation. As well, the assay was used on formalin-fixed feces collected as part of previous experiments. Additionally, the specificity of each probe was tested against B. murdochii and B. pilosicoli which are the most commonly isolated weakly-hemolytic

Brachyspira spp. at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL).

Materials and Methods

Bacterial strains and growth conditions

Isolates used in this study were obtained from the culture collection at the ISU VDL.

The “B. hampsonii” clade II isolate (EB107) was originally recovered from a clinical case of

SD in 2011 and has been previously used in multiple pig inoculation experiments.3,14 The B. hyodysenteriae isolate (B204) was originally recovered from a clinical case of SD in 1972.

The B. murdochii (KC63) and B. pilosicoli (P43/6/78) isolates were obtained from a clinical case in 2008 and from the American Type Culture Collection, respectively. Isolates were plated onto selective agars containing either colistin, vancomycin, and spectinomycin9 or pig feces extract, spiramycin, rifampin, vancomycin, colistin, and spectinomycin.10 Following

75 four days of incubation in an anaerobic environment provided by a commercial systema at 41

± 1°C, isolates were transferred to trypticase soy agar with 10% bovine blood (10% TSA) and incubated as described previously. Plate media used in this study were prepared in- house and passed the quality assurance standards of the ISU VDL.

Preparation of fecal dilutions and standard plate counts

All isolates were harvested from 10% TSA plates and rinsed into 0.85% physiological saline to a McFarland 0.5 standard. Ten-fold serial dilutions were performed on “B. hampsonii” and B. hyodysenteriae to make eight dilutions ranging from 10-1 to 10-8. One milliliter of the varying dilutions of each isolate was added to 7 ml of phosphate buffered

0.85% physiological saline which contained 2 g of homogenized normal and antibiotic free pig feces. One milliliter of McFarland 0.5 standard of B. murdochii and B. pilosicoli was added to 9 ml of phosphate buffered saline (PBS). At 0 and 48 hours after fecal dilution preparation, fecal dilutions of B. hyodysenteriae and “B. hampsonii” were plated onto trypticase soy agar with 5% bovine blood. Plates were incubated as stated above. Plates were observed and while confluent growth was observed at the concentrated dilutions, discrete colonies were observed on the more dilute plates and were counted to obtain an estimation of the colony-forming units (CFU) per ml of each dilution on days 2, 4, and 6 of incubation.

The final CFU/ml was calculated when the count was between 30 and 300 CFU/ml. Between the two time points, fecal dilutions were stored at 4°C. Isolation of any strongly beta- hemolytic, ring phenomenon positive spirochetes was considered a positive culture.

Fluorescent in situ hybridization

Custom oligonucleotide probes (Hamp1210 and Hyo1210) were purchased from a commercial sourceb and were 5’-labeled with orange fluorescentc dye. One milliliter of the

76 varying fecal dilutions of B. hyodysenteriae or “B. hampsonii” was added to 9 ml of PBS and passed through a commercially available filterd (pore size: 200 to 300 µm) from which 0.5 ml was transferred to 1.5 ml 10% neutral buffered formalin (NBF) or 1.5 ml PBS and incubated at 20 to 25°C for 20 minutes. Using a hydrophobic marking pen on positively charged slides, a 10 to 15 mm diameter circle was drawn and 20 µl of filtered fecal dilutions in 10% NBF or

PBS was placed inside the circle. Half a milliliter of a 1:10 dilution of a McFarland 0.5 standard of B. murdochii and B. pilosicoli was added to 1.5 ml 10% NBF, incubated at 20 to

25 C for 20 minutes, and then a 20 µl aliquot was placed inside a marked circle. Slides were then dried in an incubator at 42°C for 20-30 minutes. The Hyo1210 and Hamp1210 probes were reconstituted using DNase- and RNase-free water and diluted to a working concentration of 10 ng/μl in hybridization buffer (20 mM of Tris, 0.9 M of NaCl, 20% sodium dodecyl sulfate [SDS] buffer, 40% formamide, 10% dextran sulfate [pH 7.2]). Each of the dried fecal samples of B. hyodysenteriae and “B. hampsonii” and dried PBS samples of

B. murdochii and B. pilosicoli were covered with 50 µl of the hybridization solution containing the desired probe and placed in a moisture chamber. Hybridization was carried out for 6 hr at 42°C. Following hybridization, slides were washed in a wash buffer (hybridization buffer without SDS or formamide and prewarmed to the hybridization temperature) for 20 min. After washing, the slides were rinsed with sterile water, allowed to air dry, and mounted using a commercial antifade reagent.e A fluorescent microscope with a filter appropriate for

Alexa555 was used for all analyses. Ten 60X high power fields of each circumscribed area were reviewed. The identification of a total of three or more spirochetes in the reviewed fields was designated a positive sample. The most concentrated samples were reviewed first, and after two consecutive negative dilutions, additional dilutions were not reviewed. All

77 fecal dilutions were stored at 4°C for 48 hrs and processed again in the same manner. Two replicates with each isolate were performed.

Real-time polymerase chain reaction primers, probes, and protocol

One milliliter of each filtered fecal dilution in 10% NBF or PBS was submitted at 0 and 48 hours after sample preparation for qPCR. The DNA was extracted from the fecal samples according to manufacturer’s instructions using a commercial extraction kitf and an automated extraction procedure using a commercial instrument.g

The primers (forward: 5’-TTGCTACTGGTTCTTGGCCTG-3’; reverse: 5’-

GAATGCTTCTATAAGTTCAACACCTAT-3’)13 and probes (B. hyodysenteriae: FAM-5’-

CGAAGGCTTAAAACAAGAAGGA-3’-IABlkFQ;13 “B. hampsonii”: cyanine 5-5’-

CGCTAAATTATTCCAACAAGGACAGG-3’-IABkFQ) were obtained from a commercial source.h Real-time PCR was run using a commercially available master mixi and an exogenous internal positive controlj (IPC) with a VIC-labeled probe. Each Brachyspira probe was used separately, meaning a total of two reactions per DNA. Briefly, 12.5 µl of the mastermix was combined with 1 µl of each primer diluted to 10 µM and 0.5 µl of probe diluted to 10 µM, 2.5 µl of 10x IPC mix, 0.5 µl of 50x IPC DNA, and 4.5 µl of PCR-grade water. The DNA template was added at 2.5 µl per reaction for a total reaction volume of 25

µl. The qPCR was run as follows: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Resulting cycling threshold (Ct) values were reported through 45 cycles. A Ct value less than 45 was considered positive.

Preparation of samples from experimentally infected pigs

One milliliter of large intestinal luminal content was collected at necropsy during a previous inoculation trial14 (B. hyodysenteriae (B204): n = 10; “B. hampsonii” (EB107): n =

10) and placed in 9 ml of brain heart infusion broth from which 0.5 ml was added to 1.5 ml

10% NBF and stored at -80°C for 6 months. At time of collection, sample consistency varied from semi-solid (clinically unaffected pigs) to liquid (clinically affected pigs). Samples were thawed and passed through a commercially available filter.d A 20 µl aliquot of the resulting filtrate was placed onto positively charged slides and FISH was performed as described above.

Mucohemorrhagic feces were collected from the cement floor of pens containing experimentally infected pigs during a previous inoculation trial (results not yet published; B. hyodysenteriae (B204): n = 5; “B. hampsonii” (EB107): n = 3). Briefly, forty 6-week-old crossbred pigs were housed in separate rooms by inoculum (n = 20 per isolate) and inoculated and observed as previously described.14 A 1 ml sample of dysenteric feces was placed in 9 ml of 10% NBF. On the day of collection, samples were passed through a commercially available filter.d The filtrate was divided and stored at either -80°C or 20 to 25

°C. FISH was run as described above with a shortened hybridization time (3-4 hours).

Samples stored at 20 to 25 °C were run at 24 to 96 hours after fixation.

Results

Culture

The culture results of the varying dilutions at the 0 and 48 hour time points for all replicates are presented in Table 1. Briefly, culture was positive at dilutions of 10-1 to 10-7 immediately after sample preparation. After 48 hours of refrigerated storage, cultures from these same samples were positive at dilutions of 10-1 to 10-4, and there was a consistent loss of 2-3 log-dilutions between the 48 hour time point and time 0 for all replicates.

Fluorescent in situ hybridization

The FISH results on formalin-fixed feces of the varying dilutions at the 0 and 48 hour time points of all replicates are presented in Table 1. Figure 1 is a representative image of strong positive signal labeling spirochetes using a probe specific for “B. hampsonii.”

Figure 1. Photomicrograph of a formalin-fixed pig fecal sample spiked with “B. hampsonii” EB107 0 hours after sample preparation (replicate 2) and after fluorescent in situ hybridization using an orange fluorescent probe (Hamp1210) specific for “B. hampsonii.” Detection of 3 or more spirochetes was only observed in the formalin-fixed samples. At both

0 and 48 hours after sample preparation for replicate 1 and 2 of “B. hampsonii”, FISH was positive at the two most concentrated dilutions with a threshold of detection of 7.2 X 105

CFU/ml. The threshold of detection of FISH at 48 hours after sample preparation was 5.9 X

106 CFU/ml in replicate 1 of B. hyodysenteriae. Additionally, FISH failed to detect any positively labeled spirochetes at 0 hours after sample preparation for replicate 2 of B. hyodysenteriae but was positive at 48 hours. A positive signal was not identified in the

80 circumscribed area after hybridization of B. murdochii and B. pilosicoli with either the

Hyo1210 or Hamp1210 probe.

Real-time polymerase chain reaction

The Ct values of qPCR on feces not fixed in formalin of the varying dilutions at the 0 and 48 hour time points of all replicates are presented in Table 1. The internal positive control showed little fluctuation in Ct value indicating minimal qPCR inhibition (Ct 28.7 to

30.7). A total of eleven positive qPCR results were obtained, three of which were from the formalin-fixed samples. Real-time PCR was consistently positive at the first fecal dilution for both spirochetes and at both time points with the lowest level of detection for qPCR being

5.9 X 105 CFU/ml at 48 hours after sample preparation with B. hyodysenteriae.

Fluorescent in situ hybridization: Samples from experimentally infected pigs

Fluorescent in situ hybridization run on large intestinal fecal samples collected as part of a previous experimental inoculation trial was positive for all culture positive, qPCR positive, and clinically affected pigs at necropsy inoculated with either B. hyodysenteriae (n = 9) or

“B. hampsonii” (n = 5). However, FISH was negative on samples from two inoculated but clinically unaffected pigs, one of which was culture positive but qPCR negative and the other of which was culture negative but qPCR positive. Fluorescent in situ hybridization was also negative on samples from all other culture negative pigs at necropsy.

A representative image of a pen level fecal sample with strong positive signal labeling spirochetes using a probe specific for “B. hampsonii” is displayed in Figure 2. Of the five samples collected from the pen of a B. hyodysenteriae-inoculated group, three were positive when fixed in formalin for 48 hours or less. The two samples that were fixed for

Table 1. Summary of culture, fluorescent in situ hybridization (FISH), and real-time polymerase chain reaction (qPCR) results for each isolate and replicate at 0 and 48 hours after sample preparation Isolate/Count/Time Assay Result at indicated dilution Neat 10-1 10-2 10-3 10-4 10-5 10-6 10-7 EB107* Culture NA‡ TNTC§ TNTC TNTC 76 5 2 0 8.3 X 106† FISH 34 (1-6)¦ 4 (0-2) 0 1 NA NA NA NA 0 hr qPCR# NA 41.4 Negative Negative NA NA NA NA

EB107 Culture NA 4 4 5 0 0 0 0 8.3 X 106 FISH 22 (0-6) 3 (0-1) 1 0 NA NA NA NA 48 hr qPCR NA 39.7¶ Negative Negative NA NA NA NA

EB107 Culture NA TNTC TNTC TNTC 106 7 1 1 7.2 X 106 FISH 11 (0-3) 3 (0-1) 0 0 NA NA NA NA 0 hr qPCR NA 42.4¶ Negative Negative NA NA NA NA

EB107 Culture NA TNTC TNTC 110 7 0 0 0 7.2 X 106 FISH 25 (0-7) 3 (0-1) 1 0 NA NA NA NA 48 hr qPCR NA 41.6 Negative Negative NA NA NA NA

B204** Culture NA TNTC TNTC 135 52 5 3 0 5.9 X 106 FISH 3 (0-3) 0 0 NA NA NA NA NA 0 hr qPCR NA 44.1 Negative Negative NA NA NA NA

B204 Culture NA 5 8 2 0 0 0 0 5.9 X 106 FISH 5 (0-2) 0 0 NA NA NA NA NA 48 hr qPCR NA 40.2¶ Negative Negative NA NA NA NA

B204 Culture NA TNTC TNTC 129 6 0 0 0 9.3 X 106 FISH 0 0 NA NA NA NA NA NA 0 hr qPCR NA 41.2 Negative Negative NA NA NA NA

B204 Culture NA 7 19 0 0 0 0 0 9.3 X 106 FISH 7 (0-4) 0 0 NA NA NA NA NA 48 hr qPCR NA 38.1 Negative Negative NA NA NA NA * EB107 = “B. hampsonii” clade II † Reported in colony-forming units/ml ‡ Not assayed § TNTC = Too numerous to count ¦ Total number of spirochetes visualized in ten 60X high power fields of samples run on formalin-fixed feces. Numbers in parentheses represent the range of spirochetes visualized in a single 60X high power field. # Cycle threshold value of samples run on feces not fixed with formalin ¶ Real-time PCR on formalin-fixed feces was also positive at this time point ** B204 = B. hyodysenteriae greater than 48 hours were negative. One sample that was positive at 48 hours fixation was rerun at 72 hours fixation and was negative while a formalin-fixed frozen aliquot of the same

82 sample remained positive. Similarly, of the three samples collected from the pen of a “B. hampsonii”-inoculated group, two were positive when fixed in formalin for 48 hours or less.

The single sample that was fixed for greater than 48 hours was negative.

Figure 2. Photomicrograph of a formalin-fixed pig fecal sample collected from the pen of pigs inoculated with “B. hampsonii” EB107 24 hours after formalin-fixation and following 4 hours of in situ hybridization using an orange fluorescent probe (Hamp1210) specific for “B. hampsonii.” Discussion

Recently, there has been an increase in SD diagnoses in growing and finishing pigs in the United States6 and Canada12 associated with infection with B. hyodysenteriae or “B. hampsonii”. Due to the time constraints and limitations of currently available assays, this current study has developed a same-day FISH assay to detect B. hyodysenteriae and “B. hampsonii” in pig feces using previously described oligonucleotide probes Hyo1210 and

Hamp1210 targeting 23S rRNA of B. hyodysenteriae and “B. hampsonii”, respectively.

In the present study, the sample type greatly impacted the level of detection. Real- time polymerase chain reaction on fresh feces and FISH on formalin-fixed feces had similar levels of detection at both time points for both target spirochetes. Fluorescent in situ hybridization failed when used on fresh feces. Formalin-fixation is commonly used in FISH to preserve the integrity of cells, prevent cell loss, and preclude nucleic acid degradation.1

Yet, qPCR run on formalin-fixed samples was rarely positive. Although beneficial for

FISH, formalin-fixation is likely to inhibit primer access during PCR through the formation of cross-links.7 Accordingly, formalin fixation of feces when done for 48 hours or less, while the most appropriate sample for FISH, is not suitable for the qPCR assay used in the current study.

Culture was the most sensitive method overall in this study, commonly detecting both

B. hyodysenteriae and “B. hampsonii” at less than 10 CFU/ml immediately after sample preparation. This is consistent with previous studies in which qPCR of rectal swabs12 or mucosal scrapings obtained at necropsy3 detected fewer positive samples than selective culture. However, a significant reduction in the number of culturable spirochetes was observed following 48 hours of storage of fecal dilutions at 4°C. Testing samples following forty-eight hours of storage was selected to replicate the average transport time (time between sample collection and laboratory receipt) for clinical diagnostic samples shipped in the United States and received by the ISU VDL. Although this study was not a direct representation of laboratory submissions, these data suggest that culture is the best assay currently available for surveillance testing and identification of carrier animals; yet, diagnostic samples should be promptly processed and delivered to the laboratory to decrease the likelihood of false negative cultures due to prolonged transit time.

Fluorescent in situ hybridization effectively detected the target Brachyspira spp. in formalin-fixed feces from infected pigs with clinical dysentery. Labeled spirochetes were also readily detectable in pen level samples from infected groups of pigs with clinical disease. While culture remains the diagnostic assay of choice for surveillance situations where low numbers of spirochetes are anticipated, clinically affected pigs may be identified in a timelier manner using either qPCR or FISH. Although a positive signal was not identified after hybridization of B. murdochii and B. pilosicoli with either probe, it bears noting that cross-reactivity with the Hamp1210 probe and Brachyspira intermedia in FISH on formalin-fixed tissues has been observed.4 However, given the relatively high threshold of detection for FISH on formalin-fixed feces in this experiment, it seems unlikely that a clinically insignificant infection would be detected by this method. Accordingly, FISH performed on feces from clinically affected pigs and fixed in formalin for 48 hours or less will allow for same-day identification and preliminary speciation of spirochetes commonly associated with SD in North America. Concurrent culture for confirmation of hemolytic phenotype is recommended where available, and speciation from culture by molecular methods remains a more definitive assay.

Sources and Manufacturers a. BD GasPak EZ Anaerobe Container System, BD Diagnostic Systems, Sparks, MD. b. Invitrogen Custom Oligos, Life Technologies, Carlsbad, CA. c. Alexa Fluor 555, Life Technologies, Carlsbad, CA. d. Fisherbrand™ Disposable Filter Columns, Thermo Fisher Scientific, Waltham, MA. e. Invitrogen ProLong Gold, Life Technologies, Carlsbad, CA.

85 f. MagMAX™ Total Nucleic Acid Isolation Kit, Ambion, Life Technologies, Grand Island,

NY g. KingFisher™, Thermo Scientific, Waltham, MA h. TaqMan, IDT, Inc. Coralville, IA i. VetMAX™-Plus qPCR Master Mix, Applied Biosystems, Life Technologies, Grand

Island, NY j. TaqMan, Applied Biosystems, Life Technologies, Grand Island, NY

REFERENCES

1. Amann R, Fuchs BM. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol. 2008;6:339-348.

2. Boye M, Jensen TK, Møller K, et al. Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoli in porcine intestines by fluorescent rRNA in situ hybridization. Mol Cell Probes. 1998;12:323-330.

3. Burrough ER, Strait EL, Kinyon JM, et al. Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs. J Vet Diagn Invest. 2012;24:1025-1034.

4. Burrough ER, Wilberts BL, Bower LP, et al. Fluorescent in situ hybridization for detection of "Brachyspira hampsonii" in porcine colonic tissues. J Vet Diagn Invest. 2013;25:407-412.

5. Chander Y, Primus A, Oliveira S, Gebhart CJ. Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated "Brachyspira hampsonii". J Vet Diagn Invest. 2012;24:903-910.

6. Clothier KA, Kinyon JM, Frana TS, et al. Species characterization and minimum inhibitory concentration patterns of Brachyspira species isolates from swine with clinical disease. J Vet Diagn Invest. 2011;23:1140-1145.

7. Dowd SE, Gerba CP, Enriquez FJ, Pepper IL. PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. Appl Environ Microbiol. 1998;64:333-336.

8. Hampson DJ, Fellström C, Thomson JR. Swine dysentery. In: Straw BE, Zimmerman JJ, D'Allaire S, Taylor DJ, eds. Diseases of swine. 9th ed. Ames, IA: Blackwell Publishing; 2006:785 - 805.

9. Jenkinson SR, Wingar CR. Selective medium for the isolation of Treponema hyodysenteriae. Vet Rec. 1981;109:384-385.

10. Kunkle RA, Kinyon JM. Improved selective medium for the isolation of Treponema hyodysenteriae. J Clin Microbiol. 1988;26:2357-2360.

11. Rubin JE, Costa MO, Hill JE, et al. Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with "Brachyspira hampsonii" strain 30446. PLoS One. 2013;8:e57146.

12. Patterson AH, Rubin JE, Fernando C, et al. Fecal shedding of Brachyspira spp. on a farrow-to-finish swine farm with a clinical history of "Brachyspira hampsonii"- associated colitis. BMC Vet Res. 2013;9:137. 13. Song Y, Hampson DJ. Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens. Vet Microbiol. 2009;137:129-136.

14. Wilberts BL, Arruda PH, Kinyon JM, et al. Comparison of lesion severity, distribution, and colonic mucin expression in pigs with acute swine dysentery following oral inoculation with "Brachyspira hampsonii" or Brachyspira hyodysenteriae. Vet Pathol. 2014;Online ahead of print: doi:10.1177/0300985813516646.

CHAPTER 4

INVESTIGATION OF THE IMPACT OF INCREASED DIETARY INSOLUBLE FIBER THROUGH THE FEEDING OF DRIED DISTILLERS GRAINS WITH SOLUBLES ON THE INCIDENCE AND SEVERITY OF BRACHYSPIRA-ASSOCIATED COLITIS IN PIGS

Modification of a paper submitted to PLoS ONE

Bailey L. Wilberts, Paulo H. Arruda, Joann M. Kinyon, Tim S. Frana, Chong Wang, Drew R. Magstadt, Darin M. Madson, John F. Patience, and Eric R. Burrough

Abstract

Diet has been implicated as a major factor impacting clinical disease expression of swine dysentery and Brachyspira hyodysenteriae colonization. However, the impact of diet on novel pathogenic strongly beta-hemolytic Brachyspira spp. including “B. hampsonii” has yet to be investigated. In recent years, distillers dried grains with solubles (DDGS), a source of insoluble dietary fiber, has been increasingly included in diets of swine. A randomized complete block experiment was used to examine the effect of increased dietary fiber through the feeding of DDGS on the incidence of Brachyspira-associated colitis in pigs. One hundred 4-week-old pigs were divided into five groups based upon inocula (negative control,

Brachyspira intermedia, Brachyspira pilosicoli, B. hyodysenteriae or “B. hampsonii”) and fed one of two diets containing no (diet 1) or 30% (diet 2) DDGS. The average days to first positive culture and days post inoculation to the onset of clinical dysentery in the B. hyodysenteriae groups was significantly shorter for diet 2 when compared to diet 1 (P = 0.04 and P = 0.0009, respectively). A similar difference in the average days to first positive culture and days post inoculation to the onset of clinical dysentery was found when comparing the “B. hampsonii” groups. Due to the importance of the colonic mucus bilayer in health and disease, significantly different mucin expression was observed between pigs

88 inoculated with strongly beta-hemolytic spirochetes and control pigs including increased

MUC5AC (P < 0.01) and decreased sulphomucins (P < 0.005). In this study, pigs receiving

30% DDGS shed on average one day prior to and developed swine dysentery nearly twice as fast as pigs receiving 0% DDGS. Accordingly, these data suggest dietary modification with reduction in insoluble fiber should be considered an integral part of any effective disease elimination strategy for swine dysentery.

Introduction

Brachyspira spp. are a diverse group of Gram-negative, oxygen-tolerant, anaerobic spirochetes. Included in this genus is Brachyspira hyodysenteriae, a causative agent of swine dysentery (SD), which is an economically significant disease of grow-finish swine characterized by severe diarrhea with blood and mucus.11 Brachyspira pilosicoli is the only other Brachyspira spp. that has traditionally been recognized as a significant swine pathogen and is the causative agent of porcine colonic spirochetosis (PCS), a condition typically associated with mild colitis and reduced feed efficiency and rate of gain.37

By the mid-1990s, clinical SD had essentially disappeared from North American swine herds as a result of effective treatment, control, and elimination methods. However, outbreaks of bloody diarrhea have been reported in the United States and Canada since 2007 and these outbreaks have often been associated with “Brachyspira hampsonii” infection.3

Like B. hyodysenteriae, “B. hampsonii” is strongly beta-hemolytic on blood agar but can be differentiated by its lack of indole cleavage or by nox gene sequencing.3 Experimental infection with “B. hampsonii” strains has consistently resulted in disease similar to, if not indistinguishable from, B. hyodysenteriae infection.2,33,41 Clinical SD associated with infection with either B. hyodysenteriae or “B. hampsonii” is characterized by gross lesions

89 limited to the large intestine that commonly include variable mucosal thickening, hemorrhage, fibrinonecrotic exudate, and abundant mucus. Microscopically, crypt lumens are often distended with mucus; neutrophils are present in the lamina propria, and silver staining highlights spirochetes within crypt lumens and mucus-producing goblet cells.

The importance of the intestinal mucus layers in both health and disease has been elucidated with the recent understanding of the molecular nature of mucins, the main building blocks of mucus. Mucins are large glycoproteins that are responsive to environmental factors in the gut lumen4,14 including the microbiota and microbial products.18

Mucins provide both a reporting and barrier function and are divided into two subgroups: secretory mucins and cell surface mucins.13,29,36 Secretory mucins, also called gel-forming mucins, are extracellular and secreted from the apical surface of goblet cells.7 Mucin 5AC

(MUC5AC) and mucin 2 (MUC2) are both secretory mucins, MUC5AC is important in the inhibition of bacterial adherence and invasion.28 MUC2 is the major gel-forming mucin in the intestine organizing the mucus by its large net-like polymers.10 Despite the chemoattractant nature of B. hyodysenteriae and B. pilosicoli to mucus16,23,26 and the biosynthetic response of mucus to diet and/or microbiota, the immunohistochemical and histochemical characteristics of colonic goblet cells have not been extensively evaluated in pigs with SD or following dietary modification.

Coincident with the recent reemergence of SD has been a change in swine feeding practices to include feeding of ethanol byproducts, such as distillers dried grains with solubles (DDGS), which are frequently added to varying degrees in swine rations. Relatively little is known about the pathogenesis of SD associated with infection with either B. hyodysenteriae or “B. hampsonii”; however, diet is considered to play a major role in disease

90 expression.9 The effects of DDGS and other forms of insoluble fiber on the intestinal environment of pigs and other mammals have revealed possible mechanisms by which

DDGS may enhance disease expression through alteration of the intestinal environment providing pathogenic Brachyspira spp. an optimal niche in which to cause clinical colitis.

To the authors’ knowledge, the impact of increased insoluble dietary fiber resulting from the inclusion of DDGS in swine diets on the development of Brachyspira-associated colitis in swine has not described. To better characterize the potential impact of DDGS on the development of spirochetal colitis in pigs, the study described herein was performed with the following objectives: 1) to assess the clinical effect of increased insoluble dietary fiber on the time to onset of Brachyspira-associated disease in inoculated pigs, 2) to assess the effect of increased insoluble dietary fiber on colonic pH in both infected pigs and controls, 3) to describe gross lesions and compare histologic lesions within the colon of inoculated pigs, and 4) to characterize the biosynthetic responses in mucin-secreting colonic goblet cells in pigs with histologic lesions consistent with SD compared to controls.

Materials and Methods

Animals

All procedures were approved by the Institutional Animal Care and Use Committee of Iowa State University (Log Number: 1-12-7283). One hundred crossbred pigs 4 weeks- of-age were obtained from a commercial source with no known history of Brachyspira- associated disease. Upon arrival, all pigs received a starter/transition diet and were acclimated to the facility for 2 days. Pigs were then weighed and sorted into ten pens of 10 pigs each to insure even weight distribution across all pens. Each pen was assigned to group based upon the inoculum (sham, B. intermedia, B. pilosicoli, “B. hampsonii”, or B.

91 hyodysenteriae) and diet (diet 1: 0% or diet 2: 30% DDGS) they were to receive. Pigs receiving different inocula were separated by room. Pigs were acclimated to their respective diets for two weeks prior to inoculation and remained on these diets until study termination.

Diet

Diets were formulated to meet or exceed the nutrient requirements for pigs of this age27 and prepared in mash form at the Swine Nutrition Farm at Iowa State University (Table

1). Corn DDGS was included in the second diet as an additional source of insoluble fiber.

Urriola et al. (2010) reported that about 96.6% of total dietary fiber in corn DDGS is insoluble. This add rate would be considered on the high side for pigs of this age, but would not be considered high in commercial practice for early grower diets. Both diets were formulated to contain the same concentration of metabolisable energy (3.4 Mcal/kg), the same level of standardized ileal digestible (SID) lysine (1.23%), the same minimum ratios of other essential amino acids to lysine, all expressed on an SID basis, and the same levels of calcium and available phosphorus. Neutral detergent fiber increased from 7.9% in the control diet to 14.3% in the treatment diet.

Inoculum

Isolates used in this study were obtained from the culture collection at the Iowa State

University Veterinary Diagnostic Laboratory (ISU VDL). Media, isolates, and challenge inocula were prepared as previously described.2 The “B. hampsonii” clade II isolate

(EB107) was recovered from a clinical case of SD in 2011 and was 11th and 12th passage at the time of inoculation. The B. hyodysenteriae (B204) isolate was originally recovered from a clinical case of SD in 1972 and was 8 – 10th passage at the time of inoculation. The B. intermedia isolate (BR2000) and B. pilosicoli isolate (BR2001) originated from pigs with

92 clinical diarrhea and nonsuppurative catarrhal colitis and were 11th and 12th passage, respectively.

Table 1. Ingredient and nutrient composition of experimental diets Component Diet 1 (%) Diet 2 (%) Ingredients Corn, yellow dent 61.13 34.55 Corn DDGS 0 30.00 Soybean meal 20.00 17.50 Fish meal, Menhaden Select 5.66 5.06 Whey, dried 10.00 10.00 l-Lysine HCl 0.31 0.31 dl-Methionine 0.12 0.02 l-Threonine 0.12 0.03 l-Tryptophan 0.02 0.01 Monocalcium phosphate 0.48 0.10 Limestone 0.43 0.70 Salt 0.35 0.35 Vitamin premixc 0.23 0.23 Trace mineral premixd 0.15 0.15 Soybean oil 1.00 1.00

ME, Mcal/kg 1.54 1.54 Crude Protein, % 19.6 23.9 SID Lysine, % 1.23 1.23 SID Threonine, % 0.76 0.76 SID TSAA, % 0.71 0.71 SID Tryptophan, % 0.21 0.21 ADF, %a 3.0 5.7 NDF, %b 7.9 14.3 Crude Fat, % 4.6 6.4 Calcium, % 0.70 0.71 Phosphorus, % 0.65 0.67 Sodium, % 0.27 0.33 Chloride, % 0.42 0.46 aADF = Acid detergent fiber bNDF = Neutral detergent fiber c Provided per kg of diet: 7,000 IU vitamin A, 800 IU vitamin D3, 57 IU vitamin E, 3.4, menadione, 13 mg riboflavin, 64 mg niacin, 31 mg pantothenic acid, and 57 µg vitamin B12 d Provided per kg of diet: 165 mg Zn as ZnSO4, 165 mg Fe as FeSO4, 39 mg Mn as MnSO4, 17 mg Cu as CuSO4, 0.3 mg I as Ca(IO3)2 and 0.3 mg Se as Na2SeO3.

Brachyspira spp. isolation and growth

For isolation of Brachyspira spp., individual rectal swabs were collected daily starting at 5 days post-inoculation (DPI) and at necropsy. Swabs were plated onto selective agars within 6 hours of collection. Specifically, swabs were plated onto CVS selective agar containing colistin, vancomycin, and spectinomycin; and BJ selective agar containing pig feces extract, spiramycin, rifampin, vancomycin, colistin, and spectinomycin. Plate media used in this study were prepared in-house and passed the quality assurance standards of the

ISU VDL. An anaerobic environment was provided by a commercial system (BD GasPak

EZ Anaerobe Container System, BD Diagnostic Systems, Sparks, MD) and plates were incubated at 41 ± 1°C. Agar plates were observed for growth at 2, 4, and 6 DPI and, for positive cultures, the degree of beta-hemolysis and the presence or absence of ring phenomenon were recorded. The sample was reported as positive if either selective agar had growth of Brachyspira spp. The final positive culture for each pig was speciated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) as previously described.40 At necropsy, rectal swabs were also directly plated onto

MacConkey’s agar and brilliant green agar with novobiocin as well as tetrathionate broth enrichment subcultured to brilliant green agar with novobiocin and XLT4 to test for the presence of Salmonella spp.

Animal inoculation

Pigs were inoculated with an agar slurry as previously described.41 Briefly, pigs received three doses of inoculum (100 ml/dose) administered via gavage 24 hours apart with each administration preceded by a 12-18 hour fast. A 5-g sample of each inoculum was reserved from which 1 gram was vortexed for 45 seconds in tubes with 9 ml of sterile PBS

94 and a few glass beads. A standard plate count procedure was performed by titration of 1 ml of the vortexed sample into 9 ml and carried out to 10-9. The dilution series was plated on trypticase soy agar with 5% bovine blood and incubated anaerobically for 6 days with plates being observed on days 2, 4, and 6. Brachyspira spp. grew confluently at the more concentrated dilutions, but discrete colonies were observed from the more dilute plates.

Colonies were counted after 6 days incubation to obtain the inoculum titer in approximate colony-forming units per ml (CFU/ml). The approximate CFU/ml by day and isolate are presented in Table 2. Pigs in the control group received a sham inoculum consisting of agar material from non-inoculated culture plates prepared in the same manner as for the

Brachyspira inocula.

Table 2. Approximate CFU/ml of Brachyspira spp. in inocula by days post inoculation and isolate. 0 DPI* Isolate 1 DPI 2 DPI (CFU/ml) † (CFU/ml) (CFU/ml) Brachyspira hyodysenteriae (B204) 1.4 x 106 5.8 x 105 8.8 x 105 “Brachyspira hampsonii” (EB107) 1.1 x 106 1.2 x 106 1.2 x 106 Brachyspira pilosicoli (BR2001) 8.9 x 107 9.3 x 107 7.9 x 107 Brachyspira intermedia (BR2000) 9.0 x 105 3.1 x 105 8.8 x 105 *DPI = days post inoculation †CFU/ml = colony-forming units per ml

Molecular identification

The subpassaged isolates used to prepare the inocula were verified to species by PCR amplification of partial nox gene sequences using previously described primers32 followed by sequence comparison with sequences available in GenBank.

Oral fluids

Oral fluids were collected from each group daily starting 5 DPI until two pigs remained within the group or at the termination of the study 21 DPI. Briefly, a 0.75 m length

95 of 0.95 cm cotton rope was hung from a bracket in each pen for approximately 20 minutes.

Following sufficient oral fluids accumulation, ropes were placed into a 3.8 L plastic bag and wrung by hand. The oral fluids were then transferred to a sterile plastic tube and submitted for isolation of Brachyspira spp. as previously described.

Animal observations and necropsy

Throughout the study period, investigators were blinded to diet composition.

Following inoculation, animals were observed at least twice daily for feed consumption, availability of adequate water, and clinical illness. Pigs were weighed at arrival, inoculation, and necropsy. Fecal consistency was determined daily, and each pig received a score based upon the following system: 0 if normal, 1 if soft but formed, 2 if semisolid, and 3 if liquid to watery with an additional 0.5 point added each for the presence of discernible mucus and/or blood. Animals were euthanized by barbiturate overdose after 72 hours of consecutive diarrhea with blood and mucus or at the termination of the study 21 DPI. At necropsy, the entire intestinal tract was observed for gross lesions and the full length of the cecal and colonic lumens were exposed and evaluated for the severity, distribution, and location of luminal mucus, mucosal hemorrhage, and fibrinous exudate. Tissue samples were collected and placed in 10% neutral buffered formalin and included sections of ileum and apex of the spiral colon. After 24 to 48 hours of fixation, tissue samples were transferred to 70% ethanol and processed routinely for histopathology. A rectal swab was also collected from each pig for microbial culture. The pH of luminal contents taken from the apex and cecum was recorded using a calibrated commercially available portable pH meter (Thermo Scientific,

Orion Star™ A121 pH Portable Meter) within 5 minutes of euthanasia.

Histopathology

The investigator was blinded to individual pig numbers and group identifications.

Sections were cut to 4 µm and stained routinely with hematoxylin and eosin. Sections of ileum were evaluated for the presence of any lesions suggestive of Lawsonia intracellularis or Salmonella spp. infection. For sections of spiral colon, neutrophils in the lamina propria were counted in ten 40X fields and the mean for each section of the apex was determined.

Crypt depth was measured in each section using a standard eyepiece micrometer and measurements were taken from an area where the crypts were perpendicular to the mucosal surface with intact epithelium. The mean of three measurements was calculated for each section of apex of the spiral colon for each pig.

Immunohistochemistry

MUC5AC. Sections (4 μm) of the apex were cut onto positively charged slides, incubated at 57°C for 45 minutes, and deparaffinized through graded alcohol. Endogenous peroxidase was inhibited by application of 3% hydrogen peroxide for 20 minutes followed by three rinses with ultrapure water. Slides were processed for antigen retrieval by microwaving in Tris/EDTA (pH 9.0) for 1 min followed by a steam chamber for 20 minutes. Slides were then incubated with 1:10 dilution of goat serum and PBS buffer for 20 minutes at 37ºC to saturate non-specific protein binding sites. A MUC5AC (Zymed Laboratories, Invitrogen

Corporation, Carlsbad, CA) primary antibody which has previously been shown to have specificity in pig large intestine17 was diluted to 1:50 in PBS containing 0.1% Tween 20.

Slides were incubated with the primary antibody overnight at 4°C in a humidified chamber.

Following PBS washes, sections were incubated with a commercially available biotinylated secondary antibody (MultiLink, BioGenex, San Ramon, CA; 1:80); slides were incubated at

22oC for 30 minutes followed by rinsing in a bath of PBS solution for 5 minutes. Sections were then incubated with horse radish peroxidase–streptavidin conjugate (Zymed, Invitrogen,

Carlsbad, CA) at 22oC for 15 minutes followed by rinsing in a bath of PBS solution for 5 minutes. Slides were subsequently incubated with a commercial chromagen (NovaRED™,

Vector laboratories, Burlingame, CA) for 5 minutes at 22ºC followed by rinsing with ultrapure water. Finally, the sections were counterstained with Shandon’s hematoxylin, placed in Scott’s Tap water for 1 minute, rinsed with ultrapure water, dehydrated through a graded alcohol series, and mounted.

MUC2. Sections (4 μm) of the apex were processed as described for MUC5AC with a few modifications. Antigen unmasking was performed with citrate buffer (pH 6.0) and a pressure cooker (1,100-W microwave, 45 sec at 100% power followed by 5 min at 30% power). Slides were then placed in an automated cell staining system (OptiMaxPlus,

BioGenex, San Ramon, CA) and incubated with 1:10 dilution of goat serum and PBS buffer for 20 minutes at 22ºC to saturate non-specific protein binding sites. A MUC2 (Santa Cruz

Biotechnology, Santa Cruz, CA) primary antibody which has previously been shown to have specificity in pig large intestine8 was diluted to 1:100 in PBS containing 0.1% Tween 20.

Slides were incubated with the primary antibody for one hour at 22ºC and processed as described above.

Histochemistry

Sections (4 µm) of the apex were placed onto glass slides, deparaffinized, and treated with a combination of high-iron-diamine and Alcian blue (pH 2.5) (HID/AB) staining.

Slides were placed in high-iron-diamine solution for 18 hours at 37ºC, rinsed, placed in

Alcian blue solution for 5 minutes, rinsed, dehydrated, and mounted.

Image analysis

Quantitative analysis of histochemical and immunohistochemical assays was performed using a commercial software program (Photoshop CS4, Adobe Systems Inc. San

Jose, CA). Five representative 40X images were captured for each slide. The number of pixels representing positive staining was recorded for each image and the average number of pixels representing positive staining was calculated per slide.

Statistical analyses

Commercial statistical software packages (SAS 9.3, SAS Institute, Cary, NC; JMP

Pro 10, SAS Institute, Cary, NC) were utilized to perform all analyses. Average daily gain

(ADG) at 2 wks, total ADG, cecal pH, and pH at the apex of the spiral colon were analyzed using analyses of variance (ANOVA). Time to event data (days to SD, days to positive culture) were analyzed using Cox’s proportional hazard regression. Brachyspira spp. inoculated, diet type, and their interaction were used as explanatory variables in the above analyses. Wilcoxon two- sample test was performed for difference in neutrophilic inflammation, crypt depth, and mean of the log-transformed data of pixels representing positive immunohistochemical or histochemical staining. The correlation between neutrophilic inflammation and the logarithmic transformation of the pixels representing positive immunolabeling for MUC5AC was assessed using the Spearman correlation coefficient. In all circumstances, values of P ≤

0.05 were considered significant and means are reported with standard error of the mean.

Results

Animal observations

Clinical signs of dysentery were first observed 5 and 7 DPI in the B. hyodysenteriae- inoculated diet 2 group and B. hyodysenteriae-inoculated diet 1 group, respectively and 4 and

6 DPI in the “B. hampsonii”-inoculated diet 2 group and “B. hampsonii”-inoculated diet 1

99 group, respectively. The timing of onset of clinical dysentery for individual animals within each group is displayed in Tables 3 and 4. When analyzed based upon culture phenotype, the average DPI to the onset of clinical SD was significantly less in strongly beta-hemolytic- inoculated diet 2 groups than strongly beta-hemolytic-inoculated diet 1 groups (P = 0.018).

However, when analyzed based upon inoculum identity and diet, the average DPI to the onset of clinical SD remained statistically significant only when comparing the B. hyodysenteriae- inoculated diet 2 group and B. hyodysenteriae-inoculated diet 1 group (diet 1: 12.2 ± 1.5; diet

2: 6.8 ± 0.33; P = 0.0009). For the “B. hampsonii”-inoculated groups the average DPI to the onset of SD was numerically shorter in the diet 2 group (diet 1: 12.4 ± 1.45; diet 2: 8.0 ±

0.46); however, this difference was not statistically significant. Fecal scores and timing of euthanasia for each pig are summarized by group in Table 3 and 4 for B. hyodysenteriae and

“B. hampsonii” groups, respectively.

Diarrhea with mild mucus was first noted 6 DPI in both B. pilosicoli-inoculated groups. The average maximum daily diarrhea score for pigs in the B. pilosicoli-inoculated diet 1 and B. pilosicoli-inoculated diet 2 groups was 0.85 on DPI 10 and 1.75 on DPI 10, respectively. No significant diarrhea was noted in either group of B. intermedia-inoculated pigs throughout the study period with a maximum average daily diarrhea score of 0.3 on DPI

5 for diet group 1 and 0.2 on DPI 9 for diet group 2. Similarly, sham-inoculated pigs had a maximum average daily diarrhea score of 0.1 on DPI 11 and 18 for diet group 1 and 0.2 on

DPI 14 for diet group 2.

Microbial culture

Oral fluids were consistently culture positive when collected from pens containing pigs shedding strongly beta-hemolytic spirochetes, and these results are presented by group

100 in Table 3 and 4. Oral fluids collected from B. pilosicoli-inoculated pigs were positive 10 days out of the 17 days in which there was at least one culture positive rectal swab in diet group 1 and 8 days out of the 17 days in diet group 2 (daily culture data not shown). Oral fluids collected from B. intermedia-inoculated pigs were positive 8 days out of the 17 days in which there was at least one culture positive rectal swab in diet group 1 and 1 day out of the

16 days in diet group 2 (daily culture data not shown). Oral fluids collected from sham- inoculated pens were culture negative throughout the study.

Culture results for each pig are summarized by inoculation group and diet in Table 3 and 4 for B. hyodysenteriae and “B. hampsonii” groups, respectively. Days to first positive culture was shorter in both B. hyodysenteriae-inoculated diet 2 (mean ± SEM = 5.5 ± 0.30) and “B. hampsonii”-inoculated diet 2 groups (6.4 ± 0.54) when compared to B. hyodysenteriae-inoculated diet 1 (6.9 ± 0.59) and “B. hampsonii”-inoculated diet 1 (7.5 ±

1.42) groups, respectively; however, this difference was only statistically significant when comparing the B. hyodysenteriae-inoculated groups (P = 0.0443). In contrast, the average days to first positive culture was longer in both B. pilosicoli-inoculated diet 2 (6.2 ± 0.51) and B. intermedia-inoculated diet 2 (10.2 ± 1.62) groups when compared to B. pilosicoli- inoculated diet 1 (5.3 ± 0.15) and B. intermedia-inoculated diet 1 (8.56 ± 1.09) groups, respectively; however, these differences were not statistically significant. Every pig in the B. pilosicoli-inoculated groups shed at least eight days throughout the study period while only 9 and 5 pigs shed viable spirochetes in the B. intermedia-inoculated diet 1 and 2 group, respectively. Sham-inoculated pigs were culture negative throughout the study.

At necropsy Salmonella spp. were isolated from 12 out of 100 pigs with the majority of these isolates (8 of 12) occurring only after tetrathionate broth enrichment. Pigs with

101 positive cultures were distributed across multiple groups as follows: sham-inoculated diet 1

(1 of 10), sham-inoculated diet 2 (0 of 10), B. pilosicoli diet 1 (2 of 10), B. pilosicoli diet 2 (0 of 10), B. intermedia diet 1 (3 of 10), B. intermedia diet 2 (0 of 10), “B. hampsonii” diet 1 (1 of 10), “B. hampsonii” diet 2 (1 of 10), B. hyodysenteriae diet 1 (2 of 10), and B. hyodysenteriae diet 2 (2 of 10). Isolates were characterized as either serogroup B (10 of 12) or rarely C1 (2 of 12). Individual pigs with positive Salmonella culture are identified in

Tables 3 and 4.

Average daily gain

Average daily gain was not statistically different between any groups following the two weeks of diet acclimation. Average daily gain, as calculated from study initiation to study termination or euthanasia, was significantly less in pigs inoculated with strongly beta- hemolytic Brachyspira spp. than sham-inoculated or weakly beta-hemolytic-inoculated pigs

(P < 0.0001, all analyses). However, no statistically significant difference was identified between sham-inoculated or weakly beta-hemolytic-inoculated pigs (P > 0.9, all analyses).

Average daily gain is summarized by group in Table 5.

Large intestine pH

Cecal pH was more alkaline in the sham-inoculated diet 2 group (6.157 ± 0.06) than the sham-inoculated diet 1 group (5.906 ± 0.05), and this difference was statistically significant (P = 0.0025). No statistically significant difference was observed within any other group between diet 1 and diet 2 (P > 0.17, all analyses). The pH at the apex was significantly different between sham-inoculated diet 1 group (6.169 ± 0.05) and the sham- inoculated diet 2 group (6.389 ± 0.07; P = 0.01) and the B. pilosicoli-inoculated diet 1 group

(6.088 ± 0.07) and B. pilosicoli-inoculated diet 2 group (5.880 ± 0.06; P = 0.038).

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Table 3. Summary of fecal scores, Brachyspira culture results of feces and oral fluids, and timing of euthanasia of B204-inoculated pigsa,b Individual Fecal Scoresc by Days Post-Inoculation Diet 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Pig ID 0% DDGSd 88 0+ 2+ 4+ 4+ 4+ 4+ ------83 0+ 0+ 4+ 4+ 4+ 4+ ------85 0 0+ 2+ 4+ 4+ 4+ ------84 0+ 0+ 0+ 0+ 4+ 4+s 4 + ------86 0 0 0 1 0 0+ 4+ 4+ 4+ 4+s ------87 0 0 0 0 0+ 0+ 1+ 4+ 4+ 4+ ------81 0 1+ 0 1 0 1 1+ 1+ 3.5+ 4 2.5 2.5 1 1 1 0 1 82 0 1 0+ 1 0+ 0 0+ 0 1 1+ 0 0 0 0 0 0 0 89 0 0 0 0 0+ 0 0 0 0 0 0 0+ 0+ 0 0 0 0 90 0 0 0+ 0 0 0 0 0 0+ 0+ 0+ 2.5+ 1+ 4+ 4+ 3+ 4+ Oral Fluids Neg Neg Neg Pos Pos Pos Pos Pos Pos Pos Neg Pos Neg Pos Pos Neg Pos 30% DDGS 92 3.5+ 4+ 4+ 4 + ------96 0+ 4+ 4+ 4+ ------100 0+ 4+ 4+ 4+ ------91 2+ 3.5+ 4+ 4+ 4+s ------94 0+ 4+ 4+ 4+ 4+ ------95 0+ 4+ 4+ 4+ 4+ ------98 0+ 0+ 4+ 4 4+ ------99 0 0+ 4+ 4+ 4 + ------93 0 0 0 0+ 4+ 4+ 4+ ------97 0 0+ 0+ 4 4+ 4+ 4+s ------Oral Fluids Neg Pos Pos Pos Neg ------a B204= B. hyodysenteriae b Sham-inoculated diet 1 group and sham-inoculated diet 2 group had a maximum daily diarrhea score of 0.1 on days 11 and18 post-inoculation and 0.2 on day 14, respectively; never had a positive Brachyspira culture; and were euthanized at 21 days post inoculation. c A fecal score was determined for each pig daily based upon the following system: 0 if normal, 1 if soft but formed, 2 if unformed with semisolid consistency, and 3 if severely liquid to watery with an additional 0.5 point added each for the presence of discernible mucus and/or blood (max score = 4). d Distillers dried grains with solubles + Positive Brachyspira culture sPositive Salmonella culture at necropsy

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Table 4. Summary of fecal scores, Brachyspira culture results, and timing of euthanasia of EB107-inoculated pigsa,b Individual Fecal Scoresc by Days Post-Inoculation Diet 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Pig ID 0% DDGSd 70 3.5+ 4+ 4+ 4+ ------69 2+ 4+ 4+ 4+ 4+ ------68 0 0+ 1+ 2.5+ 4+ 4+ 4+s ------62 0 0 0+ 0+ 0+ 1+ 4+ 4+ 4+ 4+ ------63 1 1+ 1 1+ 0+ 1+ 2.5+ 4+ 4+ 4+ ------67 0 0 0+ 0+ 0+ 0+ 0+ 4+ 4+ 4+ ------61 0 0+ 0+ 0 0+ 0+ 0 0 0 0 0 0 0 0 1 0+ 1+ 64 0 0 0 0+ 0+ 1+ 0+ 0 0+ 0 0 0 4+ 4+ 3+ 4+ 4+ 65 0+ 0 0 0 0 0+ 0 0+ 0+ 0 0 0 0 0 0+ 0+ 0+ 66 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0+ 0+ Oral fluids Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Pos Pos Pos Pos Pos 30% DDGS 76 4+ 4 + ------79 0+ 4+ 4+ 4+s ------78 0+ 0+ 3.5+ 4+ 4+ 4+ ------75 0 0 1+ 4+ 4+ 4+ 4+ ------77 0+ 0+ 1+ 4+ 4+ 4+ 4+ ------80 0+ 0 0+ 1+ 4+ 4+ 4+ ------71 0 0 0 0 0 0+ 0 0 0 0 0 0 0 0 0 0 0 72 0 0 0 0+ 0 0+ 0 0 0 0 0 0 0 0 0 0 0 73 0 0 0+ 0+ 0 0 0 0 0 0 0 0 0 0 0 0 0+ 74 0 0 0+ 0+ 0+ 0 0 0 0 0 0 0 0 0 0 0+ 0 Oral fluids Neg Neg Pos Neg Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg a EB107 = “B. hampsonii” clade II b Sham-inoculated diet 1 group and sham-inoculated diet 2 group had a maximum daily diarrhea score of 0.1 on days 11 and18 post-inoculation and 0.2 on day 14, respectively; never had a positive Brachyspira culture; and were euthanized at 21 days post inoculation. c A fecal score was determined for each pig daily based upon the following system: 0 if normal, 1 if soft but formed, 2 if unformed with semisolid consistency, and 3 if severely liquid to watery with an additional 0.5 point added each for the presence of discernible mucus and/or blood (max score = 4). d Distillers dried grains with solubles + Positive Brachyspira culture sPositive Salmonella culture at necropsy

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Table 5. Average daily gain by group Group ADGa SEM Sham-inoculated diet 1b 0.90 0.05 Sham-inoculated diet 2c 0.85 0.04 B. intermedia-inoculated diet 1 0.90 0.04 B. intermedia-inoculated diet 2 0.90 0.04 B. pilosicoli-inoculated diet 1 0.84 0.03 B. pilosicoli-inoculated diet 2 0.90 0.03 “B. hampsonii”-inoculated diet 1 0.60* 0.09 “B. hampsonii”-inoculated diet 2 0.62* 0.09 B. hyodysenteriae-inoculated diet 1 0.57* 0.06 B. hyodysenteriae-inoculated diet 2 0.47* 0.04 a Mean of Average Daily Gain (kg/day) b Diet with 0% DDGS c Diet with 30% DDGS * Significantly different from the sham-inoculated and weakly beta-hemolytic inoculated group regardless of diet (P < 0.0001, all analyses)

Gross pathology

Gross lesions observed in the B. hyodysenteriae and “B. hampsonii” groups are summarized in Table 6. When present, gross lesions in these groups were limited to the cecum and large intestine. Lesions consisted of variable degrees of fibrinous exudate; mucosal thickening, mucosal congestion, and hemorrhage; and luminal mucus. No gross lesions were observed in the sham-inoculated groups, B. intermedia-inoculated groups, or B. pilosicoli-inoculated diet 1 group. Two pigs in the B. pilosicoli-inoculated diet 2 group had mild multifocal mucofibrinous exudate present in the centripetal portion and apex of the spiral colon.

Histopathology

Histologic evaluation of the apex of the spiral colon is summarized in Table 7. At the apex of the spiral colon there was a significant increase in neutrophilic inflammation between each strongly beta-hemolytic-inoculated group and each sham-inoculated group or each weakly beta-hemolytic-inoculated group regardless of diet (P < 0.02, all analyses). There

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Table 6. Summary of gross lesions of “B. hampsonii”-inoculated and B. hyodysenteriae- inoculated groups Mucosal Fibrinous Exudate Severity Distribution Location Group Mild M/S MF Seg D Cecum Base CP Apex CF DC “B. hampsonii”- 6/10 1/10 4/10 3/10 0/10 5/10 4/10 5/10 2/10 1/10 0/10 inoculated diet 1a “B. hampsonii”- 4/10 0/10 2/10 2/10 0/10 2/10 2/10 3/10 3/10 1/10 0/10 inoculated diet 2b B. hyodysenteriae- 3/10 4/10 4/10 1/10 1/10 5/10 6/10 7/10 6/10 3/10 3/10 inoculated diet 1 B. hyodysenteriae- 1/10 9/10 1/10 5/10 4/10 9/10 10/10 9/10 7/10 2/10 1/10 inoculated diet 2

Luminal Mucus Severity Distribution Location Mild M/S MF Seg D Cecum Base CP Apex CF DC “B. hampsonii”- 1/10 8/10 0/10 2/10 7/10 2/10 7/10 9/10 8/10 7/10 7/10 inoculated diet 1 “B. hampsonii”- 2/10 5/10 1/10 1/10 5/10 4/10 5/10 7/10 6/10 6/10 4/10 inoculated diet 2 B. hyodysenteriae- 2/10 5/10 0/10 0/10 7/10 4/10 7/10 7/10 7/10 7/10 5/10 inoculated diet 1 B. hyodysenteriae- 0/10 10/10 0/10 0/10 10/10 6/10 9/10 10/10 10/10 10/10 9/10 inoculated diet 2

Hemorrhage Severity Distribution Location Mild M/S MF Seg D Cecum Base CP Apex CF DC “B. hampsonii”- 0/10 7/10 5/10 0/10 2/10 2/10 5/10 7/10 7/10 6/10 6/10 inoculated diet 1 “B. hampsonii”- 4/10 3/10 5/10 2/10 0/10 4/10 4/10 6/10 6/10 6/10 4/10 inoculated diet 2 B. hyodysenteriae- 4/10 3/10 5/10 1/10 1/10 3/10 4/10 6/10 7/10 4/10 3/10 inoculated diet 1 B. hyodysenteriae- 3/10 7/10 10/10 0/10 0/10 5/10 9/10 10/10 10/10 9/10 8/10 inoculated diet 2 M/S = Moderate or severe MF = Multifocal Seg = Segmental D = Diffuse CP = Centripetal CF = Centrifugal DC = Descending colon a Diet with 0% DDGS b Diet with 30% DDGS was no significant difference in neutrophilic inflammation between strongly beta-hemolytic- inoculated groups (P > 0.18, all analyses). Crypt depth was statistically different when comparing the B. hyodysenteriae-inoculated groups and the “B. hampsonii”-inoculated diet 1

106 group to the sham-inoculated groups as well as each weakly beta-hemolytic-inoculated groups (P < 0.02, all analyses). There was no significant difference in crypt depth between strongly beta-hemolytic-inoculated groups or weakly beta-hemolytic-inoculated groups and sham-inoculated control groups.

Table 7. Summary of histologic lesions in the apex of the spiral colon Neutrophilic Crypt Depth (µm)b Inflammationa Group Mean SEM Mean SEM Sham-inoculated diet 1 c 0.58 0.17 466.70 12.47 Sham-inoculated diet 2 d 1.10 1.17 506.70 24.78 B. intermedia-inoculated diet 1 0.46 0.24 478.90 18.52 B. intermedia-inoculated diet 2 0.44 0.18 469.70 12.95 B. pilosicoli-inoculated diet 1 0.12 0.03 496.50 14.34 B. pilosicoli-inoculated diet 2 0.34 0.11 518.00 21.29 “B. hampsonii”-inoculated diet 1 29.38* 5.44 848.90* 57.38 “B. hampsonii”-inoculated diet 2 19.34* 6.45 696.70 79.60 B. hyodysenteriae-inoculated diet 1 24.85* 7.62 763.90* 64.78 B. hyodysenteriae-inoculated diet 2 25.17* 3.74 799.00* 32.33 aNeutrophils were counted in ten 40X fields and the mean for each section of the spiral colon was determined bCrypt depth was determined by taking the mean of three measurements for each section of apex of the spiral colon c Diet with 0% DDGS d Diet with 30% DDGS *Significantly different from the sham-inoculated and weakly beta-hemolytic inoculated group regardless of diet (P < 0.02, all analyses)

Immunohistochemistry

Image analysis results of mucin immunohistochemistry are summarized in Figure 1 with representative photomicrographs of tissue sections used for image analysis displayed in

Figures 2, 3, and 4. There was a significant increase in MUC5AC expression between each of the strongly-beta hemolytic-inoculated groups in pigs that had histologic lesions suggestive of SD and the sham-inoculated diet 1 group (P < 0.01, all analyses); however, only the pigs in B. hyodysenteriae-inoculated diet 1 group and the “B. hampsonii”-inoculated diet 2 group had significantly more MUC5AC expression than the sham-inoculated diet 2 group (P < 0.04). In contrast, MUC2 expression was not significantly different between a majority of the groups with the exception of a significant decrease in MUC2 expression in

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pigs in the B. hyodysenteriae-inoculated diet 2 group when compared to either of the sham-

inoculated groups or the “B. hampsonii”-inoculated diet 1 group (P < 0.05). The location of

MUC2 did vary being present primarily in the goblet cells of the sham-inoculated group and

the crypt lumen of pigs with histologic lesions suggestive of SD. A Spearman’s rho revealed

a statistically significant relationship between the number of neutrophils and the pixels

representing positive staining of MUC5AC (ρ(50) = 0.6193, P < 0.0001).

Figure 1. Mean ± SEM of pixels representing positive immunolabeling of mucins following log transformation of data from the apex of the spiral colon of pigs with histologic lesions consistent with swine dysentery. Means without a common letter differ (P < 0.05). * P < 0.04.

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Figure 2. Spiral colon, apex, pig 1 (sham-inoculated diet 1): (a) Lack of MUC5AC expression in goblet cells. (b) Numerous MUC2 positive goblet cells. Figure 3. Spiral colon, apex, pig 100 (“B. hampsonii”-inoculated diet 1): (a) A moderate number of MUC5AC positive goblet cells. (b) Abundant MUC2 within crypt lumens. Figure 4. Spiral colon, apex, pig 86 (B. hyodysenteriae-inoculated diet 1): (a) Many MUC5AC positive goblet cells. (b) Abundant MUC2 within crypt lumens.

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Histochemistry

Image analysis results for mucin histochemistry are summarized in Figure 5 with representative photomicrographs of tissue sections used for analysis displayed in Figure 6.

There was a significant decrease in the amount of sulphomucins at the apex of the spiral colon in pigs with histologic lesions consistent with SD. This difference was significant between each strongly beta-hemolytic-inoculated group and the sham-inoculated groups regardless of diet (P < 0.005). In addition, the sham-inoculated diet 1 group had significantly less sulphomucins than the sham-inoculated diet 2 group (P < 0.03). There was a significant increase in sialomucins when comparing each strongly beta-hemolytic-inoculated group and the sham-inoculated group regardless of diet (P < 0.05) with the exception of “B. hampsonii” diet 2 group when compared with the sham-inoculated diet 1 group (P = 0.1555).

Figure 5. Mean ± SEM of pixels representing positive histochemical staining of mucins following log transformation of data from the apex of the spiral colon of pigs with histologic lesions consistent with swine dysentery. Means without a common letter differ (P < 0.05). ** P <0.005 and * P < 0.05.

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Figure 6. Spiral colon, apex; high-iron-diamine and Alcian blue (pH 2.5) staining to reveal the presence of sulphomucins (black) and sialomucins (blue). (a) Pig 6 (sham-inoculated diet 1): Abundant sulphomucins and absent sialomucins in goblet cells and crypt lumens. (b) Pig 12 (sham-inoculated diet 2): Abundant sulphomucins and absent sialomucins in goblet cells and crypt lumens. (c) Pig 70 (“B. hampsonii”-inoculated diet 1): Diminished presence of sulphomucins and increased sialomucins in goblet cells and crypt lumens. (d) Pig 76 (“B. hampsonii”-inoculated diet 2): Diminished presence of sulphomucins and increased sialomucins in goblet cells and crypt lumens. (e) Pig 88 (B. hyodysenteriae-inoculated diet 1): Diminished presence of sulphomucins and increased sialomucins in goblet cells and crypt lumens. (e) Pig 93 (B. hyodysenteriae- inoculated diet 2): Diminished presence of sulphomucins and increased sialomucins in goblet cells and crypt lumens.

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Discussion

A resurgence of swine dysentery has recently been observed in North American swine herds after a period in which the disease was nearly eliminated. The underlying reason(s) for this increased observance is poorly understood; however, coincident with the reemergence of Brachyspira-associated disease in pigs has been increased feeding of insoluble dietary fiber through the addition of DDGS in many swine diets. Currently, relatively little is known about the precise pathogenesis of SD; however, diet is considered to play a major role in disease expression.9 In a previous study, pigs fed a highly digestible diet of cooked rice developed an increased colonic content pH and decreased total volatile fatty acid concentrations and were protected from developing SD;35 however, this was not corroborated by a later study19 where feeding cooked rice again alkalinized colonic contents yet failed to protect pigs from infection with B. hyodysenteriae and development of SD. In the current study, the pH at the cecum and apex was significantly more alkaline in the sham- inoculated diet 2 group (30% DDGS) than the sham-inoculated diet 1 group (0% DDGS) yet this alkalization of the colonic contents was not associated with protection against the development of SD as not only did pigs in the diet 2 groups inoculated with strongly beta- hemolytic spirochetes develop SD but the average days post inoculation to the onset of clinical SD was significantly shorter. Furthermore, the days to first positive culture was also significantly shorter when comparing the B. hyodysenteriae-inoculated diet 2 group and B. hyodysenteriae-inoculated diet 1 group. Such incongruence concerning the impact of colonic content pH could be attributed to the multifactorial nature of SD in which the pH of the large intestinal content is a single component amongst many that influence the clinical expression of SD including mucin expression, mucolytic enzyme activity, and the total microbiota.

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The large intestine contains a dynamic microenvironment with tremendous interplay between microorganisms. The enteric microbiome can change rapidly in a diet-specific manner6 resulting in changes in goblet cell functions and in the composition of intestinal mucus through the production of bioactive factors by epithelial and lamina proprial inflammatory cells or direct stimulation of signaling cascades. Dietary fiber has been shown to increase viscosity and production of mucins.34 Specifically, insoluble fiber increases daily excretion of fucose, galactose, and mucins25 all of which are chemoattractant to B. hyodysenteriae16 and the last of which is chemoattractant to B. pilosicoli,26 suggesting a link between increased mucus production and the propensity for colonization by Brachyspira spp.

In the current study, the impact of increased insoluble dietary fiber through the feeding of

DDGS was significant when comparing the onset of dysentery in pigs inoculated with strongly beta-hemolytic Brachyspira spp. The observed effect was most significant between the B. hyodysenteriae-inoculated diet groups, but was also apparent in the “B. hampsonii”- inoculated groups and the lack of statistical significance with this species may reflect the total number of pigs that developed disease and limitations of statistical analysis on time to event data rather than an actual difference in disease pathophysiology. The feeding of increased insoluble dietary fiber did not have any significant observable effect on pigs inoculated with either B. pilosicoli or B. intermedia under the conditions of the current study.

The recent analysis of the B. hyodysenteriae genome has revealed multiple metabolic pathways and enzymes that could be utilized as a competitive advantage with the addition of dietary insoluble fiber such as DDGS.1 Fermentation of DDGS increases short-chain fatty acids15 which B. hyodysenteriae has the capacity to use as a major energy and carbon source.1 Additionally, insoluble fiber causes a shift from carbohydrate to protein

113 fermentation in the colon22 and B. hyodysenteriae has many amino acid and oligopeptide transporters that may enhance the utilization of amino acids as a major energy source.1

Furthermore, B. hyodysenteriae possesses key enzymes for gluconeogenesis as well as fifteen proteases, all of which suggests that a shift to protein fermentation in the colon associated with feeding of DDGS may provide multiple potential energy sources for the pathogenic

Brachyspira sp.1 Future genetic analyses of the genome of “B. hampsonii” are needed to determine if similar or distinctly different pathways and enzymes are present which may account for the observations in the present study.

Diet composition alone had no significant impact on ADG over the time course of the present study. A highly significant difference in ADG was observed between B. hyodysenteriae-inoculated or “B. hampsonii”-inoculated groups compared with sham- inoculated or weakly-beta hemolytic-inoculated groups. This highly significant reduction in

ADG is consistent with what is reported historically with SD and underscores the ongoing significance of this pathogen in worldwide pig production. No significant difference was observed when comparing the B. pilosicoli-inoculated or B. intermedia-inoculated groups to the sham-inoculated groups under the conditions of this study. This was unexpected given the historical association of B. pilosicoli with PCS and the high percentage of pigs that became culture positive and shed the organism for a prolonged period. These findings would suggest that the economic impact of these spirochetes may result from infections lasting longer than the conditions of this report or from infections coincident with other factors.

In swine, oral fluids can be used to detect both viral and bacterial pathogens including porcine reproductive and respiratory syndrome virus, swine influenza virus, and Mycoplasma

114 hyopneumoniae by PCR. Results of this investigation support the use of oral fluids for the detection of Brachyspira spp. by culture.

In the present study, multiple gross and two microscopic parameters were compared and revealed minimal differences in lesion characteristics, severity, or distribution between pigs infected with either B. hyodysenteriae or “B. hampsonii” clade II which is consistent with multiple previous reports2,33,41 suggesting that DDGS may increase the incidence but not necessarily the severity of SD. No significant gross or histologic lesions were noted in the pigs inoculated with weakly beta-hemolytic Brachyspira spp. or sham-inoculated pigs.

While a few pigs had positive Salmonella cultures at necropsy, most of these cultures were from tetrathionate broth enrichment of samples set up within a few hours of collection.

This low level of recovery and requirement for enrichment suggests these were likely subclinically infected pigs and this is further supported by the lack of lesions typical of salmonellosis in these pigs. Furthermore, the Salmonella positive pigs were fairly evenly distributed across inoculated groups which should further minimize any potential confounding effects of this minor coinfection on the parameters assessed.

In light of the abundant mucus typical of SD, the chemoattraction of B. hyodysenteriae to mucus, and the important role mucins play in innate mucosal defense, the histochemical and immunohistochemical characteristics of select mucins present in the goblet cells and crypt lumens at the apex of the spiral colon were evaluated. Statistically significant differential mucin expression was common when comparing strongly beta-hemolytic- inoculated pigs and controls including increased MUC5AC and decreased sulphomucin expression which is consistent with previous work.41

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To the authors’ knowledge, MUC2 expression in pigs with SD has not been previously evaluated. In the present study, no significant difference in the average number of pixels representing positive immunolabeling was observed between a majority of pigs with histologic lesions consistent with SD and sham-inoculated pigs regardless of diet; however, the location of MUC2 staining did differ. MUC2 was commonly present in crypt lumens of pigs with SD while the majority of MUC2 in sham-inoculated pigs was present in goblet cells. The significance of this finding in not known but does suggest techniques evaluating only the quantity of a mucin may fail to identify important aspects of disease pathophysiology. Additional investigations exploring mucin mRNA expression or mucus quality might be useful to further explore the role of mucins in SD.

The structural heterogeneity of mucin oligosaccharide chains impacts the biological and physiologic properties of mucins influencing the types of bacteria that can colonize the mucus bilayer through the differential availability of binding sites and nutrients.5 Enteric pathogens bypass the defensive role of mucus by using mucolytic enzymes including sulfatases, sialate O-acetylesterases, and sialidases; motility; and induction of mucus hypersecretion21 resulting in the quantitative and qualitative alteration in mucus layers with subsequent attachment and invasion of epithelial cells. Cleavage of each glycosidic linkage begins at the outer nonreducing end of each chain and requires the presence of its linkage- specific glycosidase.4 The principal source of these exoglycosidases is a subset of normal enteric bacteria which constitutively produce the requisite glycosidases as extracellular enzymes.4

Decreased expression of sulphomucins, as observed in this study, constitutes the first step in mucin degradation.31 As such, evidence of mucin degradation suggests a mechanism

116 by which pathogenic Brachyspira spp. may circumvent protective mucus layers to access the underlying epithelial cells. Sulphatase activity has been documented in the enteric genera

Bacteroides and Prevotella.30,38 Recent review of information publicly available in the NCBI database revealed that proteins identified as sulphatases and sialidases have been identified in

Brachyspira spp. including B. hyodysenteriae, “B. hampsonii”, B. pilosicoli, and B. intermedia; however, sialate O-acetylesterases were not reported. Yet, to the authors’ knowledge the expression and function of the mucolytic enzymes present in these

Brachyspira spp. has not been evaluated. Accordingly, syntrophic associations with mucin- degrading bacteria may be required in order to induce the decreased expression of these mucins as observed in the present study. Indeed, previous work in gnotobiotic pigs has demonstrated the requirement for additional bacteria as part of the pathogenesis of SD.12 The significant increase in sulphomucins observed in the sham-inoculated diet 2 group when compared to the sham-inoculated diet 1 group may have resulted in a microenvironment in which additional binding sites and/or nutrients that could be utilized by either strongly beta- hemolytic spirochetes or essential mucin-degrading bacteria were made available leading to the reduced days to onset of SD and days to first positive culture observed. Further investigation into these potential effects is warranted and may further elucidate factors involved in the pathogenesis of SD.

Sulphatase activity may also be directly impacted by feed composition due to a shift in the colonic microbiome and/or the pH of the intestinal contents.4 Recently, a study found that when pigs were fed cooked rice or cooked rice supplemented with either 10% potato starch or 20% wheat bran the colonic bacterial community lacked the terminal restriction fragments associated with the sulphatase-producing Prevotella group.20 When fed diets

117 similar to those shown to prevent SD, pigs had a higher proportion of Bifidobacterium thermacidophilum subsp. porcinum and Megasphaera elsdenii perhaps resulting in alterations in the intestinal environment.24 These changes may decrease the sulfphatase activity in the colon preventing quantitative and qualitative alteration in mucus layers and disease initiation. Identifying mucin glycoprotein binding sites used by commensal and pathogenic microbes with concurrent analysis of the effect of native and altered mucins on the proportion of protective/pathogenic microbes in the large intestine may contribute significantly to the management of SD. Additional studies exploring these potential relationships through the analysis of the colonic microbiota in pigs with and without SD may shed light on the specific host and microbial factors involved in the pathogenesis of this disease.

In summary, the results of the present study reveal that both the time to onset of mucohemorrhagic diarrhea and days to first positive culture in the B. hyodysenteriae- inoculated group were significantly reduced with the inclusion of 30% DDGS in the diets fed under the conditions of this report. Accordingly, dietary composition should be considered an important risk factor for the development of SD and dietary modification might also be an additional consideration in any effective disease elimination strategy for strongly beta- hemolytic Brachyspira spp. infections.

Acknowledgements

This work was supported by a grant from the Innovative Swine Industry Enhancement Grant

Program administered by the Iowa Attorney General’s Office. The funding source had no involvement in data collection, analysis and interpretation of data, writing the report, and decision to submit the article for publication. The authors wish to thank Hallie Warneke for

118 her help with the Brachyspira culture work and maintenance of the isolate collection at the

ISU VDL; Diane Gerjets, Jennifer Groeltz-Thrush, and Toni Christofferson for preparation of the histologic sections; and Marie Bockenstedt for her assistance with the necropsy procedures.

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19. Kirkwood RN, Huang SX, McFall M, Aherne FX. Dietary factors do not influence the clinical expression of swine dysentery. Swine Health Prod. 2000;8:73-76.

20. Leser TD, Lindecrona RH, Jensen TK, et al. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl Environ Microbiol. 2000;66:3290–3296.

21. Lidell ME, Moncada DM, Chadee K, Hansson GC. Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel. Proc Natl Acad Sci U S A. 2006;103:9298-9303.

22. Macfarlane S, Macfarlane GT. Regulation of short-chain fatty acid production. Proc Nutr Soc. 2003;62: 67-72.

23. Milner JA, Sellwood R. Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization. Infect Immun. 1994;62: 4095-4099.

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24. Mølbak L, Thomsen LE, Jensen TK, et al. Increased amount of Bifidobacterium thermacidophilum and Megasphaera elsdenii in the colonic microbiota of pigs fed a swine dysentery preventive diet containing chicory roots and sweet lupine. J Appl Microbiol. 2007;103:1853-67.

25. Montagne L, Piel C, Lalles JP. Effect of diet on mucin kinetics and composition: nutrition and health implications. Nutr Rev. 2004;62:105-114.

26. Naresh R, Hampson DJ. Attraction of Brachyspira pilosicoli to mucin. Microbiology. 2010;156:191-197.

27. NRC. Nutrient Requirements of Swine. 2012. Washington, D.C.: The National Academies Press.

28. Raja SB, Murali MR, Devaraj H, Devaraj SN. Differential expression of gastric MUC5AC in colonic epithelial cells: TFF3-wired IL-1β/Akt crosstalk-induced mucosal immune response against Shigella dysenteriae infection. J Cell Sci. 2012;125:703-713.

29. Ramsauer VP, Pino V, Farooq A, et al. Muc4-ErbB2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand for ErbB2. Mol Biol Cell. 2006;17:2931–2941.

30. Roberton AM, McKenzie C, Scharfe N, Stubbs LB. A glycosulphatase that removes sulphate from mucus glycoprotein. Biochem J. 1993;293:683-689.

31. Robinson CV, Elkins MR, Bialkowski KM, et al. Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients. J Med Microbiol. 2012;61:1644-1653.

32. Rohde J, Rothkamp A, Gerlach GF. Differentiation of porcine Brachyspira species by a novel nox PCR-based restriction fragment length polymorphism analysis. J Clin Microbio. 2002;4:2598-2600.

33. Rubin JE, Costa MO, Hill JE, et al. Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with "Brachyspira hampsonii" strain 30446. PLoS One. 2013;8:e57146.

34. Schmidt-Wittig U, Enss ML, Coenen M, et al. Response of rat colonic mucosa to a high fiber diet. Ann Nutr Metab. 1996;40:343-350.

35. Siba PM, Pethick DW, Hampson DJ. Pigs experimentally infected with Serpulina hyodysenteriae can be protected from developing swine dysentery by feeding them a highly digestible diet. Epidemiol Infect. 1996;116:207-216.

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36. Theodoropoulos G, Hicks SJ, Corfield AP, et al. The role of mucins in host-parasite interactions: Part II - helminth parasites. Trends Parasitol. 2001;17:130-135.

37. Trott DJ, Stanton TB, Jensen NS, et al. Serpulina pilosicoli sp. nov., the agent of porcine intestinal spirochetosis. Int J Syst Bacteriol. 1996;46:206-215.

38. Tsai HH, Hart CA, Rhodes JM. Production of mucin degrading sulphatase and glycosidases by Bacteroides thetaiotaomicron. Lett Appl Microbiol . 1991;13:97–101.

39. Urriola PE, Shurson GC, Stein HH. Digestiblity of dietary fiber in distillers coproducts fed to growing pigs. J Anim Sci. 2010;88:2373-2381.

40. Warneke HL, Kinyon JM, Bower LP, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid identification of Brachyspira species isolated from swine, including the newly described "Brachyspira hampsonii." J Vet Diagn Invest. 2014;26:635-639.

41. Wilberts BL, Arruda PH, Kinyon JM, et al. Comparison of lesion severity, distribution, and colonic mucin expression in pigs with acute swine dysentery following oral inoculation with "Brachyspira hampsonii" or Brachyspira hyodysenteriae. Vet Pathol. 2014;Online ahead of print: doi:10.1177/0300985813516646.

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CHAPTER 5

GENERAL CONCLUSIONS

Conclusions

Although B. hyodysenteriae was once considered the sole etiologic agent of swine dysentery (SD),7 the studies in this report as well as other investigations have demonstrated that the characteristic clinical disease and lesions of SD can be reliably identified in field cases in which “B. hampsonii” was isolated5,11 and reproduced by experimental oral inoculation with “B. hampsonii.”3,15 Specifically, the time to onset of mucohemorrhagic diarrhea, duration of spirochete shedding, microscopic and gross changes, colonic mucin expression were similar in pigs infected with either “B. hampsonii” or B. hyodysenteriae. A cytokine analysis of the colonic mucosa from pigs with SD has not yet been published; however, it would seem that proinflammatory cytokines likely induce differential mucin expression in acute SD resulting in increased expression of MUC5AC. While the inner mucus layer is in health impermeable to bacteria, decreased expression of sulphomucins is the first step in mucin breakdown resulting in a mucus bilayer defect.14 As such, evidence of mucin degradation resulting in an inner mucus layer defect suggests a mechanism by which motile, strongly beta-hemolytic Brachyspira spp. access the underlying epithelial cells.

Definitive diagnosis of SD associated with infection by either B. hyodysenteriae or

“B. hampsonii” commonly includes culture and PCR.7 The advent of the oligonucleotide probes, Hyo12102 and Hamp12104 for the detection of B. hyodysenteriae and "B. hampsonii", respectively, provided an additional diagnostic technique that could be applied to either formalin-fixed tissue or feces. Similar levels of detection were found when comparing fluorescent in situ hybridization (FISH) on formalin-fixed feces and qPCR on

123 fresh feces. Fluorescent in situ hybridization detected both target Brachyspira spp. on formalin-fixed feces from pen level samples from infected groups of pigs with SD as well as from infected pigs with clinical dysentery at necropsy. Accordingly, pigs with SD may be identified in a timelier manner using either qPCR or FISH; although, speciation by PCR from culture is still a more definitive assay. Culture detected the target spirochetes at least two log-dilutions less when compared to FISH and qPCR 48 hours after sample preparation making it the most sensitive method; however, there was a substantial reduction in the number of culturable spirochetes following 48 hours of storage of fecal dilutions at 4°C.

While culture is the best assay currently available for surveillance testing and identification of carrier animals, prompt delivery of diagnostic samples to the laboratory is imperative to decrease the likelihood of false negative cultures.

A resurgence of SD has recently been observed in North American swine herds.5,11

Coincident with the reemergence of SD has been increased feeding of insoluble dietary fiber through the addition of DDGS in many swine diets. Diet has long been recognized to impact clinical disease expression of SD.7 This was further substantiated by the impact DDGS had on the clinical expression of SD associated with infection by either B. hyodysenteriae or “B. hampsonii.” Pigs receiving 30% DDGS shed on average one day prior to and developed swine dysentery nearly twice as fast as pigs receiving 0% DDGS. Yet, statistical significance was only observed when comparing the B. hyodysenteriae-inoculated groups in time to event data (days to SD, days to positive culture). The lack of statistical significance with “B. hampsonii” may reflect the total number of pigs that developed disease rather than an actual difference in disease pathophysiology. The analysis of the B. hyodysenteriae genome1 revealed multiple metabolic pathways and enzymes that could be utilized as a competitive

124 advantage by pathogenic Brachyspira sp. with the addition of DDGS to swine diets.

Accordingly, disease elimination strategy for strongly beta-hemolytic Brachyspira spp. infections should include dietary modification with a reduction in insoluble fiber.

Recommendations for Future Research

Currently, there is little published data regarding the pathogenesis of SD associated with infection with either B. hyodysenteriae or “B. hampsonii” providing a plethora of hypotheses yet untested. The most pressing need for future research on SD is in the area of whole genome sequencing and analysis of these species which will provide more complete information concerning pathogenicity with the potential of developing new targeted diagnostic assays. Whole genome sequencing may also help elucidate important metabolic and enzyme synthesis pathways. Specifically, sequencing of additional species and strains is needed to better characterize the presence of sulphatase genes in Brachyspira spp. and activity of the encoded proteins.

The interplay between the resident colonic microbiota and diet impacts bacterial colonization, clinical expression of SD, and colonic mucin expression. Identifying mucin glycoprotein binding sites used by both commensal and pathogenic microbes with simultaneous investigation of differential mucin expression and the proportion of protective/pathogenic microbes may contribute significantly to the management of SD through elucidating specific host and microbial factors involved in the pathogenesis of this disease. Mucin synthesis and secretion may be altered due to numerous factors including bacterial degradation, the microbiota, and/or bacterial products.13 Currently, the pathways involved in differential mucin expression, mucin mRNA expression, or mucus quality in SD are not well characterized. Specifically, a cytokine analysis of the colonic mucosa may

125 reveal likely mechanisms impacting mucin biosynthetic responses in SD when juxtaposed to the literature on pathogens, signaling pathways, and mucin expression.

While a majority of the commonly available diagnostic assays are able to detect pigs with clinical SD, there is often a significant delay in diagnosis and informed treatment implementation. Recently, loop-mediated isothermal amplification assays have been developed for the diagnosis of multiple veterinary pathogens using clinical samples providing a field assay that is simple, accurate, fast, and economic.6,8,10,12,16,17,18 The development of LAMP on a laser etched indium tin oxide (ITO) electrode-based multiplex microfluidic chip, microfluidic multiplex electrochemical LAMP (μME-LAMP) system, now allows for the simultaneous detection of multiple pathogens providing a streamlined, cost effective, and time efficient assay.9,19 The use of this system in the field could differentiate between the three most common bacterial causes of grow-finish colitis allowing for rapid and informed treatment strategies.

REFERENCES

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4. Burrough ER, Wilberts BL, Bower LP, et al. Fluorescent in situ hybridization for detection of "Brachyspira hampsonii" in porcine colonic tissues. J Vet Diagn Invest. 2013;25:407-412.

5. Clothier KA, Kinyon JM, Frana TS, et al. Species characterization and minimum inhibitory concentration patterns of Brachyspira species isolates from swine with clinical disease. J Vet Diagn Invest. 2011;23:1140-1145.

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6. Du GM, Liu MJ, Wu YZ, et al. Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis. Clin Lab. 2013;59:1363-1371.

7. Hampson DJ, Fellström C, Thomson JR. Swine dysentery. In: Straw BE, Zimmerman JJ, D'Allaire S, Taylor DJ, eds. Diseases of swine. 9th ed. Ames, IA: Blackwell Publishing; 2006:785 - 805.

8. Kinoshita Y, Niwa H, Katayama Y. Development of a Loop-Mediated Isothermal Amplification Method for Detecting Streptococcus equi subsp. zooepidemicus and Analysis of Its Use with Three Simple Methods of Extracting DNA from Equine Respiratory Tract Specimens. J Vet Med Sci. 2014. [Epub ahead of print]

9. Luo J, Fang X, Ye D, et al. A real-time microfluidic multiplex electrochemical loop- mediated isothermal amplification chip for differentiating bacteria. Biosens Bioelectron. 2014;60:84-91.

10. Nimesh M, Joon D, Varma-Basil M, Saluja D. Development and clinical evaluation of sdaA loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis with an approach to prevent carryover contamination. J Clin Microbiol. 2014;52:2662-4.

11. Patterson AH, Rubin JE, Fernando C, et al. Fecal shedding of Brachyspira spp. on a farrow-to-finish swine farm with a clinical history of "Brachyspira hampsonii"- associated colitis. BMC Vet Res. 2013;9:137.

12. Pawar SS, Meshram CD, Singh NK, et al. Rapid detection of bovine herpesvirus 1 in bovine semen by loop-mediated isothermal amplification (LAMP) assay. Arch Virol. 2014;159:641-648.

13. Pearson JP, Brownlee IA. The interaction of large bowel microflora with the colonic mucus barrier. Int J Inflam. 2010;2010:321426. doi: 10.4061/2010/321426.

14. Robinson CV, Elkins MR, Bialkowski KM, et al. Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients. J Med Microbiol. 2012;61:1644-1653.

15. Rubin JE, Costa MO, Hill JE, et al. Reproduction of mucohaemorrhagic diarrhea and colitis indistinguishable from swine dysentery following experimental inoculation with "Brachyspira hampsonii" strain 30446. PLoS One. 2013;8:e57146.

16. Soli KW, Kas M, Maure T, et al. Evaluation of colorimetric detection methods for Shigella, Salmonella, and Vibrio cholerae by loop-mediated isothermal amplification. Diagn Microbiol Infect Dis. 2013;77:321-323.

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17. Wang F, Yang Q, Qu Y, et al. Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce. Appl Environ Microbiol. 2014;80:2516-2525.

18. Yamazaki W. Sensitive and rapid detection of Campylobacter jejuni and Campylobacter coli using loop-mediated isothermal amplification. Methods Mol Biol. 2013;943:267- 277.

19. Zhou QJ, Wang L, Chen J, et al. Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals. J Microbiol Methods. 2014;104:26-35.

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APPENDIX

PERMISSIONS

Permission to reproduce in a dissertation 2 messages

Bailey Wilberts Wed, Aug 27, 2014 at 11:43 AM To: [email protected]

Dear Ms. Voyce,

I am preparing the dissertation for my PhD and would like permission to include a manuscript that has been electronically published ahead of print in Veterinary Pathology for which I am the primary author. From the instructions to authors, I understand that I retain the copyright, but that SAGE has exclusive publication rights. I contacted Jill Findlay, and she suggested I contact you; however, if there is someone else that I should contact at SAGE, please let me know and I will do that as well. The citation for the article is Vet Pathol epub ahead of print February 27, 2014; doi: 10.1177/0300985813516646. I look forward to your reply.

Thank you,

Bailey Wilberts, DVM Adjunct Instructor Department of Veterinary Pathology 2740 Veterinary Medicine Iowa State University Ames, IA 50011

Voyce, Kaitlyn Thu, Aug 28, 2014 at 7:35 PM To: Bailey Wilberts Hi Bailey,

You are able to use version 2 of your article (before typesetting and copyediting, not the final PDF version) in future works such as a thesis or a book as long as the article is properly cited. If you need written permissions, you can visit the RightsLink page for your article (the link is below) and it will provide you with the permission needed.

This should be the direct link to your RightsLink page: https://s100.copyright.com/AppDispatchServlet?publisherName=sage&publication=J645&t itle=Comparison%20of%20Lesion%20Severity%2C%20Distribution%2C%20and%20Colonic% 20Mucin%20Expression%20in%20Pigs%20With%20Acute%20Swine%20Dysentery%20Follow ing%20Oral%20Inoculation%20With%20%E2%80%9C%3ABrachyspira%20hampsonii%3A%E 2%80%9D%20or%20%3ABrachyspira%20hyodysenteriae%3A%0A%3A&publicationDate=02

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%2F27%2F2014&author=B.%20L.%20Wilberts%2C%20P.%20H.%20Arruda%2C%20J.%20M. %20Kinyon%2C%20D.%20M.%20Madson%2C%20T.%20S.%20Frana%2C%20E.%20R.%20B urrough&startPage=&contentID=10.1177%2F0300985813516646&orderBeanReset=true&public ationType=STM&trimSize=8%203%2F8%20x%2010%207%2F8©right=American%20Coll ege%20of%20Veterinary%20Pathologists&isn=0300- 9858&endPage=&volumeNum=&issueNum=&article_permission=yes

Please let me know if you have any questions.

Thank you!

Kaitlyn