Morphologic and Molecular Pathogenesis Study of Condemned Kidneys in Swine From

UNIVERSITY OF CALGARY

Morphologic and molecular pathogenesis study of condemned kidneys in swine from

Alberta

Claudia Benavente

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF MICROBIOLOGY AND INFECTIOUS DISEASES

CALGARY, ALBERTA

DECEMBER, 2010

395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-75207-4

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Abstract

Fifty condemned and 10 normal kidneys were collected from slaughtered pigs in southern Alberta and tested for pathogens as etiological agents of interstitial nephritis. A 75% of the samples were positive for bacterial isolation; bacteria caused an unspecific immune response in the host tested by western blot. In 50% of the tissues, porcine parvovirus was detected by PCR; 17.5% of samples were porcine circovirus type 2 positive; and there was no evidence of porcine reproductive and respiratory syndrome virus infection. Fungi and leptospires were not detected by histochemistry and/or immunohistochemistry. The majority of the condemned kidneys (76%) showed subacute to chronic interstitial nephritis; a 16% glomerulonephritis; and 8% had minimal histological changes. In contrast, normal kidneys (70%) were histologically normal. Taken together, these data suggest that the chronicity of the lesions did not determine the etiological agent, and manipulation of these kidneys may not represent a high human health risk.

ii ACKNOWLEDGEMENTS

First and foremost, my sincere thanks are due to Dr. Carmen Fuentealba. It is difficult to find words to express my deep gratitude to her. A deep appreciation to Dr. Glen Armstrong, he has contributed in many ways toward the completion of this study, through his support, encouragement and advice. I am grateful for the guidance of Dr. Rebakah De Vinney, who provided helpful suggestions during the course of this project. I thank Professor Oscar Illanes for his valuable discussions and providing helpful critiques in the review of this manuscript. My thanks also go to Dr. Markus Czub and the virology laboratory people for the training, use of equipment, and scientific discussions which were an integral part of my graduate experience. Gratitude is expressed for assistance provided by Dr. Anne Muckle and Dr. Carmencita Yason from the Atlantic Veterinary College, UPEI. The technical assistance needs special mention: Maureen Bukhari, Ramona Taylor, Barbara Waddell, Jan Giles, and Lorraine Lund is greatly appreciated, they helped me in many ways, for which I am thankful to them. I wish to thank Wendy Hutchins for the skills learned in the Biotechnology Training Centre at the University of Calgary. I also express my gratitude to Lisa Ashton of Canadian Food Inspection Agency for her special contribution in the collection of samples. I gratefully acknowledge the financial support provided by UCVM Studentship- Fellowship Award, UCVM Research Fellowship Department of Ecosystem and Public Health, and UCVM C. Fuentealba Start-up Grant from the University of Calgary. Finally, I am indebted to my family for always providing patience, love and words of encouragement during this challenging process.

iii DEDICATION

To my family,

my husband Eduardo and children Sebastian, Macarena and Rodrigo.

iv TABLE OF CONTENTS

Abstract ...... ii Acknowledgements ...... iii Dedication ...... iv Table of Contents ...... v List of Tables ...... vii List of Figures and Illustrations ...... viii List of Symbols, Abbreviations and Nomenclature ...... x

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Nephritis in swine ...... 2 1.1.1 Classification of nephritis ...... 2 1.1.2 Causes of nephritis ...... 3 1.2 Characteristics of emergent/re-emergent pathogens Actinobacillus suis and Actinobacillus equuli ...... 4

CHAPTER TWO: MATERIALS AND METHODS ...... 12 2.1 Study design ...... 12 2.2 Sample collection ...... 12 2.3 Classification of gross lesions in the kidneys ...... 13 2.4 Histology ...... 13 2.4.1 Classification of microscopic lesions in the kidneys ...... 14 2.5 Histochemistry ...... 14 2.6 Bacteriology ...... 16 2.7 Virology [Polymerase Chain Reaction (PCR)] ...... 17 2.8 Western blot ...... 21 2.9 Statistical Analyses ...... 23

CHAPTER THREE: RESULTS ...... 25 3.1 Classification of gross lesions in the kidneys ...... 25 3.2 Classification of microscopic lesions in the kidneys ...... 25 3.2.1 Tubulointerstitial nephritis ...... 29 3.2.2 Glomerulonephritis ...... 30 3.3 Histochemistry ...... 31 3.4 Bacteriology ...... 40 3.5 Polymerase Chain Reaction for PCV-2, PPV, and PRRS viruses ...... 41 3.6 Western blot ...... 42

CHAPTER FOUR: DISCUSSION ...... 55 Conclusions ...... 69

REFERENCES ...... 72

1. APPENDIX 1 ...... 86

2. APPENDIX 2 ...... 88

v 3. APPENDIX 3 ...... 89

4. APPENDIX 4 ...... 90

5. APPENDIX 5 ...... 91

6. APPENDIX 6 ...... 92

7. APPENDIX 7 ...... 94

LIST OF TABLES

Table 1 Bacterial growth conditions in the incubator with and without CO2 ...... 24

Table 2 Relationship between macroscopic and microscopic changes by categorization in kidney's samples of 60 market hogs from Southern Alberta...... 48

Table 3 Distribution of cases according to gross grade (0-3) and microscopic classification (normal-severe) of changes observed in swine kidneys...... 48

Table 4 Relationship between bacteria isolated from kidney's lesions and the categorization of the gross lesions in condemned and control kidneys from trial 1...... 49

Table 5 Relationship between bacterial isolation and morphological changes in the kidney's lesions from pigs of trial 1...... 50

Table 6 Relationship between bacteria isolated from 20 condemned kidneys from trial 2 and the gross classification of the lesions...... 51

Table 7 Relationship detected between presence of PCV-2 and PPV nucleotides in tissue, bacteria isolation from lesions, histological changes and diagnosis from samples of trial 1...... 52

Table 8 Immune response of the host to the bacteria isolated from the respective kidney's lesion from trial 2...... 53

Table 9 Cross reaction of sera and bacteria isolated from renal tissue of 11 pigs with nephritis...... 54

vii LIST OF FIGURES AND ILLUSTRATIONS

Figure 1 Grade 0. Normal kidney, control ...... 26

Figure 2 Grade 1. Presence of less than 10 round grey-white foci measuring 2-5 mm are scattered throughout the capsular surface of the kidney...... 26

Figure 3 Grade 2. Numerous (more than 10) grey-foci measuring between 2 to 5 mm and one focus of pale tan discoloration measuring 8 mm in the largest dimension...... 27

Figure 4 Grade 3. Multifocal to coalescing areas of pale discoloration measuring over 1 cm in dimension are scattered throughout the renal surface...... 27

Figure 5 Grade 3. Multifocal grey-white foci throughout the renal cortical surface...... 28

Figure 6 Grade 3. Diffuse grey-white foci and multifocal petechias throughout the surface of the kidney...... 28

Figure 7 Interstitial inflammatory cell infiltration composed primarily of lymphocytes and plasma cells. H&E x20 Case 4...... 32

Figure 8 Interstitial fibrosis and dilatation of renal tubules (arrow). H&E x10 Case 14...... 32

Figure 9 Mineralization of degenerated and necrotic renal tubular epithelium. H&E x40 Case 56...... 33

Figure 10 Tubular regeneration evidenced by the presence of mitosis (arrows) and enlarged tubular epithelial cells. H&E x 60 Case 42...... 33

Figure 11 Inflammatory cell infiltration extends radially from the medulla towards the cortex. H&E x2 Case 48...... 34

Figure 12 Follicular lymphoid formation within the cortex and corticomedullary junction. H&E x2 Case 5...... 34

Figure 13 Numerous syncytial giant-cells (arrows) admixed with lymphocytes, plasma cells and eosinophils in the renal cortex. H&E x40 Case 43...... 35

Figure 14 Syncytial cell within the lumen of a renal tubule. H&E x60 Case 12...... 35

Figure 15 Proliferative glomerulonephritis evidenced by increased cellularity within the glomerular tuft. H&E x40 Case 13...... 36

Figure 16 Glomerulitis characterized by fibrinoid necrosis and presence of karyorrhectic debris within the glomerulus. H&E x40 Case 12...... 36

viii Figure 17 Abundant eosinophilic proteinaceous material within Bowman's space and markedly dilated renal tubules. H&E x10 Case 51...... 37

Figure 18 Membranoproliferative glomerulonephritis with prominent periglomerular fibrosis (arrow). H&E x20 Case 51...... 37

Figure 19 Glomerulosclerosis characterized by shrinkage of the glomerulus, thickening of basement membranes and collagen deposition. H&E x40 Case 51. ... 38

Figure 20 Hyaline droplets characterized by numerous eosinophilic globular material within the tubular epithelium. H&E x40 Case 51...... 38

Figure 21 Fibrinoid necrosis (arrow) and inflammatory cell infiltration in the wall of a middle sized renal artery. H&E x10 Case 12...... 39

Figure 22 Chronic vasculitis with complete obliteration of the vascular lumen. H&E x10 Case 56...... 39

Figure 23 Serum from 6 different pigs and the 12 different bacteria isolated...... 44

Figure 24 Serum from 5 different pigs and 12 different bacteria isolated ...... 45

Figure 25 Detection of PCV-2 specific DNA extracted from frozen renal samples from trial 1. The fragment amplified was 629 bp. Lane 1, middle lane and end lane are 1-Kb DNA ladder. Samples 12 and 14 were positive; + show the lane with positive control...... 46

Figure 26 Detection of PCV-2 specific DNA extracted from frozen renal samples from trial 2. The fragment amplified was 629 bp. Lane 1, middle lane and end lane are 1-Kb DNA ladder. Samples 42, 43, and 46 were positive; + show the lane with positive control...... 47

ix LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE

Symbol Definition

UCVM University of Calgary, Veterinary Medicine PCV-2 Porcine circovirus type 2 PPV Porcine parvovirus PRRSV Porcine reproductive and respiratory syndrome virus H&E Haematoxylin and eosin stain RTX Repeats in the structural toxin CPS Capsular polysaccharides LPS Lipopolysaccharides PCR Polymerase chain reaction AVC Atlantic Veterinary College UPEI University of Prince Edward Island PAS Periodic acid-Schiff stain GMS Grocott’s Methenamine Silver stain MALDI-TOF Matrix-assisted laser desorption/ionization- time of flight MS Mass spectrometry ID Identification API Analytab Products Identification DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylenediaminetetraacetic acid ORF Open reading frame LB Lysogeny broth SDS Sodium dodecyl sulphate PAGE Polyacrylamide gel electrophoresis PVDF Polyvinylidene fluoride

x TBS Tris Buffered Saline IgG, IgM Immunoglobulin G, Immunoglobulin M ECL Enhanced chemiluminescent ELISA Enzyme-linked immunosorbent assay MAT Microscopic agglutination test PMWS Post-weaning multisystemic wasting syndrome PDNS Porcine dermatitis and nephropathy syndrome

xi 1

Chapter One: Introduction

Canada has been one of the world’s leading exporters of pork for many years. Data from July 2010 indicate that the Province of Alberta is the fourth largest producer of hogs in Canada behind Quebec, Ontario and Manitoba with 3,925.0; 2,799.2; 2,605.0 and 1,495.0 thousand heads, respectively (www.statcan.gc.ca). Also, pork is the fourth most valuable agriculture commodity in the Alberta economy behind beef, wheat and canola (www.albertapork.com). Maintaining a healthy herd and reducing economic losses due to diseases or condemned tissues during processing is of crucial importance, mainly when it involves food safety issues and potential public health consequences (9). There have been sporadic verbal reports of a condition tentatively named “septic embolic nephritis”, which has been detected relatively frequently in sows and market hogs. In 2007, a veterinarian employed by the United State Department of Agriculture contacted the College of Veterinary Medicine, University of Calgary, requesting information about these lesions in condemned kidneys in sows originating from Canada. The perception of a questionable health status of pigs grown in Canada can have serious consequences to the Alberta pork industry. Septic embolic nephritis has been detected in sows and market hogs in Alberta (Dr. Jan Bystrom, personal communication), as well as in Manitoba (Dr. Marc Swendrowski, personal communication). The pattern of the inflammatory process in this condition is unique and resembles Actinobacillus equuli infection in horses instead of common swine pathogens such as Actinobacillus suis. .A. suis traditionally has been associated with polyserositis characterized by fibrinous inflammation with a predilection for causing pericarditis, pneumonia, arthritis and very rarely septicemia. Septicemia is of importance because of the potential of bacterial emboli to lodge in small capillaries and cause further lesions such as embolic nephritis or glomerulitis. Although there are previous reports of A. equuli infections in pigs, few have originated from North America (103). The morphologic description of a case reported in 2008 included lesions consistent with a diagnosis of embolic nephritis (103).

1.1 Nephritis in swine

Renal disease which encompasses any deviation from normal renal structure or function is usually subclinical (78). Since by definition a subclinical condition is characterized by absence of clear clinical signs, disease in affected animals is not detected and treatment is not implemented. Once hogs reach market weight, they are sent to the abattoir and if lesions are detected by food inspectors the affected organs are condemned. Although the pathogenesis and etiology of renal diseases involve diverse modes and patterns of infection, all inflammatory lesions detected at the slaughter plants are classified under the broad category of “nephritis”, often called “white-spotted kidneys” (30). However, due to the anatomical characteristics of the urinary system, infections can reach the kidney, cause injury and chronically remain in this vital organ. The blood supply can provide a portal of hematogenous entry for infectious organisms which, in the case of kidneys, leads to arterial or glomerular localization (91). Specifically, the renal system is exposed to injurious agents by various routes, including: a) ascending infections, which are most common in females by an extension from lower urinary tract infections due to contamination from gastrointestinal contents, or secondary to infections of the genital tract such as in cases of pyometra b) hematogenous, and c) exposure to preformed or locally metabolized substances that are excreted through the renal tubular epithelium. 1.1.1 Classification of nephritis

Diseases of the kidney are complex and to facilitate their study the traditional approach is to divide them according to the location of injury within its four basic morphologic locations: glomeruli, blood vessels, tubules and interstitium. However, due to the anatomic and functional interdependence of the components of the kidney it is common to find that damage to one structure almost always results in secondary damage to others (3). The main general types of nephritis are glomerulonephritis and interstitial nephritis. Primary glomerular damage often occurs as a result of deposition of immune complexes, entrapment of bacterial emboli, or direct viral or bacterial infection of glomerular structures.

Hematogenous infections localize within blood vessels and are classified as septic and non-septic. Embolic nephritis is the main form of septic hematogenous spread (91). 1.1.2 Causes of nephritis

Suppurative glomerulitis, also known as embolic nephritis, is the result of bacteremia in which bacteria lodge in random glomeruli, and to a lesser extent within interstitial capillaries, and cause the formation of multiple foci of inflammation also known as microabcesses throughout the renal cortex. A specific example of embolic nephritis is Actinobacillosis of foals caused by Actinobacillus equuli. Embolic nephritis also occurs in bacteremias of pigs infected with Erysipelothrix rhusiopathiae, a zoonotic pathogen. Grossly, multifocal random, raised, tan pinpoint foci are seen beneath the renal capsule and on the cut surface throughout the renal cortex. Microscopically, glomerular capillaries contain numerous bacterial colonies intermixed with necrotic debris that often obliterate the glomerulus. Interstitial nephritis refers to inflammation mounted against the veins, arteries, lymphatics or connective tissue. The process may be of infectious or non-infectious origin and according to the duration it is further classified as acute, subacute or chronic. Interstitial nephritis is traditionally associated with inflammatory cell infiltration composed of lymphocytes and plasma cells (91). Although lesions observed in “white- spotted kidney” can be caused by a variety of bacterial and viral infections (31), an association between interstitial nephritis and leptospirosis has been described (9, 52, 76, 78). Leptospirosis is a zoonotic infectious disease caused by various serovars of Leptospira interrogans sp., which affect a large number of wild and domestic species. The incidence of leptospirosis has decreased after the widespread use of vaccines; however, kidney condemnations are still common suggesting that additional organisms may be involved in the pathogenesis of interstitial nephritis (30). Multifocal interstitial nephritis has been associated to some bacterial infections other than leptospires. Corynebacterium sp., Escherichia coli, Staphylococcus sp., Streptococcus sp., S. dysgalactiae equisimilis, S. suis., and S. salivarius have been isolated from kidneys with interstitial nephritis (76).

There are few studies on the morphologic and etiologic characteristics of “white spotted kidney”, including cases in pigs suffering from wasting at slaughter. These pigs show decreased growth rate and have concomitant chronic diseases. It is important to elucidate the specific origin of these lesions as the problem causes financial losses and poses a health risk to abattoir workers, and the consumers (9). The potential impact that sub-clinical lesions may have on animal welfare and the economic cost of renal lesions in general remain unknown. In addition to bacteria, many emergent viral diseases have tropism for renal tissue (30, 48), including porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine adenovirus (30, 92). A condition in pigs characterized by a systemic necrotizing vasculitis with marked cutaneous tropism has been reported in several countries (44). The disease, originally called porcine dermatitis and nephropathy syndrome, is observed in weaned and feeder pigs and less commonly in breeding animals (124). Reported renal lesions include multifocal non-suppurative interstitial nephritis, exudative glomerulonephritis, and presence of glomerular hyaline thrombi. There is strong evidence that PRRS virus may be involved in the pathogenesis of this syndrome, since (a) its appearance coincided with the emergence of PRRSV, (b) characteristic lesions were seen in specific-pathogen free pigs infected with PRRSV (25), and (c) PRRSV antigens were detected by immunohistochemistry in macrophages surrounding affected vessels in skin and kidney in both acute and chronic spontaneous cases (124). 1.2 Characteristics of emergent/re-emergent pathogens Actinobacillus suis and Actinobacillus equuli

Actinobacillus suis has emerged as a new threat to swine health (69, 139). Under conditions that are poorly understood Actinobacillus suis can cause disease in the colonized animal or be spread to other animals of a different age or health status (82, 139). Although little is known about the complexity of the normal upper respiratory tract microflora of swine, members of the family Pasteurellaceae and Gram positive organisms such as Streptococcus sp., and Staphylococcus aureus have been reported. Actinobacillus suis is an early colonizer of the upper respiratory tract of swine (96).

Nasopharynx and palatine tonsils are the main colonization sites (95). In a study of 50 swine herds, only 6% of them were found to be free of both A. pleuropneumoniae and A. suis, as evidenced by presence of these organisms in samples of tonsils and nostrils from weanling pigs (70). Actinobacillus suis infection can take place via the aerosol route, close contact, or breaks in the skin (71, 111). The systemic spread of A. suis involves the development of septic emboli which will reach various organs, including the kidney, and form microcolonies surrounded by areas of hemorrhage, necrosis and inflammation (96). The genus Actinobacillus are mostly commensals or pathogens of animals, but have also been described as a cause of zoonotic infection. For example, Actinobacillus suis was isolated from a wound of a farmer after a pig bite (33), and from a bone biopsy from an injured arm 3 months after a horse bite (99). A. equuli was isolated from a butcher with septicemia due to a cut in his thumb three days earlier (5). Actinobacillus suis or A. suis-like bacteria have been isolated sporadically from other species, including a Canada goose with conjunctivitis (72), an alpaca with endocarditis, embolic suppurative nephritis, pulmonary abscessation and interphalangeal arthritis (45), a cat (27), neonatal calves with septicaemia (28), a buffalo with multiple abscesses (122), a colt with ocular and nasal purulent discharge (43), horses with abscesses, respiratory and genital infections (19, 20, 50), and foals with septicaemia (90). Mice can be infected with A. suis and subsequently develop pneumonia and bacteremia comparable to that seen in pigs, suggesting that mice may be used as a model for studying infection in swine (94). Actinobacillus suis has a wide geographical distribution. Cases have been reported from United States (26, 72, 90), Canada (68, 82, 93, 111), Great Britain (43), New Zealand (19, 45), India (122), Australia (99, 135), Hungary and Croatia (77). Actinobacillus suis, a member of the family of Pasteurellaceae, genus Actinobacillus, is a small, gram-negative, oval to rod-shaped, capnophilic, non -mucoid, non-motile, nonspore producer, coccobacillary organism with short chains of bacillary and filamentous forms. India ink-crystal violet stain revealed that the organism is encapsulated (26, 28, 43, 68, 94, 121). Actinobacillus suis is catalase and oxidase positive, can grow on Mac Conkey’s agar, and is nitrate and urease positive but indole

6 negative. This microorganism produces hemolysis of 5% sheep blood agar and hydrolysis of esculin. Mannitol is not fermented by A. Suis (27, 28, 77, 122, 137, 139). These abilities can differentiate A. suis from other related bacteria as Actinobacillus pleuropneumonia. Actinobacillus suis is not easy to routinely diagnose (83), as it can be isolated along with other bacteria, and may be present in chronic cases of arthritis (139). This organism is considered an opportunistic pathogen of both swine and horses (6, 19, 82, 103), and it can cause fatal septicemia in young pigs (68, 69, 111), and pneumonia, endocarditis, polyarthritis and suppurative lesions in the kidney. Actinobacillus suis infection may also cause lesions resembling erysipelas in mature sows (82, 112). A. suis can be carried in the vagina of healthy sows and has the capacity to cause metritis, abortion and low parity (77). The pathogenicity of the A.suis disease is not well understood. Actinobacillus suis has genes that encodes pore-forming protein toxins belonging to the RTX (repeats in the structural toxin) that are very similar to ApxI and ApxII of Actinobacillus pleuropneumoniae (15, 54, 129), resulting in the development of similar lesions caused by these microorganisms (36, 50). It is likely that two RTX toxins (ApxI and ApxII), capsular polysaccharides (CPSs) (K) and lipopolysaccharides (LPSs) (O), an iron- regulated outer-membrane protein, and resistance to complement-mediated killing contribute to virulence (7, 60, 61, 94, 103, 115, 116). RTX toxins are produced by a broad range of pathogenic Gram-negative bacteria. The toxins exhibit a cytotoxic and often a hemolytic activity in vitro, suggesting that they may play a role in host specificity of certain pathogens (38). For example, Actinobacillus suis and Actinobacillus equuli have host specific specificity toxins, whereby A. suis has the apxICA and apxIICA genes, (60, 61) not present in A. Equuli (103). The cell surface (CPS) and (LPS) of Gram-negative bacteria also play key roles in bacteria-host interactions. Monteiro et al. (85) described the presence of (1→6)-β-D- glucan in the LPSs and CPSs of A. suis, and the strains expressing that homopolymer as LPS-O-chains and CPSs were designated serotype O1/K1. The presence of (1→6)-β-D- glucan is also found in common environmental fungal organisms and yeast, and some

7 pigs may have low levels of antibody resulting in false positive results in a serotyping system for A. suis by using LPS/CPS based enzyme-linked immunosorbent assay (ELISA). A study was also done with the O-antigen polysaccharide chemical structure of an O2 serogroup strain of A. suis, which is associated with severely diseased animals and produce a more complex O-chain PS. The capsule PS (K2) of these strains contains sialic acid, a carbohydrate that has been associated with many immunological events (109). A cell surface antigen-typing system was devised in order to examine the prevalence of different lipopolysaccaride (O) types in healthy and diseased pigs with A. suis, and O2/K3-reactive strains may be more virulent than O1/K1 strains (115). Although O1/K1 and O1/K2 strains were isolated from clinically “healthy” pigs (asymptomatic animals), they both have a potential to cause disease. However, differences in the histopathologic changes and the severity of peritonitis between A. suis serotypes O1/K1, O1/K2, and O2/K2 have been reported (116). Although iron is plentiful in animal hosts, it is not readily available to pathogens due to iron-withholding properties of transferrin and lactoferrin. A. suis is an iron- requiring pathogen that must be capable of acquiring iron from its host despite the iron- restricted environment. Pathogenic members of Pasteurellaceae can obtain iron directly from the host proteins, most notably from transferrins (Tfs), by means of siderophore- independent, receptor-mediated mechanisms. This mechanism involves two surface receptor proteins referred to as Tf-binding proteins A and B (7). Actinobacillus suis also has the ability to acquire iron from hemoglobins for growth and involves a single- component receptor that is up-regulated in response to iron restriction (8). Actinobacillus suis has been described to secrete metalloproteases in vitro and these proteins displayed proteolytic activity. Metalloproteases degrade pig and bovine IgG and they may have a role in diseases caused by A. Suis (88). Microbial proteases exert direct pathological effects by destroying host tissues and potentiate inflammatory processes. The ability of A.suis to survive in the environment has not been fully described. This organism is killed within 15 minutes at a temperature of 60oC, is sensitive to most disinfectants, and will die within a few days in clinical specimens (96). The presence of a

8 specific bacterial strain, environmental and management factors (excessive temperature fluctuations, high humidity, mixing of pigs with an age spread of more than 2 weeks, and overcrowding), as well as the immune status of the pig and other factors (such as genetics) may play a role in the development of Actinobacillus suis disease in swine. Despite the fact that A. suis is sensitive to a wide range of antibiotics, the rapid onset of disease makes effective treatment difficult. Clinical signs and lesions caused by A. suis are usually non-specific, and a list of differential diagnoses associated with other pathogens that can cause septicemia is necessary for the development of effective control strategies. Bacterial pathogens that can cause systemic infection in swine include Haemophilus parasuis (cause fibrinous pleuritis, pericarditis and polyserositis), Actinobacillus pleuropneumoniae (common cause of pneumonia), Erysipelothrix rhusiopathiae (typical lesions include endocarditis, arthritis and skin lesions). The most promising technology for identification of Actinobacillus spp. and other pathogens is PCR test based on species specific genetic elements including virulence genes (23). Vaccination and serodiagnostic testing are complicated by the presence of multiple serotypes, and cross-reactive antigens (65, 69, 108). Commercial vaccines are not available; the only vaccines for prevention of A. suis diseases are limited to autogenous bacterins used to stabilize antibody levels in a herd by increasing humoral immunity of animals with lower levels. Oliveira et al (96) reported that A. suis affecting North American swine herds are highly clonal and this information may be important for the selection of vaccine strains. Some commercial A. pleuropneumoniae vaccines, containing Apx toxoids, might provide some cross protection. Actinobacillus equuli is a small, nonmotile, gram-negative rod phenotypically and phylogenetically closely related to A. Suis (60). However, A. equuli differs from A. suis by being nonhemolytic, producing acid from mannitol, and not hydrolyzing esculin (111). The natural host of A. equuli is the horse and the organism is found in the alimentary tract. Actinobacillus equuli has been associated with several diseases in foals and adult horses, and the most important condition is fatal septicemia in neonatal foals which is thought to be associated with a failure of passive transfer of immunoglobulins via the colostrums (12). The disease in foals is often characterized by diarrhea followed

9 by septicemia and associated meningitis, pneumonia, embolic nephritis and septic polyarthritis. Disease in adult horses due to A. equuli is less common but the agent can cause arthritis, endocarditis, abortions, septicemia, nephritis, and orchitis (98, 132). A few cases of acute and chronic peritonitis caused by A. equuli infection have also been documented in adult horses (39, 40, 98). The pathogenesis of peritoneal infection caused by A. equuli is unclear. In addition to septicemia resulting in polyserositis, and/or peritonitis, other routes of infection are possible. Carriage of A. equuli by Strongylus sp. larvae from the intestinal tract may play a role in peritoneal invasion (39, 40). The feasibility of this route of infection is supported by the fact that A. equuli has been isolated from verminous aneurysms of the cranial mesenteric arteries caused by Strongylus vulgaris larvae (40). Mesenteric and cecocolic verminous arteritis lesions were observed in one horse with chronic A. equuli peritonitis (39). However, A. equuli peritonitis is a rare diagnosis in adult horses but because appropriate antimicrobial treatment in small numbers of horses with acute A. equuli peritonitis has resulted in clinical improvement and a return to original activity, pathologists and clinicians examining smears of abdominal fluid from equine colic cases should be aware of this diagnosis (98). It is important to determine whether there are specific strains of A. equuli with greater virulence for foals and/or adult horses or whether such strains are common inhabitants of the equine intestinal tract (98). Actinobacillus equuli was subdivided in two subspecies with distinct genotypes, disease pattern and epidemiology (24). They were designated A. equuli subsp. haemolyticus (carrying aqxA gene, that express RTX toxin) (60), which has mainly been described in horses (102), and A.equuli subsp. equuli associated with “sleepy foal disease” a fatal septicaemia in neonatal foals, and abortion and endocarditis in pigs (86, 110). The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx (12). The incidence of foal infection is reduced when foals have access to colostrum. A.

10 equuli antibodies against outer membrane proteins, and Aqx are readily transferred from mares to foals but some mares and their foals do not have antibodies to Aqx (46). Septicemic actinobacillosis in pigs is usually attributed to A. suis (82, 111) but A. equuli can produce septicemia in piglets and sows, with similar lesions in kidneys, joints and endocardium (11, 51, 103). A. equuli has also been isolated in cases of porcine abortion (133), metritis in sows, and bacterial embolism (103). Porcine A. equuli infection has been sporadically reported in Europe since 1924, and the rarity of isolating A. equuli in North American pigs could reflect the separation of pigs and horses in most farming systems (111). But a recent case of A. equuli infection in swine was reported from a farm in which pigs were raised in an environment without contact with horses (103). In this particular case, a molecular approach was used to detect and identify the bacteria in the affected tissues and the analysis of the amplified nucleotide sequences revealed 100% homology with two A. equuli isolates (GenBank AY634640 and AY465359). In addition, PCR typing of the RTX gene ruled out A. suis and A. equuli subsp. haemolyticus, and confirmed A. equuli subsp. equuli (103). Although a differential diagnosis of porcine actinobacillosis as the result of A. equuli is not commonly considered in swine, veterinarians should be aware of the importance of this pathogen in septicemic conditions of pigs. Any septicemic condition has the potential to cause embolism in small blood vessels and endothelial damage including endocarditis, condition that has been reported in natural and experimental A. equuli infections of piglets (51). The pathogenesis of thromboembolism starts with primary infection, followed by secondary bacteremia, bacterial colonization of multiple organs and culminating with thromboembolism (105). Thus, it is important to recognize that embolic nephritis is an entity with many possible causes, including emergent zoonotic pathogens such as A. suis and A. equuli. Isolation and identification of the etiologic agent are required to achieve an accurate diagnosis and to implement adequate therapeutic and management strategies. There is a lack of information regarding the prevalence of Actinobacillus suis and Actinobacillus equuli in condemned swine kidneys in the province of Alberta and there are no recently published studies about the etiology of nephritis in pigs slaughtered in this

11 province. The objective of this study was to identify Actinobacillus suis, A. equuli, and other infectious agents in condemned kidneys of market hogs and culled sows in Southern Alberta utilizing bacterial cultures, histochemistry, molecular tests and western blot technique.

Chapter Two: Materials and Methods

2.1 Study design

The original plan for this project was to sample condemned kidneys from culled sows and market hogs from two slaughterhouses in the province of Alberta, and study the prevalence of Actinobacillus suis and Actinobacillus equuli with nephritis lesions. Shortly before the study began, the access to the abattoirs was restricted due the outbreak of swine flu H1N1 declared in April of 2009, in Alberta. In response to this unforeseen circumstance, the project was modified and samples were collected from one slaughterhouse located near Calgary, Alberta, which provided access to the premises. The plant slaughters in average 250 pigs daily, twice a week. The majority of the pigs corresponded to healthy pigs aged between 4 to 7 months. A few animals were wasted pigs presenting some of the following macroscopic findings: cachexia, swollen umbilicus, dermatitis, ascites, and polyarthritis. Kidneys from these animals were not included in the study.

2.2 Sample collection

Trial 1 Forty kidneys were selected from apparently healthy slaughtered pigs, 30 with macroscopic lesions of multifocal interstitial nephritis or “white spotted” kidneys condemned by Canadian Food Inspection Agency personnel and 10 without gross lesions, the control kidneys of the study. Both condemned kidneys were taken from each of 30 pigs and one control kidney from each of 10 slaughtered pigs during five different weeks (May 2009 to August 2009) with visits made on Tuesdays and Thursdays in order to minimise a potential herd effect. The pig’s tattoo numbers were recorded, to keep track of the origin of the studied market hogs. The kidneys were placed in individual labelled plastic bags and kept at 4oC in a cooler with ice packs until they arrived at the laboratory. Clotted blood from the cardiac cavities of each pig was collected, and the serum was frozen at -80oC for further serological studies.

After the organs were collected, and before arriving at the laboratory, two pieces of tissue from the affected area of each kidney (one per pig) were wrapped in aluminum foil and placed on dry ice for further molecular analysis by polymerase chain reaction (PCR) test. These pieces were frozen at minus 80oC immediately after arrival to the laboratory.

2.3 Classification of gross lesions in the kidneys

The kidneys, one from each pig, were macroscopically classified from 0 to 3 following the number, size and distribution of the lesions in the renal cortex described by Baker et al., 1989, and Martínez et al., 2006. The lesions were visible with a careful examination of the surface of the renal cortex after the capsule was removed. Grade 0: indicated no lesion. Grade 1: presented less than 10 whitish foci between 2-5 mm in diameter. Grade 2: had equal or more than 10 foci measuring 2-5 mm in size or had the presence of at least one area of white discoloration measuring less than 1 cm in greatest dimension. Grade 3: renal cortical tissue was completely covered by multifocal to coalescent, round to irregular shaped areas of pale-tan discoloration reaching over 1 cm in dimension.

2.4 Histology

Samples of the lesions of one kidney from each pig (three to four pieces) were fixed in 10% buffered formalin for no more than four days, and embedded in paraffin for routine histology, and histochemistry procedures. One or two sections by block were cut at 5-microns, and stained with haematoxylin and eosin (H&E) for light microscope examination. Details of embedding, processing and H & E staining protocol are presented in Appendix 1. Each sample was examined microscopically and classified based on the distribution of inflammatory cells in the kidney tissue, location, duration, and severity of histological changes. The distribution was classified as focal, multifocal, coalescent,

14 diffuse; location was grouped as cortex, medulla, and corticomedullary junction; time of occurrence was grouped as acute, subacute and chronic; and severity classified as normal, mild, moderate and severe. 2.4.1 Classification of microscopic lesions in the kidneys

Normal or minimum: No changes in the parenchyma or presence of three or less small foci (less or equal to the size of a glomerulus) of interstitial inflammatory cells in cortex or medulla. Mild: Multifocal, more than 3 small foci of inflammatory cell infiltration, and/or one large focus (larger than the size of a glomerulus) of interstitial inflammatory cell infiltration in cortex and medulla. Moderate: Multifocal to coalescent inflammatory cell infiltration. Presence of multifocal histopathological changes in renal parenchyma such as fibrosis, glomerulosclerosis, necrosis of the tubular epithelium, proteinuria, proliferative glomerulitis involving up to 40% of the tissue sections. Severe: Multifocal, coalescent to diffuse inflammatory cell infiltration in the renal parenchyma. Widespread histopathological changes such as fibrosis, glomerulosclerosis, necrosis of the tubular epithelium, proteinuria, proliferative glomerulitis, vasculitis, involving more than 40% of the tissue sections.

2.5 Histochemistry

The samples were processed in the Diagnostic Services Unit of the Atlantic Veterinary College (AVC), University of Prince Edward Island (UPEI). Gram stain, Periodic acid-Schiff stain, and Ziehl-Neelsen stain were done in an automatic Tissue-Tek DRS 2000 slide stainer. Grocott’s Methenamine Silver stain and Warthin-Starry stain were processed manually. 1) Gram stain, the objective is to differentiate two groups of bacteria based on the chemical and physical properties of the bacterial wall, and classified as Gram-positive and Gram-negative. The purple crystal violet stain is trapped by the layer of peptidoglycan in Gram-positive bacteria, which forms the outer layer of the cell, as a result Gram positive bacteria stain purple-blue. In Gram-negative bacteria, the outer

15 membrane prevents the stain from reaching the peptidoglycan layer in the periplasm, but the outer membrane is then permeabilized by acetone treatment to the pink safranin or carbol fuchsin counterstain, which is trapped by the peptidoglycan, as a result Gram negative bacteria stain pink. Details of the Gram staining protocol are in Appendix 2. The controls slides were bacteria from the lumen of the intestine of a mink.

2) Periodic acid-Schiff stain (PAS) is used as a routine diagnostic tool to detect fungal organisms based on the high carbohydrate content of the organism cell wall. Periodic acid oxidizes glycols to aldehydes, which are then rendered visible by reaction with Schiff’s reagent. It is positive with structures containing glucose, galactose, mannose, and/or sialic acids resulting in pink to bright purplish red colour, as a result basement membranes, collagen fibres, glycolipids and phospholipids can be demonstrated (57). Details of the PAS staining protocol are in Appendix 3. Controls slides were obtained from a bovine liver with mycotic hepatitis.

3) Grocott’s Methenamine Silver stain (GMS), utilized for the detection of fungal elements in tissue sections. Carbohydrates in the cell wall of the organisms are oxidized to generate aldehyde groups, and the aldehydes are detected by reduction of a silver complex. Other carbohydrates that yield aldehydes on oxidation are also stained. These include cellulose, starch, chitin, glycogen, collagen and many types of mucus. These substances are also stainable by the periodic acid-Schiff method. Silver stains have the potential to show individual fungal hyphae in black against a pale background (57). Details of the GMS staining protocol are in Appendix 4. The controls slides were obtained from bovine liver with mycotic hepatitis.

4) Ziehl-Neelsen stain is used for detection of acid fast bacteria (genera Mycobacterium and Nocardia ) and inclusion bodies. Mycobacteria produce mycolic acids, which covalently link to peptidoglycan in the bacterial cell wall. Mycolic acids uniquely withstand alcohol decolouration following staining with hot carbol-fuchsin. Acid-fast bacteria stain bright red or fuchsia, and the background stains light blue. Details

16 of the Ziehl-Neelsen staining protocol are in Appendix 5. Control slides were lung tissue of birds infected with Mycobacterium avium. 5) Warthin-Starry method for spirochaetes. They include important pathogens in such genera as Borrelia, Helicobacter, Leptospira and Treponema. Although this technique stains neurofilamentous material, numerous other objects are stained, notably the nuclei of all cells and the cytoplasm of many endocrine cells. The reason for the specificity of the method remains obscure. Details of the Warthin-Starry staining protocol are in Appendix 6. The control slides were tissue from the intestine of a pig infected with Lawsonia intracellularis. Additionally, paraffin blocks were submitted for immunohistochemical detection of Leptospira spp. to Prairie Diagnostic Services, Saskatoon, Saskatchewan. (Routine protocol not included).

2.6 Bacteriology

For routine bacteriological studies, a portion of the surface of each single kidney with lesions was seared with a hot spatula, opened with a sterile surgical blade exposing cortex and medulla, and a swab sample was obtained and placed into a transport medium for aerobes and anaerobes (Amies without charcoal, Copan). The swab samples were sent at room temperature to the Diagnostic Services Unit of the AVC, UPEI, the same day they were taken, and the bacteriological analysis included a standard culture for aerobic and facultative anaerobic bacteria. The media selected for the culture of the bacteria were sheep blood agar formulated to give improved haemolytic reactions, Mc Conkey agar a selective and differential medium used for the isolation of gram-negative organisms, and Thioglycolate broth an enriched liquid medium used to support the growth of microaerophilic and anaerobic organisms, including fastidious organisms. The samples were analysed also for growth in anaerobic conditions. The bacterial growth was checked daily and the cultures were held for 72 hours or 1 week depending of the growth of bacteria in the different media. After that period the cultures were discarded.

The identification of the bacteria isolated in the cultures was done through Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI- TOF MS) measuring high-abundant stable proteins, such as ribosomal proteins, with mass ranged between 2,000 and 20,000 Da. Also, some colonies were identified using conventional biochemical tests, and miniaturised methods like the rapid ID 32 Strep system and API Staph (bio-Merieux). Swab samples collected from cultures of Escherichia coli BL 21 and E. coli top 10 (Courtesy Dr. Rebekah De Vinney) and Clostridium difficile (Courtesy Dr. Glen Armstrong) were sent to the Diagnostic Unit of AVC for routine diagnosis, in order to check the efficacy of the transport medium for aerobic and anaerobic bacteria survival and the bacterial identification methods. None of the staff of the diagnostic service knew the origin of these samples which were sent with the same protocol as those collected from renal tissue.

2.7 Virology [Polymerase Chain Reaction (PCR)]

DNA extraction

A piece of the frozen kidney samples 3 mm3 of each pig were put into a 1.5 ml microcentrifuge tube with 180µl of buffer ATL, following the tissue protocol(QIAamp DNA Mini Kit), and mixed using a Vortex mixer for 15 sec. Then, 20µl of Proteinase K was added and the samples were incubated at 56oC overnight with mixing movement in order to ensure efficient lysis. Next day, the 1.5 ml microcentrifuge tubes were briefly centrifuged, added 200 µl of buffer AL, mixed vigorously using a Vortex mixer for 15 sec, and incubated at 70 oC for 10 min. 200µl of absolute ethanol was added, mixed using a Vortex mixer for 15 sec and centrifuged to remove drops from inside the lid. Then, the mixture was carefully applied to the spin column (Qiagen Inc.) in a 2 ml collection tube, and was centrifuged at 8,000 rpm for 1 min. The spin column was placed in a clean 2 ml collection tube and the tube containing the filtrate was discarded. A buffer AW1 (500 µl) was added to the spin column and centrifuged again following the same procedure described before. Next step, the spin column was added carefully with 500 µl of buffer AW2 and centrifuged at

14,000 rpm for 3 min, and the collection tube containing the filtrate was discarded. Finally, the spin column was placed in a clean 1.5 ml microcentrifuge tube, 200 µl of buffer AE was added, and the assembly was incubated at room temperature for 5 min, and then it was centrifuged at 8,000 rpm for 1 min. This last step was repeated twice and the last collection tube was labelled, which contained the DNA eluting in buffer AE. This time the spin column was discarded. The specification of the content of the buffers ATL, AW1, AW2, and AE is not revealed by the manufacturer. The eluted DNA was quantified using the NanoDrop 1000 spectrophotometer, then was stored at -20oC.

Conventional PCR for detection of porcine circovirus type 2 (PCV-2): DNA amplification

The PCR mixture in 50µl contained a concentration of 5µl of 10 X Hot Start PCR buffer with (NH4)2SO4 (Fermentas), 4µl of MgCl2 (25mM), 1µl deoxynucleoside triphosphates (dNTPs) (10mM each), 2.5µl of forward primer, 2.5µl of reverse primer, 0.5µl of Taq DNA Polymerase 5U/µl (Fermentas), 29.5µl of water nuclease-free, and 5µl of template DNA. The PCR conditions were as follow: 5min at 94oC, followed by 35 cycles of 94oC for 45 sec, 56oC for 1 min, and 72oC for 1 min, with a final extension step of 7 min at 72oC, yielding amplicons of 629 bp in a Gene Amp PCR System 9700. The resultant fragments were visualized by SafeView nucleic acid stain (Applied Biological Materials Inc.) following electrophoresis in a 1.5% agarose gel in 1X TAE (40mM Tris- aceate pH 8.0, 1 mM EDTA). The forward and reverse primers used in this study were (5’-TAA TCC TTC CGA AGA CGA GC-3’) at position (116-135) and (5’-CGA TCA CAC AGT CTC AGT AG-3’) at position (726-745), respectively. The region amplified was a conserved section of the replication gene from PCV-2 strain 05-32650 (GenBank accession number EF394779). Sixty samples were tested for (PCV-2) in pig kidneys, 40 from trial 1 and 20 from trial 2 (mentioned below). To ensure quality of data, negative and positive reference samples were always applied in each PCR reaction. The positive control was a plasmid

19 with the full length genome of PCV-2 ligated to it. The negative controls used were: negative pig tissue to PCV-2 infection and master mix with water DNAse free as the template. This PCR technique for PCV-2 was developed at the virology research laboratory of the University of Calgary (Courtesy Dr. Markus Czub). But in order to have the PCR results for three different viruses from a diagnostic centre, frozen kidney tissue samples (-80oC) of the trial 1 were sent on dry ice to the Diagnostic Services AVC, UPEI, for detection of porcine reproductive and respiratory syndrome virus (PRRSV), (PCV-2), and porcine parvovirus (PPV) in condemned and control kidneys. The protocol for extraction of DNA from the samples used in UPEI was QIAamp DNA Mini Kit (Qiagen) for (PCV-2) and (PPV), while QIAamp Viral RNA Mini Kit (Qiagen) was used for RNA extraction of (PRRSV) and reverse transcription PCR in accordance with the manufacturer’s instructions. PCR conditions and primers for PCV-2 were designed and established in the Diagnostic Services AVC, UPEI. Briefly, a 5µl template was added to 45μl of mastermix prepared as follow: 3.0 mM Mg Cl2, 200 μM each dNTP (Applied Biosystems), 0.1µM each primer, 2.5 U Amplitaq Gold DNA Polymerase (Applied Biosystems) and appropriate amount of dilution buffer and molecular grade water. After denaturation at 95oC for 5 min, the reaction mixtures were subjected to thermal cycling for 35 cycles each of 95oC for 30 sec, 54oC for 60 sec and 72oC for 60 sec, followed by a final extension at 72oC for 7 min. A second PCR test using the amplification conditions and primers developed by Caprioli at al., 2006 (17) with minor modifications was performed.

Briefly, 5µl of template was added to 45μl of mastermix, prepared with 3.0 mM Mg Cl2, 200 μM each dNTP (Applied Biosystems), 0.1µM each primer, 2.5 U Amplitaq Gold DNA Polymerase (Applied Biosystems) and appropriate amount of dilution buffer and molecular grade water. After denaturation at 95oC for 5 min, the reaction mixtures were subjected to thermal cycling for 39 cycles each of 95oC for 30 sec, 55oC for 30 sec and 72oC for 30 sec, followed by a final extension at 72oC for 7 min. The sequence of the PCV-2 primers (ORF2) was: F5’-CAC GGA TAT TGT AGT CCT GGT-3’, and R5’- CCG CAC CTT CGG ATA TAC TGT C-3’. They amplified the section from sense (1093-1113) and antisense (1565-1586).

The primers for conserved non-structural protein gene and amplification conditions that were used for PPV were modification of the protocol described by Soares et al., 1999 (118). The mastermix (45µl) was the same described above, with 5 μl of template. The condition of the amplification was performed with an initial denaturation at 95oC for 5 min, 35 cycles at 95oC for 30 sec, 54oC for 60 sec and 72oC for 60 sec, followed by a final extension at 72 oC for 10 min. Finally, the primers and amplification conditions used for PRRSV were modifications of the protocol developed by Mardassi et al., 1994 (74). Briefly, 10µl of template was added to 40μl of mastermix using Totan One Tube RT-PCR System (Roche Diagnostics Corp) prepared as follow: 0.1µM each primer, 200μM each dNTP, 2.5 U of enzyme mix, 0.24 mM DTT, 2.5 mM MgCl2 and appropriate amount of dilution buffer and molecular grade water. After an initial heating at 58oC for 30 min, the amplification was done by 35 cycles at 95oC for 25 sec, 58oC for 20 sec and 68oC for 45 sec, with a final extension at 68oC for 7 min. All the PCR tests were performed in a GeneAmp PCR System 9700 termocycler (Applied Biosystems) and the PCR products were analysed by electrophoresis on agarose gel and visualized by staining with ethidium bromide. Additionally, paraffin blocks were submitted for immunohistochemical detection of PCV-2 antigens to Prairie Diagnostic Services, Saskatoon, Saskatchewan. (Routine protocol not included). Trial 2

Sample collection

In order to perform a study of the bacteria isolated from the lesions of the kidneys and their relation with an immune response in the respective host, and to increase the sample size of the project, a second group of 20 condemned “white-spotted” kidneys were collected with the same method described in trial 1, during March 2010 to April 2010. Twelve positive cultures isolated from 11 different pigs were conserved in blood agar tubes and sent at room temperature from the Diagnostic Services Unit of AVC, UPEI, to the University of Calgary. Upon receipt, the samples were removed from the

21 biohazard casing, and some colonies of each sample were streaked and cultured in a new sheep blood agar medium, then the plates were incubated at 37oC for 24 hours. Following incubation, the entire culture was placed in a10% sterile skim milk medium and frozen in different labelled containers at -80oC for further immunological test. The only difference of treatment with this group of samples and the first 40 samples from trial 1, was that the two pieces of tissue collected from the lesion of one kidney from each pig were submerged in RNAlater (Qiagen) instead of being placed on dry ice for further PCR studies. This procedure was changed to obtain an immediate stabilization and protection of the cellular RNA in situ, and subsequent easy transport and storage of the samples. After the incubation of the tissue overnight in the reagent at room temperature, the pieces of tissue were removed from the reagent and transferred them to minus 80oC for storage. In the PCR protocol, these samples required a mechanical homogenizer, a sterile pestile, to homogenize the sample.

2.8 Western blot

Twelve samples of frozen bacteria in 10% skim milk medium from the trial 2 were removed from the freezer -80oC and ice crystals were collected from the surface of each specimen. The sample was cultured on Sheep Blood Agar (BA) (Dalynn, Calgary, o AB) plates, and incubated at 37 C with 5% CO2 for 48-72 hours. (Six of the 12 isolated o bacteria grew better at 37 C without CO2, and the results are summarized in Table 1). The spot of sample was spread over the surface of the BA plate with a 10µl inoculation loop to ensure the formation of isolated colonies. A group of bacterial colonies from each cultured sample were diluted in 100µl of Lysogeny broth (LB) and the number of collected colonies was standardized at 0.487 units of absorbance, the smaller value obtained from the studied samples, using a Microplate Manager program version 5.2 and measured in a sterile multiple well plate, flat bottom with lid (Sarsted, Inc.). Then, sodium dodecyl sulphate buffer (SDS buffer) (Appendix 7) was added in a relation of 1:4 to each sample solution and the samples were

22 boiled at 105oC x 5 minutes to denature the protein molecules to their primary amino acid structure. The gel for the electrophoresis was prepared in a concentration of 10% to separate the bacteria’s proteins according to their size, and no other physical feature. The stacking gel had a concentration of 5% acrylamide. The wells of the gel were loaded with 10µl of a pre-stained protein ladder (BenchMark, Invitrogen) which reveals ten bacterial protein sizes from 180 kDa to 6 kDa (listed in Appendix 7), and three different amounts of each sample solution (5µl, 10µl, 20µl). When the SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done, the proteins were transferred onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. After transfer was completed, the membrane was removed from the sandwich (fiber pad, filter paper, SDS-PAGE gel, transfer membrane, filter paper, and fiber pad), labelled and placed into a block solution (50 ml Tris Buffered Saline (TBS), 0.1% Tween pH7.5, and 5% (w/v) milk powder 0% fat), and incubated at 4oC overnight without shaking. The following day, the blocking solution was removed, the membranes were labelled according the sera in study, cut depending on the sample number, and put inside a labelled tube. The respective primary antibody (porcine serum) was added to each sample at a dilution of 1:100 to the block solution and incubated with mixing in a Barnstead/Thermolyne Labquake Shaker for 1 hour at room temperature. Then, the membranes were washed 3 times with TBS, 0.1% Tween pH 7.5 (15 minutes each wash in a shaker GX-1000 Labnet International, Inc.). The secondary antibody (peroxidise-conjugated AffiniPure goat anti-swine IgG (H+L), Jackson ImmunoResearch Laboratories, Inc.) was added to a block solution at 1:10,000 dilution (1.5µl/15 ml of block solution), and the membranes were immersed in this solution and incubated with mixing for 1 hour at room temperature. Finally, each blot was washed twice with TBS, 0.1% Tween pH 7.5, during 15 minutes, in a shaker. Parallel to the detection of bacterial proteins using antibodies from the host serum, a duplicate blotting of each sample as a negative control was done. The only

23 difference between both blotting assays was that the negative control membranes were not exposed to the primary antibody (host serum), only the block solution was used in that stage, but all the other steps were done in the same way. An entry-level enhanced chemiluminescent (ECL) substrate was prepared with

0.1 M Tris pH8.5, luminol, coumaric acid and H2O2 (Appendix 7), and the membranes were submerged in this solution for 1 minute. Then, they were exposed 10 seconds and 30 seconds to a high performance chemiluminescence film, and developed in a Kodak OMAT 2000 machine for detection of the horseradish peroxidise-conjugates.

2.9 Statistical Analyses

All data were entered and analyzed using statistical software Stata 10 for Windows. Confidence intervals and tests for significance were based on the Mantel- Haenszel χ2 test and the Fisher exact two-tailed test according to sample size. A value of P<0.05 was considered significant.

Table 1 Bacterial growth conditions in the incubator with and without CO2

Sample Pig number Bacteria isolated from the Growth Growth

# kidney with CO2 without

CO2 1 76 Arcanobacterium pyogenes + 2 68 Stenotrophomonas rhizophila + 3 66 No ID Hemolytic colonies + 4 80 Escherichia coli + 5 72 Staphylococcus epidermidis + 6 69 Acinetobacter spp. + 7 79 Streptococcus suis + 8 81 No ID Streptococcus group C + 9 84 Streptococcus suis (probable) + 10 78 Streptococcus suis + 11 75 No ID Gram + bacilli + 12 75 No ID haemolytic + Streptococcus No ID: No identification

Chapter Three: Results

The samples for this study were obtained from 17 swine farms as determined by the tattoo number recorded in the plant. However, the samples were not randomly selected, as there was a deliberate attempt to collect organs condemned under the general category of “white-spotted” kidneys. 3.1 Classification of gross lesions in the kidneys

In trial 1, 10 kidneys without gross abnormalities were used as controls and grouped in grade 0 (Fig.1). From the 30 condemned kidneys, 11(36.6%) were classified as grade 1 (Fig.2); eleven cases (36.6%) were classified as grade 2 (Fig.3); and seven cases (23.4%) were classified as grade 3 (Fig.4, 5, 6). The 20 condemned kidneys of trial 2 maintained a similar distribution of gross lesions with 7 (35%) classified as grade 1, six (30%) as grade 2, and 7 (35%) as grade 3.The combined distribution of gross lesions of the 50 condemned kidneys of the study was: Grade 1= 18 cases (36%) Grade 2= 17 cases (34%) Grade 3= 15 cases (30%) 3.2 Classification of microscopic lesions in the kidneys

According to the severity of microscopic findings, the condemned and control kidneys of this study were classified microscopically in four groups: normal or minimum, mild, moderate and severe.

Figure 1 Grade 0. Normal kidney, control

Figure 2 Grade 1. Presence of less than 10 round grey-white foci measuring 2-5 mm are scattered throughout the capsular surface of the kidney.

Figure 3 Grade 2. Numerous (more than 10) grey-foci measuring between 2 to 5 mm and one focus of pale tan discoloration measuring 8 mm in the largest dimension.

Figure 4 Grade 3. Multifocal to coalescing areas of pale discoloration measuring over 1 cm in dimension are scattered throughout the renal surface.

Figure 5 Grade 3. Multifocal grey-white foci throughout the renal cortical

surface.

Figure 6 Grade 3. Diffuse grey-white foci and multifocal petechias throughout the surface of the kidney.

Four out of 50 condemned kidneys (8%) had minimal or non-detectable histopathological changes. Lesions in twelve out of 50 affected kidneys (24%) were diagnosed as mild, characterized by the presence of small areas of focal or multifocal periglomerular, perivascular interstitial inflammatory cell infiltration in cortex and/or medulla, primarily composed of lymphocytes and plasma cells. In twenty out of 50 condemned kidneys (40%), lesions were classified histologically as moderate and were characterized by the presence of a wide range of lesions that affected up to forty percent of the total area of the tissue section. Lesions included multifocal to coalescent interstitial nephritis, tubulointerstitial nephritis and/or focal glomerulonephritis. Fourteen out of 50 condemned kidneys (28%) were categorized as severe. Observed microscopic changes were similar to those found in the moderate category but they were more extended in the tissue section, involving more than forty percent of the surface examined. Control kidneys, classified grossly in grade 0, had minimal or non-detectable microscopic changes in the parenchyma and were classified histologically as normal in 7 out of 10 cases (70%). Three of the control samples had moderate histological changes characterized mainly by multifocal to coalescent mononuclear interstitial inflammatory cells infiltration (lymphocytes, plasma cells and occasional macrophages) located in the cortex and corticomedullary junction. One of these cases showed variable numbers of neutrophils within the lumen of some renal tubules. Another case had a focal proliferative glomerulonephritis in addition to multifocal interstitial nephritis, and was therefore included in the group classified as glomerulonephritis. From the 50 condemned kidneys, 38 had interstitial nephritis, 8 glomerulonephritis, and 4 were classified with minimal histological changes characterized by focal to multifocal small interstitial inflammatory cells infiltration in the cortex and/or in the corticomedullary junction. Forty out of 60 collected kidneys (67%), 10 normal and 50 condemned, showed interstitial nephritis which varied widely in intensity and distribution. 3.2.1 Tubulointerstitial nephritis

Inflammatory changes in this group of samples involved both the interstitium and renal tubules, they were subacute or chronic and were associated with a wide range of

30 microscopic findings that included loss of parenchyma, fibrosis, atrophy of tubular segments, degeneration, necrosis and regeneration of tubular epithelium (Fig.7). Some renal tubules appeared moderately dilated and lined by flattened epithelium; others, surrounded by areas of interstitial fibrosis were notably atrophic and with collapsed lumen (Fig.8). Mineralization (calcification) of degenerated and necrotic tubular epithelial cells was occasionally seen (Fig.9). Degeneration and necrosis of the tubular epithelium evidenced by increased cytoplasmic eosinophilia, nuclear pyknosis, karyorrhexis, was often accompanied by concomitant regenerative changes within affected or adjacent renal tubules. Tubules undergoing regeneration had an increased mitotic rate and were lined by enlarged cells with slightly basophilic cytoplasm, large nuclei and prominent nucleoli (Fig.10). Lesions of interstitial nephritis involved principally the cortex and the corticomedullary junction, and in 6 cases appeared to extend radially from medulla to cortex (Fig11). In seven out of 38 condemned kidneys diagnosed with tubulointerstitial nephritis, the inflammatory cell infiltrate had mainly cortical distribution and was composed primarily of lymphocytes and plasma cells arranged in variably sized aggregates resembling lymphoid follicles (Fig.12). In two cases, large multinucleated giant cells, interpreted as syncytial cells were detected within the cortical tubular epithelium and tubular lumina (Fig.13, 14) 3.2.2 Glomerulonephritis

Cases of glomerulonephritis were classified as severe (75%) and moderate (25%) microscopically, and were characterized by multifocal to diffuse increase of cellularity of the glomerular tufts. Affected glomeruli were often enlarged, mainly due to increased numbers of mesangial cells (Fig.15). Influx of neutrophils, foci of hemorrhage, and necrosis of the glomerular endothelium were also detected in few cases (Fig.16). Additional microscopic findings included moderate to marked dilatation of Bowman’s spaces and renal tubules filled with homogeneous eosinophilic (proteinaceous) fluid (Fig.17). In chronic cases, variable degrees of periglomerular (Fig.18) and glomerular fibrosis leading to glomerulosclerosis was also detected. This latter lesion was characterized by hypocellularity, shrinkage and hyalinization of the glomerulus due to an

31 increase in collagen and mesangial matrix deposition with loss of glomerular capillaries (Fig.19). Proximal tubular epithelial cells of some cases had numerous, variably-sized, brightly, microscopic eosinophilic intracytoplasmic globules referred as hyaline droplets, interpreted as accumulations of intracytoplasmic protein absorbed from the filtrate (Fig.20). Severe vasculitis, characterized by fibrinoid necrosis and inflammatory cells infiltration within the tunica media and adventitia was present in a medium sized artery located at the corticomedullary junction in case 12 (Fig.21). In another case (#56), similar vascular lesions were also present in small and medium sized muscular arteries at the corticomedullary junction. In this case, lesions resulted in occlusion of the vascular lumina and neo-vascularization in the surrounding of the particular area (Fig.22). Inflammatory cells, mainly neutrophils, were seen within renal tubules in some cases of glomerulonephritis. Table 2 and Table 3 list the macroscopic and microscopic lesions of condemned and normal kidneys of 60 market hogs selected for this study. There was no significant statistical evidence of association between the distribution of the gross lesions in the kidneys and the severity of the histological changes in the renal parenchyma using χ2 test (Pearson chi2=28.6678>3.841 with p-value=0.003). 3.3 Histochemistry

In this study, no fungal or spirochaete agents were detected in the kidney tissues of these pigs utilizing acid fast, periodic acid Schiff (PAS), Grocott’s methenamine (GMS), and Warthin-Starry silver stains. However, in only one case (#49) Gram’s stain revealed Gram-negative and Gram-positive cocci in a sample classified microscopically as severe. This sample was sent for bacteriological examination and was reported negative to bacterial growth. Additionally, Leptospiral organisms were not detected by immunohistochemistry (data not shown).

Tubulointerstitial nephritis

Figure 7 Interstitial inflammatory cell infiltration composed primarily of lymphocytes and plasma cells. H&E x20 Case 4.

Figure 8 Interstitial fibrosis and dilatation of renal tubules (arrow). H&E x10 Case 14.

Figure 9 Mineralization of degenerated and necrotic renal tubular epithelium. H&E x40

Case 56.

Figure 10 Tubular regeneration evidenced by the presence of mitosis (arrows) and enlarged tubular epithelial cells. H&E x 60 Case 42.

Figure 11 Inflammatory cell infiltration extends radially from the medulla towards the cortex. H&E x2 Case 48.

Figure 12 Follicular lymphoid formation within the cortex and corticomedullary junction. H&E x2 Case 5.

Figure 13 Numerous syncytial giant-cells (arrows) admixed with lymphocytes, plasma cells and eosinophils in the renal cortex. H&E x40 Case 43.

Figure 14 Syncytial cell within the lumen of a renal tubule. H&E x60 Case 12.

Glomerulonephritis

Figure 15 Proliferative glomerulonephritis evidenced by increased cellularity within the glomerular tuft. H&E x40 Case 13.

Figure 16 Glomerulitis characterized by fibrinoid necrosis and presence of karyorrhectic debris within the glomerulus. H&E x40 Case 12.

Figure 17 Abundant eosinophilic proteinaceous material within Bowman's space and markedly dilated renal tubules. H&E x10 Case 51.

Figure 18 Membranoproliferative glomerulonephritis with prominent periglomerular fibrosis (arrow). H&E x20 Case 51.

Figure 19 Glomerulosclerosis characterized by shrinkage of the glomerulus, thickening of basement membranes and collagen deposition. H&E x40 Case 51.

Figure 20 Hyaline droplets characterized by numerous eosinophilic globular material within the tubular epithelium. H&E x40 Case 51.

Figure 21 Fibrinoid necrosis (arrow) and inflammatory cell infiltration in the wall of a middle sized renal artery. H&E x10 Case 12.

Figure 22 Chronic vasculitis with complete obliteration of the vascular lumen. H&E x10 Case 56.

3.4 Bacteriology

The study included samples from two different periods, trial 1 from May 2009 through to August 2009, and trial 2 from March 2010 to April 2010. All of the samples were submitted for bacteriologic examination. In total, 60 swabs were collected 40 from trial 1 and 20 from trial 2. In trial 1, bacteria were isolated more frequently from thioglycolate medium (28 of 40 samples) than blood agar medium (16/40) and Mc Conkey medium (8/40), indicating that bacteria isolated from kidneys of this study had a fastidious growth. From the five different visits to the slaughterhouse from May to August, the kidneys collected in June 25th presented the largest amount of different bacteria isolated (31) in the three media mentioned above, when it was compared with 5 isolations in May 28th, two in June 1st, 10 in June 11th, and 15 in August 24th. This result was not associated with the origin of those animals (throught the tattoo number) by using statistical analyses. The original objective of this study was to determine the prevalence of Actinobacillus suis and A. equuli in condemned kidneys from market hogs and culled sows. None of these bacteria were found in this study, but other microorganisms were isolated and are summarized in Table 4 for trial 1, and Table 6 for trial 2. Only 11 out of 40 (27.5%) samples resulted negative to bacteriological culture, including 4 of the control kidneys (40%) and 7 condemned kidneys (23%). The bacteria more frequently isolated were Acinetobacter spp. (18 samples), Empedobacter brevis (7 samples), “no identified” (7 samples), Streptococcus spp. (6 samples), Macrococcus caseolyticus (5 samples), and Stenotrophomonas maltophilia (4 samples). Bacteria were present individually (one genus) or mixed (different genera or species) in the same sample. Twelve samples reported positive to bacterial growth and negative to the presence of other microorganisms showed different degrees of histopathological changes which are described in Table 5. Half of these samples had minimum to mild histological changes, but the other six showed moderate to severe pathological changes in the parenchyma of the kidney where some uncommon bacteria, not previously reported in the literature,

41 were isolated individually: Kocuria kristinae, Brevundimonas nasdae and Lactococcus garvieae. Association between presence of bacteria and renal lesions led to further investigation to determine if a particular bacterium caused an immune response in the host and therefore was responsible for the pathogenesis of the nephritis. Unfortunately, the samples were kept only for one week in the diagnostic laboratory and a new group of sample collection was performed in order to make this latter study. In this second group of pigs where the samples were collected during the period March-April 2010, four out of 20 (20%) condemned kidneys were negative to isolation of bacteria, even though they were classified macroscopically as grade 1, grade 2, and grade 3. This value is similar to the data found in trial 1 (23%). Bacteria isolated from 20 condemned kidneys of trial 2 are summarized in Table 6. Sixteen samples resulted positive for growth in thioglycolate medium (80%), seven (35%) in blood agar medium and none in McConkey medium. From these bacteria isolated, 12 different samples were sent from the diagnostic laboratory in PEI to the University of Calgary for western blot study. The bacteria were Arcanobacterium pyogenes, Stenotrophomonas rhizophila, Escherichia coli, Staphylococcus epidermidis, Acinetobacter spp., Streptococcus suis (2), and 5 no identified bacteria isolated from 11 different pigs. Escherichia coli, two different samples, and Clostridium difficile were cultured and identified by mass spectrometry utilizing the same method of routine diagnosis in the Diagnostic Services Unit of the AVC, UPEI. Even though the samples had a delay for delivery of 24 hours in the shipment from Calgary to PEI, the transport media allowed these aerobic and anaerobic bacteria to survive. 3.5 Polymerase Chain Reaction for PCV-2, PPV, and PRRS viruses

Frozen tissue samples from trial 1, encompassing 30 condemned and 10 normal kidneys, were sent to the Diagnostic Services Unit of AVC, UPEI, for PCR analysis which results are shown in Table 7. Fifty percent of the tissues were infected with porcine parvovirus, in 17.5% of the samples porcine circovirus type 2 was detected, and all the 40

42 samples resulted negative for porcine reproductive and respiratory syndrome virus infection. Three samples showed infection with PPV and PCV-2 in the same tissue. In order to obtain training and familiarization with the conventional PCR technique, PCR tests for PCV-2 in samples from trial 1 and trial 2 were done at the University of Calgary (Courtesy Dr. Markus Czub). It was used a different protocol for the amplification of viral DNA from that utilized at the Diagnostic Services Unit of AVC, UPEI. The protocol developed in the University of Calgary detected 2 of the seven positive cases reported by the Diagnostic Services for the samples from trial 1, and three positive cases in the samples from trial 2 (Fig.25 and Fig.26). This latter group of samples was not sent to the diagnostic services. In addition, one positive case from trial 1 and 2 cases from trial 2 (also detected by PCR test) were found by immunohistochemistry analysis with small amount of antigen in tiny foci, and tubular epithelium (data not shown). Seventeen out of 40 samples (42.5%) had bacterial growth and the kidney tissue, from where the samples were collected were also positive for porcine circovirus type 2 and/or porcine parvovirus by PCR tests. The summary is in Table 7. The eight severe cases from trial 1 were associated with bacterial isolation from the lesions of the respective kidneys in 25%, with porcine viruses (PCV-2 and/or PPV) detection in the same proportion, but in 4 out of 8 (50%), the lesions were associated with bacterial and viral infection. In the moderate group of samples 13 out of 30 (53%) were associated as well as with the presence of both microorganisms, bacteria and viruses. A difference between the means of the concentration of DNA extracted from the samples of trial 1 and trial 2 was observed, 19.1 ng/µl vs. 14.6 ng/µl, respectively. Both trials differed in the method of conservation of the samples before they were frozen to minus 80oC for PCR test. The t- test statistic revealed that the difference in the mean of the concentration of DNA of 4.444 ng/µl was not statistical significant between the two groups of samples from trial 1 and trial 2 (p-value=0.1627). 3.6 Western blot

Each serum from ten out of eleven pigs from trial 2 reacted with the proteins of the bacterium isolated from the lesion of the respective kidney showing different patterns

43 of bands. The positive reaction was evaluated by the number of bands and the intensity of some of them. Positive samples showed equal or more than 10 different bands, negative samples did not show any band. A strong positive response of the immune system was observed in the pigs that were infected with Arcanobacterium pyogenes, Stenotrophomonas rhizophila, Escherichia coli, Acinetobacter spp., Streptococcus group C (no identified), and Streptococcus suis (sample#10). Otherwise, a weak response was observed in those pigs where haemolytic colonies (no identified), Staphylococcus epidermidis, Streptococcus suis (sample#7), and both bacteria from pig number 75. These bacteria were isolated from their respective kidney lesions, showing only two to five bands in the membrane of western blot. The results are summarized in Table 8. All of the negative controls, those which were not exposed to the pig serum (primary antibody) did not react with the bacterial proteins. A cross reaction was performed between the antibodies of each serum (11 sera) and the 12 bacteria isolated from the respective lesions of the kidneys to determine if the immune response observed was a specific reaction to the bacteria in study or a response to bacteria from the environment. Each serum was tested with all the bacteria in the same membrane, and it was detected that the majority of the sera reacted in different degrees with the bacteria isolated in the laboratory, and isolated from lesions of the different kidneys (Fig.23 and Fig.24). Stenotrophomonas rhizophila (2), Escherichia coli (4), and Acinetobacter spp. (6) produced an immune response in all the 11 pigs from trial 2. The results are summarized in Table 9.

Detection of antibodies in 11 different sera to antigens (proteins) of 12 different bacteria

Figure 23 Serum from 6 different pigs and the 12 different bacteria isolated.

Positive= > or equal 1 band. Negative= no bands showed in the column.

P is an orientation band pink in colour an indicate a protein sized ≈64 kDa (Appendix 7) Column 1=Arcanobacterium pyogenes; column 2= Stenotrophomonas rhizophila; column 3= No ID; column 4= Escherichia coli; column 5= Staphylococcus epidermidis

Western blot result was interpreted by the account of the total number of bands produced and the intensity of specific bands

Figure 24 Serum from 5 different pigs and 12 different bacteria isolated Positive= > or equal 1 band. Negative= no bands showed in the column.

Column 6= Acinetobacter spp.; column 7= Streptococcus suis; column 8= No ID; column 9= S. suis; column 10= S. suis; column 11 and column 12= No ID.

Figure 26 Detection of PCV-2 specific DNA extracted from frozen renal samples from trial 2. The fragment amplified was 629 bp. Lane 1, middle lane and end lane are 1-Kb

DNA ladder. Samples 42, 43, and 46 were positive; + show the lane with positive control.

Table 2 Relationship between macroscopic and microscopic changes by categorization in kidney's samples of 60 market hogs from Southern Alberta. sample Gross Histology sample Gross Histology sample Gross Histology grade grade grade grade grade grade 1 2 Moderate 27 1 Moderate 51 3 Severe 2 2 Severe 28 1 Mild 52 3 Moderate 3 1 Mild 29 2 Severe 53 2 Moderate 4 1 Severe 30 3 Mild 54 1 Moderate 5 2 Severe 33 3 Mild 55 2 Moderate 6 2 Mild 34 2 Minimum 56 3 Severe 9 2 Mild 35 3 Moderate 57 1 Mild 10 2 Minimum 36 3 Moderate 58 2 Mild 11 1 Minimum 38 1 Minimum 59 1 Moderate 12 3 Severe 40 2 Moderate 60 1 Mild 13 3 Moderate 41 3 Moderate 7 0 Normal 14 3 Severe 42 3 Severe 8 0 Minimum 17 1 Moderate 43 3 Severe 15 0 Normal 18 1 Moderate 44 2 Moderate 16 0 Minimum 19 2 Mild 45 2 Moderate 23 0 Minimum 20 2 Moderate 46 1 Moderate 24 0 Normal 21 1 Severe 47 1 Moderate 31 0 Moderate 22 3 Moderate 48 3 Severe 32 0 Moderate 25 1 Mild 49 2 Severe 37 0 Moderate 26 1 Severe 50 1 Mild 39 0 Normal

Table 3 Distribution of cases according to gross grade (0-3) and microscopic classification (normal-severe) of changes observed in swine kidneys.

Normal Minimum Mild Moderate Severe TOTAL Grade 0 4 3 0 3 0 10 Grade 1 0 2 6 7 3 18 Grade 2 0 2 4 7 4 17 Grade 3 0 0 2 6 7 15

Table 4 Relationship between bacteria isolated from kidney's lesions and the categorization of the gross lesions in condemned and control kidneys from trial 1.

Bacteria isolated Grade 0 Grade 1 Grade 2 Grade 3 Kocuria kristinae 1 Peptostreptococcus magna 1 Streptococcus spp.(5 species) 1 3 1 1 Acinetobacter spp.(8 species) 1 10 4 3 Sphingobacterium multivorum 1 Pseudomonas putida 1 Enterobacter kobei 1 Macrococcus caseolyticus 2 2 1 Klebsiella oxytoca 1 Brevundimonas nasdae 1 Empedobacter brevis 1 4 1 1 Lactococcus garvieae 1 1 Stenotrophomonas maltophilia 1 1 1 2 Escherichia coli 1 Enterococcus faecalis 2 Staphylococcus saprophyticus 1 No Identification (NoID) 3 2 2 Streptococcus alactolyticus, S. uberis, S.porcinus, S.dys. equisimilis, S. dys. dysgalactiae. Acinetobacter parvus,A. haemolyticus, A. johnsonii, A. hebeiensis, A. junii, A. towneri, A. lwoffi, A. grimontii.

Table 5 Relationship between bacterial isolation and morphological changes in the kidney's lesions from pigs of trial 1.

# of Bacteria isolated from kidney’s lesions Gross Microscopic Sample classification classification 4 Kocuria kristinae 1 severe 11 Acinetobacter parvus 1 minimum 18 Brevundimonas nasdae 1 moderate 19 Acinetobacter haemolyticus 2 mild 29 Acinetobacter spp., Empedobacter brevis, Lactococcus garvieae 2 severe 30 Macrococcus caseolyticus, Empedobacter brevis, Streptococcus uberis 3 mild 31 Lactococcus garvieae 0 moderate 32 Macrococcus caseolyticus, Empedobacter brevis, Streptococcus uberis 0 moderate 33 No ID, Acinetobacter spp., Stenotrophomonas maltophilia 3 mild 34 No identification 2 minimum 39 Acinetobacter hebeiensis 0 normal 40 Stenotrophomonas maltophilia, Streptococcus dys. dysgalactiae, mixed 2 moderate flora (Acinetobacter haemolyticus, A. johnsonii, A. grimontii)

Table 6 Relationship between bacteria isolated from 20 condemned kidneys from trial 2 and the gross classification of the lesions.

Bacteria isolated Grade 1 Grade 2 Grade 3 Streptococcus dys. dysgalactiae 1 Streptococcus suis 1 2 Acinetobacter spp. (2 species *) 2 Pseudomonas spp. 2 Stenotrophomonas rhizophila 1 Stenotrophomonas acidaminophila 1 Arcanobacterium pyogenes 1 Macrococcus caseolyticus 2 Staphylococcus epidermidis 1 Escherichia coli 2 1 No identification (NoID) 3 2 3 *Acinetobacter johnsoni, A. lwofiii

Table 7 Relationship detected between presence of PCV-2 and PPV nucleotides in tissue, bacteria isolation from lesions, histological changes and diagnosis from samples of trial 1.

Sample Histology Diagnosis Bacteria PCV-2 PPV

1 moderate Tubulointerstitial nephritis + 0 + 2 severe Tubulointerstitial nephritis 0 0 + 3 mild Tubulointerstitial nephritis + 0 + 4 severe Tubulointerstitial nephritis + 0 0 5 severe Tubulointerstitial nephritis + + 0 6 mild Tubulointerstitial nephritis 0 0 0 7 normal 0 0 0 + 8 minimum 0 0 + + 9 mild Tubulointerstitial nephritis 0 0 + 10 minimum 0 0 0 0 11 minimum 0 + 0 0 12 severe Glomerulonephritis + + 0 13 moderate Glomerulonephritis 0 0 + 14 severe Glomerulonephritis 0 + + 15 normal 0 0 0 + 16 minimum 0 0 0 0 17 moderate Tubulointerstitial nephritis + 0 + 18 moderate Tubulointerstitial nephritis + 0 0 19 mild Tubulointerstitial nephritis + 0 0 20 moderate Tubulointerstitial nephritis 0 0 0 21 severe Tubulointerstitial nephritis + 0 + 22 moderate Tubulointerstitial nephritis + 0 + 23 minimum 0 + 0 + 24 normal 0 + 0 + 25 mild Tubulointerstitial nephritis + 0 + 26 severe Tubulointerstitial nephritis + + 0 27 moderate Tubulointerstitial nephritis + 0 + 28 mild Tubulointerstitial nephritis + 0 + 29 severe Glomerulonephritis + 0 0 30 mild Tubulointerstitial nephritis + 0 0 31 moderate Glomerulonephritis + 0 0 32 moderate Tubulointerstitial nephritis + 0 0 33 mild Tubulointerstitial nephritis + 0 0 34 minimum 0 + 0 0 35 moderate Tubulointerstitial nephritis + + 0 36 moderate Tubulointerstitial nephritis + + + 37 moderate Tubulointerstitial nephritis + 0 + 38 minimum 0 + 0 + 39 normal 0 + 0 0 40 moderate Tubulointerstitial nephritis + 0 0

Table 8 Immune response of the host to the bacteria isolated from the respective kidney's lesion from trial 2.

Sample # Pig Bacteria isolated from the kidney Reaction with bacterial protein number 1 76 Arcanobacterium pyogenes Positive (20 bands) 2 68 Stenotrophomonas rhizophila Positive (13 bands) 3 66 No ID Hemolytic colonies Weak (5 bands) 4 80 Escherichia coli Positive (17 bands) 5 72 Staphylococcus epidermidis Weak (5 bands) 6 69 Acinetobacter spp. Positive (11 bands) 7 79 Streptococcus suis Weak (4 bands) 8 81 No ID Streptococcus group C Positive (10 bands) 9 84 Streptococcus suis (probable) Negative 10 78 Streptococcus suis Positive (10 bands) 11 75 No ID Gram + bacilli Weak (5 bands) 12 75 No ID haemolytic Streptococcus Weak (5 bands)

Positive > and equal 10 bands, Negative=0 bands, Weak>0<10 bands

Table 9 Cross reaction of sera and bacteria isolated from renal tissue of 11 pigs with nephritis.

Sample Pig Bacteria isolated from Immune reaction of serum from sample# and # number the kidney others sera Positive Weak Negative 1 76 Arcanobacterium *1,2,3,4,5,6 7,8,9,10,12 11 pyogenes 2 68 Stenotrophomonas 1,2,4,5,6,8,12 3,7,10,11 9 rhizophila 3 66 No ID Hemolytic 2,4,6 3,5,8,10,11,12 1,7,9 colonies 4 80 Escherichia coli 2,4,6,8,10 1,3,5,7,9,11,12 5 72 Staphylococcus 1,2,3,4,6,8,10,12 5,7,9,11 epidermidis 6 69 Acinetobacter spp. 2,4,5,6,8,12 1,3,7,10,11 9 7 79 Streptococcus suis 1,2,4,6 3,5,7,8,9,10,11,12 8 81 No ID Streptococcus 1,2,4,6,8 3,5,7,9,10,11,12 1 group C 9 84 Streptococcus suis 1,2,4,5,6 3,8,10,12 7,9,11 (probable) 10 78 Streptococcus suis 1,2,4,6,8 3,5,7,9,10,11,12 11 75 No ID Gram + bacilli 2,3,4,6 5,7,8,9,10,12 1,11 12 75 No ID haemolytic 2,3,4,6 5,7,8,9,10,12 1,11 Strep.

*1 to 12 are the number of each serum that represents each different pig.

Chapter Four: Discussion

Nephritis in food animals is a common cause of condemnation during food inspection and usually occurs as a result of bacterial, viral disease or following consumption of toxic substances (4). There are few studies describing causes of nephritis in food animals (107, 128), and although Alberta is one of the main swine producers in Canada, there is a little information about causes of nephritis in condemned kidneys of this species. The present study provides baseline data regarding a common cause of kidney condemnation: the condition known as “white-spotted kidneys”. Macroscopic and microscopic changes of condemned pig kidneys at a local meat processing plant were classified, and bacteriological and virological examination was performed to identify specific pathogens involved in nephritis in market hogs. There was no statistically significant association between the classification of gross lesion and the severity of the renal lesions observed microscopically in the same kidney. Only a correlation of r =0.50 was detected between gross and microscopic findings. Grade 0 included 3 samples with moderate histological changes, grade 1 and grade 2 grouped samples with minimal to severe histological changes; grade 3 encompassed samples with mild to severe microscopical changes. The most frequent morphological lesions found in this study were described as tubulointerstitial nephritis involving primarily the interstitium and tubules with inflammation and fibrosis. Severe inflammatory and degenerative diseases of the interstitium almost always impair tubular function. Tubulointerstitial nephritis can be caused by a vast array of agents, including infections, toxins, immunologic disorders, chemicals, and therapeutic drugs. The port of entry for the pathogenic agents of interstitial nephritis is usually hematogenous and even though infectious etiologies are most common, the causative agents, especially in chronic cases, are often not identified (78). Knowledge of the normal renal blood supply is important in understanding the pathogenesis and distribution of renal lesions, mainly in those associated with

56 haematogenous infections. The kidneys receive blood primarily through the renal artery which divides in the pelvic region to form interlobar arteries that run between lobes up to the corticomedullary junction, where they branch and give rise to arcuate arteries. These arteries run along the corticomedullary junction parallel to the capsule and terminate by radiating interlobular arteries into the cortex. Because the renal artery and its branches are end-arteries, occlusion of any branch leads to infarction. Interference with glomerular capillary flow markedly alters peritubular blood flow, especially in the medulla (78, 91). Evidence of infarction was not detected in this study. In this study, bacteria were recovered from a large number of samples, 76% in average between both trials, and these microorganisms could have reached the kidney either through hematogenous route, or through extension of an inflammatory process in the lower urinary tract, the ascending route. Embolic nephritis has been occasionally associated with Actinobacillus equulis infections in sows and market hogs (Dr. Bystrom, Dr. Swendrowski, personal communications). In the present study, however, bacteria lodged in glomerular and peritubular capillaries causing embolic suppurative nephritis were not detected. A case of A. equuli with septicaemia, metritis, embolic glomerulonephritis, and valvular endocarditis was reported in a gilt from the United States, and was confirmed by sequencing of amplified genes extracted from paraffin sections of heart valve sections (103). Over the last two decades, Actinobacillus suis has emerged as an important pathogen of high-health-status swine. Although, A. suis can persist as a commensal in the upper respiratory tract under conditions which are poorly understood, it can reach the bloodstream and cause septicaemia, abortion, arthritis, embolic nephritis, myocarditis, and cutaneous lesions resembling those caused by Erysipelothrix rhusiopathiae, a primary pathogen of swine and turkeys as well as a sporadic cause of disease in humans and other species (95). Actinobacillus suis or Actinobacillus equuli were not isolated from the kidneys of any of the animals in the present study. Arcanobacterium pyogenes, Escherichia coli, Staphylococcus spp., Streptococcus spp., and Acinetobacter spp. were isolated from severe renal lesions of this group of market hogs. Their pathogenic role in swine embolic nephritis should be carefully

57 considered because they are often involved in cases of septicemia (31, 119)and multifocal interstitial nephritis (47). Moreover, one case where Streptococcus suis and Escherichia coli were isolated showed a thromboembolic lesion in an artery near the corticomedullary junction, but bacteria were not detected in the kidney sections examined. Martinez et al. 2006 (76) also found discrepancies between lesions and bacteriological findings, and they attributed them to “a mild and/or random distribution of the bacterial infection within the kidney; a lack of sensitivity or specificity in identifying the gross lesions at the slaughter chain; or an insufficient collection of tissue for bacteriological or histological examination”. Arcanobacterium (Actinomyces, Corynebacterium) pyogenes, isolated from a severe case of glomerulonephritis in this study, is a rare cause of infection in humans, mostly related to living in rural areas and contact with animals. A. pyogenes is perhaps the most common opportunistic pathogen of domestic animals, can act as a primary pathogen, but is more commonly isolated in mixed infections. Recently, three cases of A. pyogenes-associated with soft tissue infection in humans were reported from India (56). The Acinetobacter genus consists of more than 30 species and is mostly associated with clinical environment and nosocomial (hospital acquired) infections in humans. While A. baumannii is frequently associated with outbreaks, lesser-known species, such as A. lwoffi, A.parvus, A. johnsonii, A. haemolyticus are considered to represent emerging pathogens as they are regularly encountered among clinical isolates, and have been associated with serious infections including bacteremia, pneumonia, meningitis, peritonitis, endocarditis, infections of the urinary tract and skin (126) . Endocarditis caused by A. lwoffi can be fulminant, accompanied by septic complications resulting in death (120), whereas bacteremia in immunocompromised catheterized patients is associated with a low risk of death (123). A. haemolyticus was reported as a Shiga toxin-producing microorganism associated with bloody diarrhea (42). Interestingly, some bacteria cultured in this study have not been previously mentioned in the literature in association with renal abnormalities. Specifically, Kocuria kristinae, Brevundimonas nasdae, and Lactococcus garvieae were isolated from kidneys

58 with severe microscopic lesions in which no other microorganisms were detected by any of the tests performed in this study. Kocuria kristinae is generally considered a non-pathogenic commensal bacterium which may cause opportunistic infections in immune-compromised patients with severe underlying diseases, often related to use of catheters. Specifically, it has been described in a case of recurrent bacteremia in a patient with ovarian cancer (10), a case of septicaemia in a patient with acute leukaemia (75), and three patients with catheter related bacteraemia, and one patient with bacterial endocarditis (62). Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans (1). Recently, intestinal disorders in humans have been associated with the consumption of raw fish contaminated with this pathogen (131), and it has been identified as a pathogen in cases of bacterial endocarditis (35), liver abscess (84), and osteomyelitis (49). These reports suggest that L. garvieae could be considered as a potentially zoonotic bacterium. Opportunistic infections occur when commensals or other non-adapted organisms are provided an advantage in the form of compromised host defences (119). It is interesting to note that in this study, opportunistic bacteria were isolated in cases with severe tissue damage and concurrent viral infection with porcine circovirus type 2. Bacteria isolated included Sphingobacterium multivorum and Stenotrophomonas maltophilia, which have been reported as potential pathogens in humans, especially in nosocomial infection. S. multivorum was cultured with no other organism from a peritoneal fluid in a patient with peritonitis (29), it was reported as the cause of septicaemia in a hemodialyzed patient (101), and in a man undergoing chemotherapy for a lymphoma (37). The bacterium has also been associated with respiratory tract infection in patients with cystic fibrosis (63). S. maltophilia, has been reported in a case with recurrent ventilator-associated pneumonia and inherent resistance to multiple antibiotics (138), in pneumonia with a high mortality rate and concomitant polymicrobial colonization or infection (125), in central venous catheters-associated bacteraemia in blood and marrow transplant patients (21), and meningitis in a preterm baby after a neurosurgical procedure (106). In animals, S. maltophilia has been associated with

59 chronic lower airway disease in horses and it has been suggested that it may act as a secondary opportunist agent in mixed infections, or as a primary pathogen when it is isolated in pure culture (136). Within clinical specimens isolation of bacteria not commonly associated with known diseases is often interpreted as suggestive of contamination. In order to verify if individual proteins of bacteria isolated from the renal lesions caused an immune response in the host, a western blot test was performed for the detection of IgG antibodies in the serum of each pig. Results indicated that the pigs studied had been exposed in the past to the majority of the isolated bacteria, therefore, the possibility that these microorganisms may have been the result of sample contamination was unlikely. A weak reaction of some sera may have been a reflection of the time of the infection when the sample was collected, where IgG antibodies may have not been high enough to be manifested as recognising the different proteins of the bacteria, or due to a low production of specific antibodies caused by a status of immune deficiency in the particular animal. IgG antibodies have been detected from 20- 49 days PCV-2 post infection in pigs (Podgorskal et al., 2008 cited by (100)). In future studies, it would be interesting to test the presence of IgM in the serum of these pigs, because IgM are the first antibodies to be produced in the body in response to an infection (7-14 days PCV-2 post infection; Podgorskal et al., 2008 cited by (100)). When IgM are present in high levels, they may represent a new active infection or an existing infection that has become reactive in the host. Over time, the number of IgM antibodies will decline (they persist up to 42 days PCV-2 post infection in pigs; Podgorskal et al., 2008 cited by (100)) as the active infection is resolved. The immune response observed in the pigs of the present study was not specific to the bacteria isolated from the lesion in the respective kidney, and we cannot conclude with certainty whether or not these microorganisms were indeed responsible for the macroscopic and microscopic renal lesions. What is clear, however, is that our results provide evidence of environmental exposure of the market hogs studied to those specific bacteria. The fact that some pigs may have been undergoing subclinical infections or

60 received antibiotic therapy at the farm may explain the subtle lesions, or negative bacterial findings by histochemistry. A positive result of western blot with IgG detection was defined by the presence of numbers of bands and not by the presence of specific bands as is performed in serological diagnosis with western blot assay in Lyme borreliosis (32, 34) and human immunodeficiency virus type 1 infection (73), which allow the determination of the antigenic specificity of the antibodies in the patient’s serum. Immunoblotting was used to determine the size of immunogenic PRRS viral proteins by Nelson et al., 1993 (89). In 29%, 13 out of 45 samples which were positive to bacterial growth, the specific bacteria was not identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI), even though is a powerful tool for the accurate mass determination of peptide mixture providing molecular weight information on intact molecules (66). These results can be explained at least by two ways. The first possibility is that the samples in question, in this case bacterial colonies selected were not pure and could have been contaminated with other bacteria from the same culture (mixed flora). The second possibility is that the information for that specific bacterium was not included in the database of the MALDI-TOF program used with this technique. Leptospirosis is a zoonotic disease found throughout the world. Before the widespread use and success of vaccination, Leptospira serovar pomona was endemic in many swine herds. Leptospirosis continues to be a potential public health threat because of the transmission by various feral and domestic animals that serve as reservoirs of infection. Some authors support that most cases of chronic interstitial nephritis in pigs, lesions often observed at slaughter, are leptospiral in origin, particularly associated with serovar Pomona (9, 78). On the other hand, Jones et al., 1987 (52) and Boqvist et al., 2003 (13)suggested that the presence of white spots in kidneys was an unreliable indicator of the presence of renal leptospirosis. In the present study, leptospires were not detected in the renal tissue of any of the animals using histochemistry and immunohistochemistry techniques (data not shown).Similar results were obtained by Martinez et al., 2006 (76) using histochemistry, immunohistochemistry, and bacterial culture methods. In addition, no leptospires were isolated from swine kidneys collected at

61 a slaughterhouse in Quebec, 2002 (30). Some authors suggest that by the age in which animals are slaughtered, leptospires would have been already cleared from the kidneys since they could have been infected at an early age, and/or received antibiotic therapy in the farm (76). Chappel et al., 1992 (22)found that Warthin-Starry technique, although specific, was of low sensitivity in their study, detecting only 20% of infected animals versus a high sensitivity of 95% of the microscopic agglutination test as a diagnostic procedure. Further serological tests from these pigs using microscopic agglutination test (MAT) and detection of leptospiral IgM-class antibodies by an ELISA test could confirm the prevalence of leptospires infection of these market hogs. It is not possible to distinguish between natural infection and vaccine-induced humoral immunity in swine on basis of results of the MAT, but it is generally accepted that a titre of >=400 on the MAT reflects natural infection. Antibody titers from immunization are short lived, ordinarily decreasing to <400 approximately 60 days after vaccination. It is important to mention that although vaccination of pigs decreases the severity of illness, it does not prevent infection or provide a cure for chronically infected pigs (16). The 9 cases with glomerulonephritis detected in this study implied a primary glomerular disease, which is usually originated by immune-complex deposition within the basement membranes of glomerular capillaries (14). One of the histologic changes in glomerulonephritis, the hypercellularity of the glomerular tuft, may be the result of proliferation of endothelial, epithelial, and/or mesangial cells. By light microscopy alone the assessment of these glomerular changes is difficult and somewhat subjective, and it is greatly improved by the combined use of immunofluorescence and transmission electron microscopy (78, 91). Glomerulonephritis is not often diagnosed in swine but does occur occasionally as a sporadic event at young age, and has been associated as a sequel to chronic infectious diseases such as hog cholera, and African swine fever (14). Glomerular lesions have been attributed to post-streptococcal infections and associated with vasculitis in multiple organs including skin (117, 134). The prevalence of glomerulonephritis in domestic animals has increased overtime. This increase could be due to the use of modified live virus vaccines, or to infections of low

62 pathogenicity able to produce persistent antigenemia and therefore resulting in immune- complex disease (78). Porcine dermatitis and nephropathy syndrome (PDNS) in feeder pigs is associated with porcine circovirus type 2 infection, and is characterized by a systemic necrotizing vasculitis that appears to be immune-mediated, glomerulonephritis and interstitial nephritis. Fibrin within glomeruli, necrotic inflammatory cells and erythrocytes within Bowman’s space are microscopic changes associated with this disease (114). Two out of five cases of glomerulonephritis from trial 1 were positive to PCV-2 nucleic acid in tissue, and they were associated with bacterial or PPV detection showing severe morphologic changes. The case associated with bacteria included necrotizing vasculitis. Morphologic evidence of pyelonephritis, inflammation of both pelvis and renal parenchyma, was occasionally seen in this study. Pyelonephritis was characterized by areas of tubulointerstitial subacute to chronic inflammation and necrosis, extending radially from the medulla towards the cortex, often along the medullary rays. Affected renal tubules contained variable amounts of detritus admixed with inflammatory cells within the lumen. Most cases of pyelonephritis arise from ascending urinary infections. Organisms involved in urinary tract infection are usually endogenous bacteria of the bowel and skin. In this study, microscopic examination of a case of pyelonephritis (case 73) with an abscess in the renal parenchyma revealed the presence of Gram-positive and Gram-negative bacteria by histochemistry, but no organisms were cultured from the lesions. Minimal lesions found in the 70% of the control kidneys and 8% of condemned kidneys characterized by focal accumulation of mononuclear inflammatory cells, are common findings in kidneys. Causes are probably not specific as some lesions may be primarily inflammatory and some may be in response to foci of antigen persistence in scars of other origins (78). Interstitial nephritis has been interpreted as a nonspecific lesion that could potentially be induced by many bacterial and viral pathogenic organisms in pigs (31). Porcine circovirus (PCV) is a member of the genus Circovirus of the family Circoviridae. The genomes of these small viruses without a lipid envelope replicate

63 through a rolling-circle mechanism, and show high recombination and nucleotide substitution rates. They have a single-stranded DNA genome that replicates through double-stranded intermediates. The genome contains 1767-1768 nucleotides, and proteins seem to be expressed only by ORF1 (replicase protein) and ORF2 (capsid protein). Two genotypes of PCV have been identified, PCV-1 and PCV-2, the latter has been associated with porcine circovirus-associated diseases, which can manifest as a systemic disease known as postweaning multisystemic wasting syndrome (PMWS), respiratory disease complex, enteric disease, porcine dermatitis and nephropathy syndrome (PDNS) or as reproductive problems, causing great losses in the pork industry (114). Porcine circovirus type 2 nucleic-acid was found within renal lesions in 7 out of 40 samples (17.5%) of trial 1, and in all of these cases concomitant porcine parvovirus and/or bacteria were also detected. Tubulointerstitial nephritis with no lesions in glomeruli were reported in pigs with naturally occurring PMWS with detection of PCV-2 nucleic acid in the cytoplasm of tubular epithelial cells as well as the cytoplasm of inflammatory cells. There was no obvious relationship between the severity of histological lesions and the amount of PCV- 2 genome in the kidney (113). In the present study, one case from trial 1 (case 43) which was positive with PCV-2 nucleic acid in tissue, showed lymphoplasmacytic tubulointerstitial nephritis and the presence of multinucleated giant cells in the cortex. The use of immunohistochemistry technique for detecting specific cell markers could classify different cell phenotypes present in the inflammatory lesions, as macrophages present in granulomatous interstitial nephritis. The method is described by Sarli et al., 2008 (113). A pattern of follicular nephritis, characterized by interstitial lymphoid follicle formation of inflammatory cells, was observed with more frequency in the kidneys of slaughtered pigs from Quebec than in the present study (30). Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Porcine circoviruses with 99% overall genome nucleotide similarity to those detected in most U.S. pork products have been found in the stools of U.S. adults. Frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for circovirus

64 transmission. It remains unknown whether PCVs simply pass through the human gastrointestinal tract or are capable of replication in it (67). There has been evidence of co-infection with PCV-2 and PPV in experimental and natural cases of PMWS, but PCV-2 would be the primary infectious viral agent causing this systemic disease in pigs, suggesting that in conjunction with exposure to PCV-2, exposure of piglets to undefined environmental factors and/or infectious agents that facilitate stimulation of the piglets’ immune system may be necessary to produce clinical disease (2, 58, 59). Clearly, this intriguing and unique synergistic relationship must be explored further. Porcine parvovirus nucleic acid was detected in the renal lesions of 20 out of 40 samples from trial 1(50%). Porcine parvovirus (PPV) belongs to the family Parvoviridae. Infection of swine with PPV occurs worldwide, and is endemic in most herds of pigs. PPV infection is a major cause of reproductive failure, resulting in foetal death and mummification, still births, and delayed return to oestrus, usually in the absence of outward maternal clinical signs. The most common routes of infection are oronasal and transplacental. PPV replicates autonomously and has physicochemical properties and genomic sequences which resemble those of minute virus of mice, H-1 rodent parvovirus, canine parvovirus, feline panleukopenia virus and B19 parvovirus (140). The viral genome is single-stranded linear DNA of approximately 4 to 6 kb, non-enveloped, with four virus-specific proteins described: three capsid protein and one non-structural protein (104). Virus has been isolated from kidneys, testicles, and seminal fluid of pigs that have been infected during gestation period after up to 8 month of age. Infection is usually subclinical in postnatal pigs, but the virus can be located in many tissues and organs, especially in lymphoid tissues. The virus had been identified in the feces of pigs with diarrhoea; however, there is no experimental evidence to suggest that PPV causes enteric disease, as do parvoviruses of several other species (80). Currently available vaccines against PPV are based on inactivated whole-virus preparations. Vaccinated primiparous sows showed a similar degree of serological response as non-vaccinated sows after infection with a heterologous PPVstrain, and a similar pattern of virus shedding as well.

Although low antibody titres apparently prevent disease, they will not stop the distribution of the virus in a vaccinated herd (53). In the present study, all the samples taken from the lesions of the kidneys and sent for porcine reproductive and respiratory syndrome virus detection by PCR test were reported negative. Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-strand RNA virus that belongs to the Arteriviridae family. The genomic RNA is approximately 15 kb, and encodes the RNA replicase (RNA-dependent RNA polymerase), the glycoproteins GP2 to GP5, the integral membrane protein M, and the nucleocapside protein N. A comparison of nucleotide sequences of different strains indicates that European and North American strains represent two distinct antigenic types (81). PRRSV causes abortion in sows and respiratory problems in piglets; it was first isolated in 1991 in the Netherlands and in the United States and Canada in 1992. PRRSV replicates primarily in pulmonary alveolar macrophages and intravascular macrophages in the lung and lymphoid tissues. The largest amount of PRRSV viral antigen and/or nucleic acid is observed in lungs and lymph nodes, but is also consistently observed in perivascular and intravascular macrophages in heart, brain, and kidney. In contrast, in stillborn and congenitally infected live born pigs, viral antigen and nucleic acid is in largest amounts in lymphoid organs, but not lung. Infection with PRRSV renders pigs more susceptible to some bacterial and viral diseases and has an additive or synergistic effect with some other bacteria or viruses to create more severe disease than either agent alone (142). In this study, one of the interesting lesions detected in twelve affected kidneys was vasculitis which has been described in the literature associated with virus infections (2, 25, 30), systemic porcine circovirus-associated disease (64) and septic thromboembolism (78). Five out of 12 cases of vasculitis observed in the present study were associated with a concomitant isolation of bacteria, 3 without detection of any microorganism, 2 with the detection of PCV-2 and bacteria, one with PPV, and one with PPV and bacteria. Vasculitis is an important component of a variety of infectious diseases in animals, but its pathogenesis is often unknown (87). Direct injury of endothelial cells by the infectious agents, or indirect damage caused by an immune-mediated inflammatory response, are

66 two mechanisms proposed to explain the development of vasculitis. Langohr et al., 2010 (64)suggested that the acute vasculitis often observed in PCV-2 infected pigs was directly caused by the virus since viral antigen was detected in endothelial cells, smooth muscle cells, and infiltrating inflammatory cells in blood vessels in pigs experimentally infected with PCV-2 serogroup b. No viral nucleic acid was observed in blood vessels of animals of the same experimental study which developed chronic arteritis. Vasculitis in chronically infected pigs may be the result of a persistent cell mediated immune reaction following viral infection. Renal lesions in pigs inoculated intranasally with PRRS virus were characterized by moderate to marked lymphohistiocytic interstitial nephritis, and mild to severe vascular changes associated with fibrinoid necrosis and leukocytic infiltrates at the junction of the tunica media and adventitia (25). Similar findings were observed in two cases of this study even though all kidneys were negative to PRRSV. On the other hand, as was the case in the present study, multifocal non-suppurative vasculitis has been reported as a frequent finding in the renal parenchyma of pigs infected with PCV-2 (2, 30). Considering the similarities of the vascular lesions described in PRRSV and PCV-2 infected pigs, immune-mediated involvement should be considered in both diseases. Several methods have been used for detection of porcine circovirus: virus isolation in cell cultures, electron microscopy, in situ hybridization, and the visualization of viral antigens by immunohistochemistry or indirect immunofluorescence. Although these techniques have a good sensitivity and specificity, they must be performed on postmortem specimens and can be time-consuming (17). Alternatively, PCR have been reported to be a useful tool for detecting porcine circovirus in clinical specimens (tissue and body fluids) from naturally and experimentally infected pigs with a high specificity ensured by sequencing the amplification products (97). PCR is fast, has high sensitivity, and can be performed on samples from live pigs (18, 79). However, it is important to keep in mind that viral DNA can be found not only in affected pigs but also in those with sub-clinical infections. Furthermore, the number of genome copies is not equivalent to the number of infectious virus particles determined by titration on tissue cultures (18); for this reason, the use of PCR alone is currently considered not sufficient for laboratory

67 diagnosis of porcine circovirus associated diseases. In the present study, PCR results detected by the Diagnostic Services Unit of AVC, UPEI, differed slightly from those obtained in the virology laboratory of the University of Calgary using different primers, and protocols. The presence of small amount of antigen of PCV-2 in tubular epithelium and tiny foci by immunohistochemistry method confirm one of the samples diagnosed as positive by PCR tests from trial 1, and two cases of the samples from trial 2. Porcine parvovirus causes reproductive failure in pregnant sows and can potentiate the effects of PCV-2 infection. PCR test from organs of aborted foetuses is a method of detection of PPV in tissue, but taken together the results of different diagnostic methods would be the best way to confirm a diagnosis of PPV infection. Serological test, PCR, isolation, and electron microscopy provided sufficient proof that PPV was the agent associated with abortions in a domesticated herd of wild boars and the authors suggested that could be a risk for pig farmers in potential contact with these susceptible wild suids (141). Porcine reproductive and respiratory syndrome virus can cause either reproductive failure or respiratory disease. Monocytes/macrophages are the likely principal target cells and vehicles for the virus dissemination. Reverse transcription PCR in milk whey and cellular fractions and immunohistochemistry in mammary glands and other tissues from experimentally infected sows were used to localize the virus (55). In summary, the lesions found in the kidneys of this study consisted of tubulointerstitial nephritis and glomerulonephritis. Bacteria were isolated from the lesions of the renal tissue in a high percentage of cases; some of these bacteria have been reported in the literature as etiologic agents of septicaemia in pigs and others had not been previously associated with renal lesion or septicaemia in swine. In order to study the immune response of the host to these bacteria isolated, a western blot test was performed and the results indicated that the majority of the pigs had been exposed to them in the past. In the kidneys of the market hogs studied, there was no evidence of acute embolic nephritis. The majority of the cases were diagnosed histologically as subacute or chronic nephritis. Some bacteria isolated from these pigs have been associated with nosocomial infections in humans and a few of them can be considered as potential zoonotic bacteria.

Porcine parvovirus was detected in half of the samples of trial 1, suggesting that the infection could be endemic in the local porcine herds. PPV has been associated with embryonic and foetal death in swine, but also has been described to potentiate the effects of porcine circovirus type 2 in dually infected pigs. PCV-2 was detected in a low percentage of the samples in association with bacteria or PPV and causing mainly moderate to severe microscopic changes in the renal parenchyma. Vasculitis was a relatively common finding in the present study, and in swine it is described as being the result of septic thromboembolism or viral infections, especially PCV-2 associated disease. In order to properly diagnose PCV-2, PPV, and PRRS virus infections, it is recommended to use more than one method of virus detection in tissue samples and serum in addition to a thorough gross and microscopic examination of different tissues from the same individual. The objectives of the meat inspection are: a) to ensure that only apparently healthy, physiologically normal animals are slaughtered for human consumption and that abnormal animals are separated and dealt with accordingly, and b) to ensure that meat from animals is free from disease, wholesome and of no risk to human health. These objectives are achieved by ante-mortem and post-mortem inspection procedures and by hygienic dressing with minimum contamination (www.fao.org). The role of the meat inspector is to determine whether the carcass or part of the carcass is fit for human consumption, given the macroscopic evidence of lesions presented. Of greatest importance are zoonotic diseases. Kidneys are condemned when they show external abnormalities during the meat inspection, but it is necessary to look for other systemic changes in the animal in order to determine the condemnation of the carcass with affected kidneys (41). In this study, 7 out of 10 (70%) of grade 0 or control kidneys were classified as normal by microscopic examination; 3 of them with moderate histological changes were associated with positive bacterial culture and PPV detection. On the other hand, 34 out of 50 (68%) of condemned kidneys were characterized with moderate and severe histological changes, 24% of them were classified with mild microscopical lesions, and only in 4 out of 50 (8%) had minimal histopathological changes.

Conclusions

Organ condemnations are cause of concern to hog producers, slaughterhouse workers, food inspectors and consumers because of financial loss and potential health risks or perceptions. Kidney condemnations due to lesions of multifocal interstitial nephritis, so called “white spotted kidneys” are important because pigs are reservoirs of zoonotic bacteria (Leptospira, Erysipelas, Streptococcus suis) and a source of persistent and latent virus infections with unknown public health consequence. Leptospira has been recognized as an emerging global public health problem because of its increasing incidence in both developing and developed countries (130). The disease in humans has probably been undiagnosed and misinterpreted as influenza infection or as a mild illness which recover without any complication; the infrequency of diagnosis is partially caused by a lack of availability of fast, reliable diagnostic assays (16). However, a small proportion develops various complications due to involvement of multiple organ systems. Although interstitial nephritis was commonly detected in condemned kidneys, in this study, evidence of Leptospira organisms was not found using histochemistry and immunohistochemistry. The actual incidence of Leptospira spp. in these pigs, however, is not known as serological testing was not performed. One of the original objectives of this study was to determine the incidence of infection by pathogens such as Actinobacillus suis and Actinobacillus equuli and to determine their role in embolic nephritis in swine. These agents were not detected in the kidneys in the present study, but bacteria which are not commonly described as pathogens were identified using MALDI-TOF mass spectrometry in addition to routine biochemical tests; Lactococcus garvieae, Arcanobacterium pyogenes, Kocuria kristinae, Acinetobacter lwofii are potential zoonotic pathogens. Moreover, Sphingobacterium multivorum and Stenotrophomonas maltophilia have been described as significant pathogens in immune-compromised patients with intrinsic resistance to multiple antimicrobial agents used to treat Gram-negative infections. The role of theses microorganisms as potential zoonotic pathogens, and the significance of their finding in

70 swine kidneys merit further studies in order to analyse their prevalence and mechanisms of infection. Antibiotics are still deemed necessary for the treatment and prevention of infectious diseases in farm animals intended for food production and to protect public health from food-borne diseases. Antibiotic use, mainly in swine and poultry by oral treatment of a large number of animals for prolonged periods of time and risk of underdosing might favour the selection of bacterial resistance (127). The immune response from the pigs studied to different bacterial proteins isolated from the renal lesions detected by western blot technique was an indicator that these animals were exposed to different microorganisms during their growth. It would be interesting to study in the future the role of those different proteins in the virulence of the pathogens, as well as to identify bacteria non recognized by mass spectrometry through sequencing proteins from the PVDF membrane. Bacteria were not detected in renal tissue by histochemistry, but viruses associated with nephropathy and reproductive problems in pigs, causing great losses in the pork industry, were found by PCR test and immunohistochemistry technique. Porcine circovirus type 2 was detected in 17.5% (7/40) of the tissue samples associated with severe microscopically changes, and porcine parvovirus in 50% (20/40) of tissue samples associated with normal to severe histological findings. Apparently, these viruses are host- specific, but their implication in human health remains unknown. The public health implications of subclinical infections in food animals should be emphasized. The lack of clinical signs of illness may give the people who manipulate live animals or handle tissues during processing a false sense of security and assume that animals are healthy and free of diseases. It is essential to educate employees working with animals or animal tissues, and to train them about the correct and efficient use of protective clothing to reduce the risk of exposure to many zoonotic pathogens. Kidneys are organs that indicate the stage of a past infection in the host, as well as presence of acute infection. The carcass can be approved for human consumption in cases of chronic nephritis or pyelonephritis, whether there is involvement of the renal lymph nodes or not, providing there are no other systemic changes on the antemortem and postmortem examinations (for example: emaciation, multiple abscesses in both

71 kidneys, evidence of septicaemia, toxemia or uremia). In cases classified as “white spotted kidneys” only the kidneys are condemned (Meat Inspection Manual, Alberta Agriculture, Food and Rural Development). In this study, the majority of cases of tubulointerstitial nephritis were in the chronic stage and etiologic agents were not specifically linked to the disease process. These results suggest that the manipulation of these kidneys at the slaughterhouse may not represent a high human health risk. The results of this project were obtained from a small sample size and included only market hog individuals, so they are not representative for the swine population of the Province of Alberta. Further studies including culled sows and sampling more than one slaughterhouse in Alberta will be suggest in order to have a prevalence of agents associated with interstitial nephritis or “white spotted kidneys” and their human health implications.

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1. APPENDIX 1

Histology

Haematoxylin and Eosin Stain

Procedure:

1. Immerse slides in xylene 5 min. to remove the wax.

2. Place slides in second xylene 5 min.

3. Place slides in 100% alcohol for 5 min. to remove xylene.

4. Place slides in a second absolute alcohol for 5 min.

5. Wash slides in 95% alcohol 3 min.

6. Wash slides in 70% alcohol 3 min.

7. Rinse in tap water 1 min and distilled water.

8. Stain slides in Haematoxylin for 8 min. (8-15 min.)

9. Wash thoroughly in distilled or tap water to remove excess Haematoxylin.

10. Differentiate in 1% Acid –Alcohol 4 to 8 dips.

11. Wash in tap water well to remove differentiator.

12. Blue in Saturated Lithium Carbonate around 10 sec. or less.

13. Wash well and check bluing microscopically.

14. Quick dip in 95% alcohol.(optional)

15. Stain 2 min. or less in Eosin.

16. Rinse in 95% alcohol 2 min.

17. Rinse in 95% alcohol 2 min.

18. Rinse in 100% alcohol 2 min.

19. Rinse in absolute alcohol 2 min. so last traces of water are removed.

20. Place in xylene 3 min.

21. Place in xylene 3 min. until last traces of alcohol are removed.

Mount with synthetic mountant.

2. APPENDIX 2

Histochemistry

Gram stain

Procedure: Step 1 to 7 is the same described in appendix 1(Deparaffinize and hydrate to distilled water) 8. Place slides on staining rack. Drop Crystal Violet onto the section for 1 min. 9. Rinse in tap water. 10. Drop Iodine onto slide for 1 min. 11. Rinse briefly in tap water. 12. Decolorize by gently rinsing with Acetone until purple stain is removed, usually 5 sec. 13. Rinse quickly in tap water. 14. Counterstain, 1 min. with basic fuchsin. 15. Rinse quickly in tap water. 16. Dip individually in acetone to start reaction (1 quick dip). 17. Differentiate each slide immediately with picric-acetone until sections are yellowish pink (7-8 dips). 18. Rinse quickly in acetone (1 dip), then in acetone-xylene (3 dips). 19. Clear in xylene, 2 min. 20. Mount with synthetic mountant. Results: Gram-positive bacteria blue Gram-negative bacteria red Nuclei red Other tissue elements yellow

3. APPENDIX 3

Periodic Acid-Schiff,s Stain (PAS) Procedure: Steps 1 to 7 identical for the stains described before. 8. Place slides in 0.5% periodic acid for 5 min. 9. Rinse in tap water. 10. Place slides in Schiff’s reagent for 15 min.

11. Wash slides in running tap water for 10 min.

12. Counterstain in light green for 1-2 min.

13. Rinse in tap water.

14. Rinse in 70% alcohol 3 min.

15. Rinse in 95% alcohol 3 min.

16. Rinse in 100% alcohol 2 min.

17. Rinse in absolute alcohol 2 min. so last traces of water are removed.

18. Place in xylene 3 min.

19. Place in xylene 3 min. until traces of alcohol are removed.

20. Mount with synthetic mountant.

Results:

Nuclei= blue

Fungi= red

Background= pale green

4. APPENDIX 4

Grocott’s Methenamine Silver

Procedure:

Steps 1 to 7 identical to deparaffinise and hydrate to distilled water.

8. Oxidize in 4% chromic acid for 1 hour.

9. Wash in tap water a few sec.

10. Sodium bisulfite for 1 min.

11. Wash in running water for 5 to 10 min.

12. Rinse in distilled water, 3 changes.

13. Place in freshly mixed methenamine-silver nitrate-at 58oC to 60oC for 50-60 min, until sections turn yellowish brown.

14. Rinse in 3 changes of distilled water.

15. Tone in gold chloride for 2-5 min.

16. Rinse in distilled water.

17. Sodium thiosulfate for 2 to 5 min.

18. Wash thoroughly in tap water.

19. Counterstain with working light green for 1-2 min.

Results:

Fungi= sharply delineated in black

Mucin= taupe to dark gray

Inner parts of mycelia and hyphae= old rose

Background= pale green

5. APPENDIX 5

Acid Fast (Ziehl-Neelsen Stain)

Procedure:

Steps 1 to 7 identical for the stains described before.

8. Stain in Carbol fuchsin for 1 hour at room temperature or in preheated 60 oC Carbol fuchsin for 15 min.

9. Wash slides in running tab water.

10. Decolorize the slides in 0.1% acid alcohol until the slides run clear.

11. Wash slides in running tab water 8 min.

12. Counterstain in 0.5% Methylene Blue for 50 sec.

13. Rinse in tap water and then rinse in distilled water.

14. Rinse in 70% alcohol 3 min.

15. Rinse in 95% alcohol 3 min.

16. Rinse in 100% alcohol 2 min.

17. Rinse in absolute alcohol 2 min. so last traces of water are removed.

18. Place in xylene 3 min.

19. Place in xylene 3 min. until last traces of alcohol are removed.

20. Mount with synthetic mountant

6. APPENDIX 6

Warthin-Starry for Spirochetes (modified)

Procedure:

Steps 1 to 7 identical for the stains described before.

8. Prepare buffer solution:

Walpole’s buffer

Acetic acid (11.8 ml acetic acid/litre) 18.5 ml

Sodium acetate (16.4 g sodium acetate/litre) 1.5 ml

Distilled water 480 ml

9. Bring sections through xylene, alcohols to buffer.

o 10. Place in 58 C oven in 1% AgNO3 (0.5g AgNO3 in 50 ml buffer) for 45 min.

11. Put slides face up on staining rod and put freshly mixed developer, and let stand until golden brown. (2-6 min. Depending on temperature of the developer) Developer:

2% AgNO3 in buffer 3 ml

5% gelatine in hot buffer 15 ml

3% hydroquinone in buffer 1 ml

12. When developer starts turning brownish black, pour off and rinse with warm tap water, then distilled water.

13. Counterstain with hematoxylin and eosin.

14. Rinse in 95% alcohol 3 min.

15. Rinse in absolute alcohol 2 min.

16. Rinse in 100% alcohol 2 min. so last traces of water are removed.

17. Place in xylene 3 min.

18. Place in xylene 3 min. until last traces of alcohol are removed.

19. Mount with synthetic mountant.

Results:

Spirochaetes= black

7. APPENDIX 7

Western blot

Sodium dodecyl sulphate (SDS)Buffer X5

5 ml 0.5M Tris pH6.8

10 ml 10% SDS

10 ml 50% Glycerol

2.5 ml 0.5% Bromophenol blue

22.5 ml H2O

10% Gel from 40% Acrylamide Stock

6.9 ml H2O dd

4 ml 40% Acrylamide

3.8 ml 1.5 M Tris pH8.8

150µl 10% SDS

150µl 10% APS

6µl TEMED

Isopropanol on the top

5% Stacking gel

4.4 ml H2O dd

750µl 40% Acrylamide

750µl 1.0 M Tris pH6.8+SDS

60µl 10% APS (Ammonium peroxydisulfate)*

6µl TEMED (Tetramethylethylenediamine)*

*catalyst for acrylamide gel polymerization

BenchMarkTM Pre-Stained Protein Ladder

Band 1 ≈180 kDa

Band 2 ≈115 kDa

Band 3 ≈82 kDa

Band 4 ≈64 kDa (orientation band pink in colour)

Band 5 ≈49 kDa

Band 6 ≈37 kDa

Band 7 ≈26 kDa

Band 8 ≈19 kDa

Band 9 ≈15 kDa

Band 10 ≈6 kDa

Enhanced chemiluminescent substrate

A B

5ml H2O 5ml H2O

50µl 1M Tris pH8.5 50µl 1M Tris pH8.5

50µl Luminol 5µl H2O2

22µl p-Coumaric acid