Canine gastric pathology. Helicobacter spp. Infection in dogs - an epidemiological and molecular study

IRINA FERRAZ AMORIM Canine gastric pathology Helicobacter spp. infection in dogs - an epidemiological and molecular study Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador - Professora Doutora Maria de Fátima Gärtner Categoria - Professora Catedrática Afiliação - Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto (ICBAS-UP). Co-orientador - Professor Doutor Celso Albuquerque Reis Categoria - Professor Auxiliar Convidado Afiliação - Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto (ICBAS-UP). Funding * Financiamento PhD Fellowship (SFRH/BD/76237/2011) provided by the Portuguese Foundation for Science and Technology (FCT) of the Portuguese Ministry of Science, Technology and Higher Education. Bolsa Individual de Doutoramento (SFRH/BD/76237/2011) da Fundação Portuguesa para a Ciência e a Tecnologia (FCT) do Ministério da Ciência, Tecnologia e Ensino Superior. II DECLARATION The author of this thesis declares that, in accordance with “nº 2, alínea a, do Art.º 31º do Decreto-Lei nº 230/2009”, afforded a major contribution to the design and technical execution of the work, interpretation of the results and manuscript preparation resulting in the following accepted and submitted articles: - Scientific publications Amorim I, Freitas DP, Magalhães A, Faria F, Lopes C, Faustino AM, Smet A, Haesebrouck F, Reis CA, Gärtner F (2014). A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. binding to canine gastric mucosa with defined gastric glycophenotype. Helicobacter. 19(4):249-59. Amorim I, Taulescu MA, Ferreira A, Rêma A, Reis CA, Faustino AM, Catoi C, Gärtner F (2014). An immunohistochemical study of canine spontaneous gastric polyps. Diagnostic Pathology. 18;9(1):166. Amorim I, Taulescu MA, Day MJ, Catoi C, Reis CA, Carneiro F, Gärtner F (2014), Unravelling canine gastric pathology. The Journal of Comparative Pathology (accepted with minor revisions). Amorim I, Smet A, Alves O, Teixeira S, Saraiva AL, Reis CA, Haesebrouck F, Gärtner F (2014). Epidemiological characterization of the Helicobacter spp. present in canine stomach. (In preparation) Taulescu MA, Valentine BA, Amorim I, Gärtner F, Dumitraşcu DL, Gal AF, Sevastre B, Catoi C (2014). Histopathological features of canine spontaneous non-neoplastic gastric polyps - a retrospective study of 15 cases. Histology and Histopathology. 29(1):65-75. This thesis also includes unpublished results. III Don’t dive in just to test the waters… Do it to make waves! V ACKOWLEDGMENTS First, I would like to thank my mentor and thesis advisor Fátima Gärtner; for her kindness, support, wisdom and guidance throughout my young career. She made me become a scientist and gave me a chance to do what I like most. I apologize for my mistakes, although they were proof that I was trying. Thank you! To my co-supervisor Celso Reis for keeping me calm and giving me peace of mind during the rough times without even noticing it. He led me on a path which I never imagined to be on and for that, I am very grateful. I hope I did not disappoint you. I owe deep thanks to Annemieke Smet and Freddy Haesebrouck. Without your help and collaboration, this thesis wouldn’t be possible. I would like to thank Augusto Faustino for his pertinent and helpful critiques. Professor Sobrinho Simões: I thank you for every kind smile that you gave me in the hall, reminding me that a great mind can also be down-to-earth and human like each one of us. Marian Taulescu for sharing his passion of gastric pathology with me and for showing me that we cannot do science alone. I thank my colleagues from the Glycobiology in Cancer and Expression and Regulation in Cancer groups who made me feel so welcome and encouraged. A special word to Ana Magalhães, Joana Carvalho e Daniela Freitas who nearly turned me into a biochemist. Three honest and hard-working friends who shared with me the feelings of anxiety, expectation, and happiness associated with the different lab results. To all the co-authors for their valuable suggestions and comments. I thank my genuine friends Caroline Esteves, Cristina Bacelar and Odete Alves. You were my support during any and every hour… in many different ways. Fátima Carvalho and Alexandra Rema for always being willing to do everything in their power to help me with whatever was necessary. Your support was priceless. To my entire family who never doubted that I could achieve this goal. VII Thank you Nuno for being by my side and for making me happy. I thank my mother… my best critic, yet my strongest supporter. Nuna… I hope someday you’d be proud of your mummy. And daddy… se tu estivesses aqui... sei que estarias feliz. VIII PREFACE It was just another busy day at the microscope… Prof Fátima Gärtner and I were calmly performing the routine diagnostic examination in that small, and often too warm, room in the old building of ICBAS where the multiheaded microscope was located. I remember we were delightfully examining a gastric biopsy of a cat, in which small organisms with the “shape of a telephone wire” were stacked in the superficial mucus. We were, as they say, “enjoying” that perfect pink tinted image of haematoxylin and eosin stain, in which two different worlds were colliding… Those small bacteria seemed to cling so strongly to the cell, just as a person hanging from a window would cling to the window sill... and it seemed that in that small instant, from a simple gesture, important decisions were being made! Just as a person hanging from the window sill must choose between letting go and falling into the abyss or swing back to the safety of the margin… It appeared that those bacteria were also struggling with a similar decision on that “precipice”... Climb up or not climb? Colonize or not colonize? Infect or not infect? I guess pathologists are like that. They can spend hours contemplating a simple image that is both immensely complex and seductively beautiful. And they can create a whole background story, a whole movie script if you will, from that small instant. And it was here that it all began… Before I knew it, I was completely enthralled in this mission. Throughout the course of my doctoral studies, I had the luck of being able to choose my subject matter and the privilege of doing something that I love and something that completes me. Of course there were some not so happy moments….quite a few in fact! When we are in the necropsy room, we are confronted with a lifeless corpse, the smell is rotten, and there are no feelings. As we “stir” the cold innards, the work is serious and tranquil. The void of emotion is filled by the enthusiasm to discover and learn, by the enthusiasm of the students, by my enthusiasm of their enthusiasm. The necropsies in the kennel were not like this. There, there are remnants of life. Your hands shift through once pulsating organs that are still warm. The intense smell of blood seems to penetrate and…..the sensation is different! One’s concentration is diminished and one tries to hurry the process because of the “waiting line.” We try to be professional but we can feel for the waiting animals whose destiny is one of silence. They can sense it too… They know what will happen next. There are barks and groans in the background and our heart breaks. IX IFN - Interferon Ig - Immunoglobulin IL - Interleukin IM – Intestinal metaplasia K - Potassium Le - Lewis antigen LPS - Lipopolysaccharide M - Molar MALT - Mucosa-associated lymphoid tissue MCP – Methyl-accepting chemotaxis proteins MG – Modified Giemsa min - Minute ml – Milliliter MUC – Mucin µM - Micromolar µl - Microliter Na - Sodium NA – Not applicable NAP - neutrophil-activating protein NHPH - Non-Helicobacter pylori Helicobacter ND - Not determined NSAID - Non-steroidal anti-inflammatory drug Oip - Outer inflammatory protein OMP - Outer membrane protein PAS - Periodic acid schiff PCR - Polymerase chain reaction RBC – Red blood cells RER - Rough endoplasmic reticulum RNA - Ribonucleic acid Sab - Sialic acid binding adhesin Ser - serine Th - T-helper Thr - Threonine Vac - Vacuolating cytotoxin VIP – Vasoactive intestinal polypeptide WSAVA – World Small Animal Veterinary Association WHO – World Health Organization U - Unit XVI TABLE OF CONTENTS CHAPTER 1 General Introduction - Histology of the stomach - Glycophenotype of gastric mucosa - Gastric Helicobacter species in humans and dogs - Gastric Helicobacter species related disease in humans and dogs - Pathogenesis of Helicobacter infections References Paper I: Unravelling canine gastric pathology Aims and Objectives CHAPTER 2 Helicobacter spp. adhesion to canine gastric mucosa Paper II: A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. binding to canine gastric mucosa with defined gastric glycophenotype CHAPTER 3 Epidemiological characterization of Helicobacter spp. present in the canine stomach Paper III: Epidemiological characterization of Helicobacter spp. present in the canine stomach CHAPTER 4 Molecular features of canine gastric lesions Paper IV: An immunohistochemical study of canine spontaneous gastric polyps CHAPTER 5 General Discussion References Summary and Conclusions Future Work and Perspectives APPENDIX XVII 1 5 10 17 19 22 29 41 94 97 99 113 115 133 135 143 145 152 156 158 159 - General Introduction - Aim and Objectives - General Introduction - Aim and Objectives The original illustrations of this chapter were created by the students David Lopes and Hugo Fernandes from the Faculdade de Belas Artes da Universidade do Porto (FBAUP-UP) and by Dr. Carlos Diogo Paulo, researcher at the Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP). General Introduction - Histology of the stomach - Glycophenotype of gastric mucosa - Gastric Helicobacter species in humans and dogs - Gastric Helicobacter species and associated disease in humans and dogs - Pathogenesis of Helicobacter infections Paper I - Unravelling canine gastric pathlogic Aim and Objectives 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 - Histology of the stomach The stomach is an expanded organ of the digestive system located between the oesophagus and the small intestine. Four different anatomical regions are identified in the stomach: cardia, fundus, body and antrum (or pylorus) (Dellman and Eurell, 1998). The cardia is a narrow band near the opening of the oesophagus. The fundus is the dome-shaped region of the stomach. The body accounts for the major portion and narrows distally as it merges with the antrum. The antrum is the distal part of the stomach and includes the pyloric sphincter (Wilcock, 2013) (Figure 1). The anatomic boundaries separating these regions are not macroscopically distinct, at least not in the unopened specimen. Figure 1 – Schematic representation of the different stomach regions. (Illustration by David Lopes) 5 45 General Introduction CHAPTER 1 Grossly, the stomach presents a number of folds or ridges, called rugae, formed by the submucosa and mucosa, which serve to accommodate the filling and expanding of the stomach. The stomach wall is composed of layers. From inside to outside, the first main layer is the mucosa. This consists of an overlying epithelium, supported by the lamina propria and underneath a thin smooth muscle layer called the muscularis mucosae. The second layer is the submucosa composed of dense irregular connective tissue. The following smooth muscle layer consists of a circular and longitudinal part lined by the serosa (Dellman and Eurell, 1998). The fundus and the body present identical microscopic structure and therefore only three regions are histologically considered. The other gastric regions are histologically distinct and are mainly characterized according to the nature of the glands present (Junqueira and Carneiro, 2005). Histologically, the surface of the stomach is lined by a simple columnar epithelium whose cells are called surface mucous cells. These produce a cloudy, viscous and alkaline mucus that forms a thick gel-like coat that adheres to the surface epithelium. There are numerous invaginations of the surface epithelium into the lamina propria. These invaginations are called gastric pits or foveolae and are also lined by surface mucous cells. The cardia region contains cardiac glands which are heavily branched tubular structures composed almost entirely of mucous-secreting cells, with the odd neuroendocrine cells (described below) present. Their secretion protects the oesophagus against gastric reflux. Gastric pits in the cardiac region are fairly shallow (Wilcock, 2013). The gastric or fundic glands present in the fundic region extend all the way to the muscularis mucosae. In this region, almost the entire lamina propria is occupied by glands. The lumina of the glands are usually not identifiable and they usually appear more like cords of cells. The only “typical” lamina propria can be seen in the areas between the foveolae and around the bases of the glands (Figure 2). The following cells types can be seen in the glands of the fundic region: mucous-neck cells, parietal (or oxyntic), chief (zymogenic) and neuroendocrine cells (Dellman and Eurell, 1998; Junqueira and Carneiro, 2005) (Figure 3) . The mucous neck cells are located in the neck region and secrete a soluble mucous only under vagal stimulation (not in the resting stomach). They have an irregular shape, with nuclei placed at the basal pole of the cell and the secretory granules close to the apical surface. The type of secreted mucin is different from that of the surface mucous epithelial cells (Junqueira and Carneiro, 2005). 45 General Introduction CHAPTER 1 6 45 General Introduction CHAPTER 1 Figure 2 – Schematic representation of the anatomy and histology of the carnivorous stomach. (Illustration by Hugo Fernandes). Parietal (or oxynctic) cells are found predominantly in the upper part of the gland interspersed among the mucous neck cells. They are large in size, rounded or pyramidal, present spherical nucleus that occupies the central position and cytoplasm intensely eosinophilic due to the presence of numerous mitochondria and of extensive intracellular canaliculi (Junqueira and Carneiro, 2005). The latter remain important structures in the mechanism of hydrochloric acid production as parietal cells secrete the hydrochloric acid (HCl) of the gastric juice and intrinsic factor (a glycoprotein that binds to vitamin B12, 7 45 General Introduction CHAPTER 1 Previous studies demonstrated that, in normal human gastric mucosa, type 1 antigens Lea and Leb are mainly expressed in the surface of the gastric epithelium. On the contrary, type 2 antigens are found deeper in the gastric glands (Lopez-Ferrer et al., 2000). Canine blood groups and canine secretory alloantigen alloantibody system (CSA) The first studies on canine blood groups date from 1910 by Von Dungren (as cited by (Swisher and Young, 1961) and since that, more than 20 different blood groups have been reported in the dog. As dogs do not develop clinically significant naturally occurring alloantibodies most of this filed research have been performed through laboratorial induction of antibodies by mismatched transfusion (Hohenhaus, 2004). Canine blood groups are currently denominated by the acronym DEA (dog erythrocyte antigen) followed by the numerical designation of the blood group. Seven different groups have received international standardization however, typing sera is only available for five of them (DEA1, DEA3, DE4, DEA5, DEA7/Tr). The DEA7 is also known as Tr which is a soluble antigen that is not produced by the RBC but is absorbed at the cell membrane as similar to the human Lewis antigen (Bull et al., 1975). The biochemical structure of specific blood group antigens has not been identified but it was determined that the sphingoglycolipids present in the canine erythrocytes membrane contain sialic acid residues (Hohenhaus, 2004). The described blood groups in dogs have no antigenic or serologic relationship to human blood groups; however, like human intestinal cells, canine intestinal cell fucolipids have human blood group activity. Canine intestinal fucolipids with blood group H activity have an oligosaccharide portion (fucose) identical to that from human erythrocytes (Smith et al., 1975). Blood group A fucolipids are found in canine intestinal glycolipid profiles. Qualitative analysis of sugar composition showed canine intestinal fucolipids with glucose, galactose, glucosamine, galactosamine, and fucose with blood group A activity (Smith et al., 1975). Antisera against Tr (DEA 7) cross reacts with human group A cells; consequently, the Tr antigen appears to have similarity with the A antigen of human A1 red blood cells (Bowdler et al., 1971). Additionally, the saliva of Tr positive dogs contains anti-A and anti-H activity (Bowdler et al., 1973). Despite the presence of anti-H activity in canine saliva, no H substance has been identified on canine erythrocytes, but it has been identified in canine intestine cells (Smith et al., 1975). 45 General Introduction CHAPTER 1 14 45 General Introduction CHAPTER 1 The canine secretory alloantigen alloantibody system (CSA) is closely related to the human ABH-Le system being considered equivalent to the major human ABO exocrine glycophenotype. The CSA was first described in 1966 by Zweibaum and colleagues (Zweibaum et al., 1966). The genetic polymorphisms of the CSA were later analysed (Zweibaum et al., 1974) and the structure of canine polymorphic antigens was further characterized in four phenotypes: A, X, Y, and AY whose structures and chemical linkages were proposed based on inhibition tests observed with oligosaccharides (Oriol et al., 1975) (Figure 6). The same authors reported the presence of only type 2 chains in dog intestinal secretions. No further information is available about the glycosylation profile of the canine gastric mucosa. Figure 6 - Schematic representation of canine secretory alloantiagens (CSA) polymorphisms. (Adapted from Oriol et al., 1975) Lectins Lectins are natural carbohydrate-binding proteins often found in extracts of seeds from plants, which react specifically with certain carbohydrate antigens. Two common and relevant forms are the Ulex europaeus or lectin H, which agglutinates cells that have H antigen; and another that has been used in the present study is Dolichos biflorus, lectin DBA or A1, which agglutinates cells with A1 (Bird, 1952). Mucins Mucins are a family of high molecular weight, heavily O-glycosylated proteins expressed by several epithelial tissues that can be produced either as membrane bound or as secreted proteins (Kufe, 2009). In the human gastric mucosa, the mucus gel that covers the mucosal surface represents the interface between the epithelial cell layers and the exterior environment and is composed of a high carbohydrate content of mucins (Linden et al., 2008b). The mucus 15 45 General Introduction CHAPTER 1 gel has a thickness of approximately 300 µm and can be divided in two layers: one firmly attached to the mucosa whose thickness increases, from the body to the antrum regions, with values ranging from 80 to 154 µm (Linden et al., 2008b; Moore et al., 2011; Phillipson et al., 2008) and other layer more loosely adherent. The mucus gels acts as a physical barrier that lubricates and defends the epithelial cells from external aggressions displaying important protective properties. Furthermore, it allows the establishment of a pH gradient with the bicarbonate ions secreted by the surface epithelial cells counteracting the diffusion of luminal acid, leading nearly to a neutral pH at the cell surface while the gastric lumen displays acidic properties (Bhaskar et al., 1992). The human mucin (MUC) family includes 21 members (MUC1 to MUC21) (Kufe, 2009). In a healthy gastric mucosa, the mucins produced are MUC1, MUC5AC and MUC6. The membrane-associated MUC1 is expressed in foveolar cells and, to a lesser extent, in mucous glands. The secreted MUC5AC mucin is restricted to the foveolar epithelium and is a major constituent of the surface mucous gel layer, whereas the expression of the secreted MUC6 is limited to the glands (Reis et al., 2000; Reis et al., 1997; Reis et al., 1998; Teixeira et al., 2002). This mucin distribution determines the gastric glycosylation pattern since expression of MUC5AC is accompanied by similar distribution of fucosyltransferases leading to co-expression of type 1 Lewis a (Lea) and Lewis b (Leb) blood group antigens, while MUC6 expression is associated with the type 2 Lewis x (Lex) and Lewis y (Ley) antigens (Magalhaes and Reis, 2010). Mucins are frequently regarded as preferential binding sites for infectious agents, mainly because of their high glycans content (Linden et al., 2008b). This may be beneficial for the host as they act as releasable decoys in which the pathogens are trapped and them removed and eliminated (Linden et al., 2008a; Moore et al., 2011). In contrast, the binding to specific glycans in mucins may represent a threat to the host since it allows the pathogens to establish a more intimate contact with epithelial cells favoring the colonization. Virtually all interactions between microbial pathogens and their hosts are mediated by cell surface expressed glycans. Therefore it is expected that both the host and the pathogen glycans landscape will define the pathogen tropism for a particular organ, tissue or specific cell. The glycan-receptor interactions may be essential for the initial colonization of host epithelial cells and establishment of infection, but can also favour microbe recognition by the host immune cells, stimulating an immune response. An example of a bacterium that exploits the glycans receptors expressed by the host cells to survive and successfully colonize a tough niche is the human gastric pathogen Helicobacter pylori (Magalhães and Reis, 2010),. 45 General Introduction CHAPTER 1 16 45 General Introduction CHAPTER 1 - Gastric Helicobacter spp. in humans and dogs The first description of a gastric member of the Helicobacter genus was reported by Marshall and Warren in 1984 (Marshall and Warren, 1984). The discovery of the association between the presence of these bacteria in the human stomach and ulcer diseases awarded Marshall and Warren the Nobel Prize of Medicine and Physiology in 2005. The causative agent, Helicobacter pylori (H. pylori), has also been associated with gastritis, peptic ulcer disease, gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT) lymphoma (Parsonnet et al., 1991; Stolte and Eidt, 1993). H. pylori is a microaerophilic, Gram-negative, spiral-shaped bacterium 0.5-1.0 µm wide and 2.4-4.0 µm long, with 5 to 7 unipolar sheathed flagella that colonizes the human stomach of one half of the human population worldwide (Forman et al., 1993). In developing countries and low socio-economic classes the bacterium is usually acquired in infancy or early childhood and the infection prevalence is high with more than 80% of the population being infected (Holcombe et al., 1992; Segal et al., 2001). On the other hand, in developed countries, the prevalence of H. pylori usually remains under 40% and is considerably lower in children and adolescents than in adults and elderly people (Pounder et al., 1995; Kusters et al., 2006). In most individuals, H. pylori infection is asymptomatic. About 25% to 30% of infected individuals will, however, one day experience the disease. In addition to H. pylori, another non-Helicobacter pylori Helicobacter (NHPH) organism was found in the stomach of patients suffering from dyspepsia (McNulty et al., 1989). This tightly coiled organism, with 10 to 20 bipolar sheathed flagella, was clearly distinguishable from H. pylori. This bacterium was first referred to as “Gastrospirillum hominis”. However, following 16S rRNA sequencing, it was designated to the Helicobacter genus and the name “H. heilmannii” was proposed (Heilmann and Borchard, 1991; Solnick et al., 1993). NHPHs generally referred to as “H. heilmannii” were further subdivided in two taxa, types 1 and 2 (Haesebrouck et al., 2011). Microorganisms referred to as H. heilmannii type 1 are identical to H. suis, a species colonizing the stomachs of pigs (De Groote et al., 1999). The former H. heilmannii type 2 represents a group of species known to colonize the gastric mucosa of dogs and cats, which includes H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis and a bacterium which was given the provisional name of ‘‘Candidatus H. heilmannii’’ in 2004 because, at that time, it could not be cultured in vitro (O’Rourke et al., 2004). In 2012, a description of H. heilmannii as a novel species was achieved (Smet et al., 2012). The name H. heilmannii sensu stricto (s.s.) was proposed 17 45 General Introduction CHAPTER 1 to refer to the novel Helicobacter species and the term H. heilmannii sensu lato (s.l.) to refer to the whole group NHPH (Haesebrouck et al., 2011). Since the description of H. pylori, the number of species belonging to the genus Helicobacter rapidly expanded and currently it is composed of at least 32 species with validly published names (Haesebrouck et al., 2009). A large number of NHPH species have been recognized in humans and in a wide variety of animals, including dogs. H. felis was first cultured by Lee and colleagues (Lee et al., 1988) from the stomach of a cat and later, the group of Paster also isolated this organism from canine gastric mucosa (Paster et al., 1991). This organism is tightly coiled and possesses 14 to 20 bipolar sheathed flagella. The presence of periplasmic fibrils that encase the bacterium was thought to be a unique feature of this gastric Helicobacter species until the isolation of H. cynogastricus from the canine mucosa (Van den Bulck et al., 2006). Later, another spiral organism morphologically distinct from H. felis was isolated from the canine mucosa (Hanninen et al., 1996). This organism lacked periplasmic fibrils around its thin body and had 10 to 20 bipolar flagella. It was named as H. bizzozeronii after Guilio Bizzozero confirmed by DNA-DNA hybridisation that it was a new Helicobacter species. Then, H. salomonis was isolated from dogs and it was named in honour of Hugo Salomon (Jalava et al., 1997). It has 10 to 23 bipolar sheathed flagella and no periplasmic fibrils. Several genome sequences of H. pylori are available and in the last few years, the genomes of other dog-related gastric Helicobacter spp. were also published. The genomes of the type strain of H. felis isolated from a cat (Arnold et al., 2011); of H. bizzozeronnii strain CIII-1 obtained from the human stomach (Schott et al., 2011b) and of H. heilmannii s.s. isolated from the gastric mucosa of a kitten with severe gastritis (Smet et al., 2013) were reported. Additionally, the H. salomonis genome has been recently completed, but results have not yet been published. The comparative genome analysis between H. pylori and these dog-related NHPH have provided new insights into the comprehension of the biology of these particular gastric organisms belonging to the Helicobacter genus (Arnold et al., 2011; Eppinger et al., 2006; O’Toole et al., 2010; Schott et al., 2011b; Smet et al., 2013). 45 General Introduction CHAPTER 1 18 45 General Introduction CHAPTER 1 - Gastric Helicobacter spp. related disease in humans and dogs Helicobacter pylori is the main cause of gastric inflammation in virtually all infected subjects (Farinha and Gascoine, 2005). Most of the infected individuals show few or none symptoms, and only a small percentage will develop severe gastric disease (Amieva and El-Omar, 2008). The first and acute phase of H. pylori human infection consists of polymorphonuclear cells infiltration into the gastric epithelium and lamina propria, accompanied by oedema. This step is very brief and is gradually replaced by a chronic active inflammatory response characterized by progressive mononuclear cells infiltration, mucosa-associated lymphoid tissue, as well as damage to the epithelial cells. The pattern and distribution of chronic gastric inflammation is associated with the type of lesions observed: individuals with bodypredominant gastritis and normal or reduced acid production are more likely to develop gastric ulcers, gastric atrophy, gastric intestinal metaplasia and ultimately, gastric carcinoma while individuals with antral-predominant gastritis, which is the most common form, show increase acid production and increased risk to develop duodenal ulcers (Kusters et al., 2006; Lochhead and El-Omar, 2007). Additionally, H. pylori has been responsible for 7085% of gastric ulcers and 90-95% of duodenal ulcers (Kusters et al., 2006). The next phase of human H. pylori infection is dominated by alterations of the epithelial cell cycle, especially increased rates of apoptosis and cell proliferation. These changes may be responsible for the multifocal atrophy that characterizes the type of gastritis associated with an increased risk of cancer. In this more advanced phase nuclear and architectural abnormalities become notice, which may represent progressive mutational events as expected in classical molecular models of carcinogenesis (Correa, 2004). Although only a small proportion of patients with H. pylori will eventually develop malignant disease, the widespread high prevalence of this bacterium explains that gastric cancer remains the fifth most common cancer worldwide (Globocan, 2012). Thus, based on epidemiological evidences, in 1994 the International Agency for Research on Cancer classified H. pylori as a class I carcinogen (IARC, 1994). In fact, H. pylori infection has a pivotal role in the development of two different gastric malignancies: gastric adenocarcinoma and gastric MALT lymphoma (Farinha and Gascoyne, 2005; Parsonnet, 1994; Parsonnet et al., 1991; Stolte et al., 2002) Gastric adenocarcinoma is subclassified into intestinal and diffuse types (Lauren, 1965). The sequence of pathological changes leading to the development of an intestinaltype start with gastritis, followed by gastric atrophy and progressing to intestinal metaplasia, 19 45 General Introduction CHAPTER 1 dysplasia and ultimately, carcinoma (Correa, 2004). Most of the individuals affected by this particular subtype are middle aged or older. The diffuse-type of gastric cancer tends to occur in younger individuals and has a stronger genetic component (Carneiro, 2012). The association between MALT-lymphoma and H. pylori was first reported in 1991 (Wotherspoon et al., 1991). H. pylori is responsible for 92 to 98% of gastric MALTlymphomas (Mbulaiteye et al., 2009). Based on recent knowledge about the existence of genetic abnormalities in this kind of lesion, a model of multistep pathogenesis was proposed: on the background of the chronic inflammation caused by H. pylori not only reactive B-cells are stimulated but also activated neutrophils, which can lead to production of oxygen species. As a result, this genotoxins provoke DNA damages that are responsible for mutations and transformations of genetic material and consequently, genetic instability (Farinha and Gascoyne, 2005; Witkowska and Smolewski, 2013). The diagnosis of H. pylori infection can be done by invasive methods such as endoscopy followed by biopsy (in order to perform histology or culture) or non-invasive tests like urea breath test and serological tests (Correa, 2004; Kusters et al., 2006; Suerbaum and Michetti, 2002). On histology, H. pylori are identified on the basis of their typical localization and their characteristic slightly curve-shaped morphology. Although less often, gastric NHPH are also able to cause disease in humans. In the human stomach, NHPH infections may be accompanied by acute gastritis (Lavelle et al., 1994; Yoshimura et al., 2002), active chronic gastritis (Haesebrouck et al., 2009), erosions mainly located in the antrum (Boyanova et al., 2003; Debongnie et al., 1998; Dieterich et al., 1998; Seo et al., 2003) and duodenal ulcers (Borody et al., 1991; Goddard et al., 1997; Iwanczak et al., 2012; Jhala et al., 1999). Glandular atrophy and intestinal metaplasia of the fundic gastric mucosa were reported. However, all these lesions seem to be less common and less severe than those associated with H. pylori (Yoshimura et al., 2002). Human NHPH infections have also been associated with low-grade MALT lymphoma of the stomach, and the risk of developing this disease is higher with NHPH than with H. pylori (Morgner et al., 1995; Morgner et al., 2000a; Morgner et al., 2000b). They have been reported to resolve after clearance of the NHPH, thus emphasizing a causal relationship (Morgner et al., 2000b; Regimbeau et al., 1998; Thomas-Marques et al., 2005). Some human patients are asymptomatic (Mazzucchelli et al., 1993) while others refer atypical complaints such as acute or chronic epigastric pain and nausea, hematemesis, recurrent dyspepsia, irregular defecation frequency and consistency, vomiting, heartburn, dysphagia and loss of appetite (Dieterich et al., 1998; Goddard et al., 1997; Heilmann and 45 General Introduction CHAPTER 1 20 45 General Introduction CHAPTER 1 Borchard, 1991; Iwanczak et al., 2012; Kaklikkaya et al., 2002; Mention et al., 1999; Oliva et al., 1993; Roehrl et al., 2012; Seo et al., 2003; Sykora et al., 2003; van Loon et al., 2003; Wuppenhorst et al., 2013; Yang et al., 1998). Histologically, the presence of NHPH in human gastric mucosa is mainly characterized by lymphocytic infiltration into gastric foveolae, sometimes accompanied by plasma cells. Lymphoid aggregates may be present and in some cases, superficial mucus was depleted (Flejou et al., 1990; Ierardi et al., 2001; Joosten et al., 2013; Kaklikkaya et al., 2002; Oliva et al., 1993). Human infections with NHPH most likely originate from animals. Indeed living in close proximity to animals has been identified as a risk factor (Lavelle et al., 1994; Meining et al., 1998; Thomson et al., 1994). The intensity of this contact also seems to be important since a higher incidence of these infections was noted in farmers, staff of slaughterhouses, veterinarians, pet owners and children having intense contact with pets (De Bock et al., 2007; Joosten et al., 2013). In agreement, the prevalence of porcine, canine and feline helicobacters in human gastric biopsies was documented and H. suis, H. felis, H. bizzozeronii and H. salomonis were present in 39.6%, 14.6%, 4% and 21%, respectively (Van den Bulck et al., 2005). Recently, the relationship between pet ownership or frequent exposure to dogs and infection with different gastric Helicobacter species was assessed (Chung et al., 2014). A significant correlation was found between human and canine infection for H. felis and to a lesser extent for H. bizzozeronnii. On the other hand, no clear association with animal contact was found in a study who reported a 0.2% of incidence of gastric NHPH infection in dyspeptic Polish children (4–18 years of age) (Iwanczak et al., 2012). Although H. pylori and these dog-related NHPH colonise similar niches, an important difference between both is the later capability to move from a dog to a human (Haesebrouck et al., 2009). Thus, when transferring from dogs to humans, NHPH necessarily undergo intensive changes to adapt to a new host. This NHPH ability for ongoing host-adaptation is probably related to their high level of genome plasticity (Schott et al., 2011a). In summary, NHPH are of zoonotic significance and the dog remains a natural reservoir for many species (Haesebrouck et al., 2009). Further information concerning the presence of these bacteria in the canine stomach and their pathogenic implications are described ahead in this chapter (paper I). 21 45 General Introduction CHAPTER 1 - Pathogenesis of Helicobacter spp. infections Most of the research concerning the pathogenesis of Helicobacter infections is focus on H. pylori. The success of long-term gastric mucosa colonization by H. pylori is dependent on several bacterial factors and of specific properties of this bacterium (Amieva and El-Omar, 2008): 1) its urease activity enables it to survive in the extreme acidic conditions of the human stomach (Eaton et al., 1991); 2) its high motility allows it to move easily in the viscous mucus of the stomach (Eaton et al., 1992); 3) its capability to adhere to the gastric epithelium due to the presence of several adhesins (Boren et al., 1993; Hessey et al., 1990 and 4) its ability to escape to the immune host response. The clinical outcome of H. pylori infection is diverse and dependent on the H. pylori strain virulence characteristics, host genetic susceptibility, environmental factors and their interactions. In contrast, very little is known about the specific direct and indirect pathogenic effects of other gastric NHPH and about differences in pathogenicity between different strains within the same species of NHPH (Haesebrouck et al., 2009). Acid Acclimation An important mechanism of survival that is required for the colonization of the stomach and that is share by all gastric Helicobacter species is the production of urease (Haesebrouck et al., 2009). This enzyme, which hydrolyzes urea to ammonia and carbon dioxide leading to a local pH increase, allows bacteria to survive and to multiply and consists of two subunits: UreA and UreB, which are encoded by two genes ureA and ureB (Hu and Mobley, 1990). Urease enzyme activity is tightly controlled by a pH-gated urea channel (ureI), which is open at low pH and closed at neutral pH conditions, allowing the bacterium a precise level of control over its pH environment. Urease is mainly localised in the cytoplasm but becomes associated with the surface of the viable bacteria after autolysis of surrounding Helicobacters (Krishnamurthy et al., 1998; Marcus and Scott, 2001; Phadnis et al., 1996). However, the contribution to acid resistance is solely the result of the cytoplasmic urease (Scott et al., 1998). The two subunits UreA and UreB of H. felis urease are considered homologous but not identical to those of H. pylori (Ferrero and Labigne, 1993; Gootz et al., 1994). A second urease system was also identified in this species - the UreA2B2 - but its function and regulation are unknown (Pot et al., 2007). High homology between the urease gene cluster of H. bizzozeronnii and those of H. felis and H. pylori is also documented (Zhu et al., 2002). 45 General Introduction CHAPTER 1 22 45 General Introduction CHAPTER 1 However, H. bizzozeronnii lacks the additional urease gene (Schott et al., 2011a). The presence of urease in the stomach is the basis of the most widely used noninvasive test for human H. pylori diagnosis, the urea breath test. A recent study concluded that this method is also quite useful for the detection of gastric Helicobacter spp. infection in dogs (Kubota et al., 2013). Motility and Chemotaxis Motility is fundamental for colonization of the mucosa by gastric Helicobacter species (Fischer et al., 2009). Gastric helicobacters possess monopolar (H. pylori) and bipolar (H. felis, H. bizzozeronnii, H. salomonis, H. heilmannii s.s., H. cynogastricus) bundles of 2 to 23 flagella. The flagella is composed of a body, hook and flagellar filament. The complex flagellar filament is covered by a sheath and is composed of two flagellin subunits: the more abundant is the FlaA and the minor is the FlaB. It was verified that both subunits are necessary for full motility in vitro (Josenhans et al., 1995) and in vivo (Eaton et al., 1996). Additionally, the sheath is suspected to play a role in acid protection, masking of antigens and possible adhesion (Jones et al., 1997). Josenhans et al. (1999) cloned H. felis flaA and flaB genes and mutants were constructed. These mutants were described as poorly motile in vitro and the loss of the ability to colonize the mouse stomach of the flaA mutant was reported. Thus, it was assumed that any impairment in motility would have consequences for gastric colonisation by H. felis (Josenhans et al., 1999). The basal body of the flagella is embedded in the bacterial cell wall and contains the proteins required for rotation and chemotaxis. Bacteria use chemotaxis to migrate towards environments that are better for growth. Chemoreceptors detect changes in attractant levels and signal through two-component systems to control the swimming direction (Porter et al., 2011). 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Van den Bulck, K., Decostere, A., Baele, M., Driessen, A., Debongnie, J. C., Burette, A., Stolte, M., Ducatelle, R. and Haesebrouck, F. (2005). Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats. J Clin Microbiol, 43, 2256-2260. Van den Bulck, K., Decostere, A., Baele, M., Vandamme, P., Mast, J., Ducatelle, R. and Haesebrouck, F. (2006). Helicobacter cynogastricus sp. nov., isolated from the canine gastric mucosa. Int J Syst Evol Microbiol, 56, 1559-1564. 39 45 General Introduction CHAPTER 1 remain in the body of the stomach moving intermittently into the pylorus (Sullivan and Yool, 1998; Wilcock, 2013) The International GI Standardization Group of the World Small Animal Veterinary Association (WSAVA) has provided guidelines for the normal histology of the stomach (Day et al., 2008). The guidelines suggest that neutrophils should not be present in the normal canine gastric mucosa, so their presence generally suggests an acute inflammatory process which may or may not involve mucosal ulceration. However, there is one category of acute gastritis commonly found in the dog: acute non-erosive gastritis, in which diagnosis relies on the successful response to the symptomatic treatment. 2 – Gastric erosion or ulceration Gastric ulcers are rarely primary. No breed, age or sex predilections are reported for canine gastric ulcer disease (Parrah et al., 2013). If the ulcer is located in the stomach or in the proximal duodenum it is also called a “peptic ulcer” due to it being constantly bathed in pepsin. While erosions involve only the mucosa (Fig. 1A), ulcers can reach at least the level of the muscularis mucosa (Sullivan and Yool, 1998). However, for some pathologists this distinction is irrelevant, being merely indicative of the lesion depth. Therefore, an ulcer means “epithelial loss” and depending upon the depth, it can be superficial or deep and range from erosion to full thickness bleeding or perforating ulcer (Parrah et al., 2013). The classification of gastric ulcers as acute or chronic is more related to their clinical presentation than to their histological features. In animals, the aetiopathogenesis of gastric ulceration is often not identified. In many cases, gastric ulcers are associated with surprisingly little inflammation and most cases of gastritis do not have ulceration at all. Thus, the unequivocal classification of these lesions as acute or chronic is difficult from the histological perspective. The pathophysiology of gastric ulceration is multifactorial and includes physical damage to the gastric mucosa, impairment of mucosal defence and chemical changes in the mucosa and in its repair process (Parrah et al., 2013). The causes of canine gastric ulceration are reviewed in Table 1. In dogs, mechanical abrasion is the most common cause of ulceration. Normally it is associated with the ingestion of a wide range of materials such as abrasive foods, household chemicals, common garden and woodland plants, items of clothing or household decorations. This type of ulceration is usually shallow and transient, completely healing 46 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 after a few hours (Wilcock, 2013). A few neutrophils may be found in the superficial lamina propria or intermingled with some fibrin and mucus covering the surface of the ulcer (Wilcock, 2013). Some substances have been implicated in causing damage to the canine gastric mucosa including glucocorticoids such as dexamethasone (Rohrer et al., 1999; Stanton and Bright, 1989; Valin and Allard, 2012) and a number of NSAIDs such as ketorolac (Mathews et al., 1996). Cyclooxygenase (COX)-2 selective inhibitors (e.g., carprofen, etodolac, deracoxib, meloxicam) have been developed to minimize the ulcerogenic effect of NSAIDs in the GI tract. Although they minimize the risk, they do not completely eliminate it and many dogs have developed gastric ulceration following the administration of these putatively safer NSAIDs (Enberg et al., 2006; Lascelles et al., 2005). Some NSAIDs are especially well known for their ulcerogenic potential in dogs, including naproxen and flunixin meglumine (Dow et al., 1990) or ibuprofen (Godshalk et al., 1992; Smith and Taylor, 1993; Stanton and Bright, 1989). The ulcerogenicity of NSAIDs is related to their inhibition of the enzyme COX in the prostaglandin synthesis pathway, resulting in the loss of the gastric protective effects of prostacyclin and prostaglandin E (Enberg et al., 2006). The gross and microscopic appearance of ulcers varies with their severity and duration. Macroscopically, an ulcer is a round to oval mucosal defect, with a smooth base and perpendicular borders. Ulcers that develop acutely have regular and slightly raised borders (Fig. 1B). Progression to perforation is rare and usually requires persistence of the injurious stimulus (Fig. 1C) (Wilcock, 2013). In the chronic form, the borders are still regular, but become elevated and there are signs of inflammation in the surrounding mucosa (Fig. 1D). Ulcers that occur as a consequence of administration of NSAIDs are concentrated in the antropyloric area (Stanton and Bright, 1989). They are often small (<2cm in diameter) with a ‘punched out’ appearance and the walls are only slightly raised from the surrounding mucosa (Sullivan and Yool, 1998). Additionally, the adjacent mucosa may appear to have radial folds, as a result of parietal scarring. Distinctively, in the ulcerative cases of gastric cancer (GC) these lesions may have thickened and irregular mucosal margins, scirrhous change at the base and perhaps serosal thickening. Microscopically, the base and margins of subacute to chronic ulcers are composed of granulation tissue of variable thickness and maturity, infiltrated by a mixed inflammatory cell population, and covered by a thin layer of necrotic debris. Chronic ulcers can fluctuate in size. Depending on the relative dominance of the reparative processes and the severity of ulceration, the layer of granulation tissue can be thick and mature or thinner and less mature, with superficial evidence of recent necrosis (Brown et al., 2007). 47 45 General Introduction CHAPTER 1 Dogs subjected to a regular and intense exercise have a higher incidence of gastric ulcers (Chatelain et al., 2014; Davis et al., 2003b; Dennis et al., 2008; Ritchey et al., 2011) and gastric disease has been identified as a causative factor in cases of sudden death during dog sled races (Chatelain et al., 2014; Davis et al., 2003a; Dennis et al., 2008). Possible explanations for this occurrence relate to the diet commonly used to support the extraordinary calorific demands of these athletes and in the stress of strenuous competition. A high-fat diet can lead to delayed gastric emptying and gastric hyperacidity, which, in isolation or together with the physiological response to stress (reflected in high levels of circulating cortisol) may predispose these animals to GI ulceration (Davis et al., 2003a). Gastric stress ulcers are poorly defined in the dog. Gastroduodenal ulceration has been reported in dogs in conjunction with severe burns, heat stroke, multiple trauma, head injuries and spinal cord disease (Dowdle et al., 2003; Neiger and Simpson, 2000). However, these changes could be more related to the drugs used for the pain management and treatment of the inflammatory conditions than to the physiological response to stress under hospitalization. Gastric ulceration is a frequent complication in dogs with hypoadrenocorticism. Systemic hypovolemia with an attendant decrease in gastric mucosal blood flow, loss of the permissive effect of glucocorticoids in mucosal defence, and significant electrolyte abnormalities are all likely contributors to the gastritis seen with this endocrinopathy (Henderson and Webster, 2006). It should be noted that, unlike people, dogs with renal failure rarely show gastric necrosis and ulceration (Peters et al., 2005). In contrast, liver disease is considered one of the two most common risk factors for gastroduodenal ulceration in dogs and its pathogenesis is probably related to alter gastric blood flow due to portal hypertension, delayed epithelial turnover, gastric hyperacidity and hypergastrinemia (Henderson and Webster, 2006). Animals with mastocytosis or mast cell tumours may have gastric ulcers (Howard et al., 1969; Ozaki et al., 2002) that can be multiple (Stanton and Bright, 1989). These tumours produce and release histamine, which binds to H2 receptors on gastric parietal cells constituting a powerful stimulant to acid secretion. Forty years ago, gastric ulcers were reported to occur in up to 80% of dogs with mast cell tumours (Howard et al., 1969). However, in the authors opinion this value is no longer accurate and the actual incidence is much lower. Infestation with Physaloptera spp. nematodes, including P. rara and P. canis, may be an infrequent cause of gastric ulceration associated with chronic vomiting in dogs (Burrows, 1983). The worms may be free in the lumen causing minimal inflammatory reaction (Pohlit, 2014), but more commonly attach to gastric mucosa where they feed on blood. They 48 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 occasionally change their attachment site resulting in multiple small bleeding wounds that become inflamed (Clark, 1990). Despite the importance of Helicobacter pylori (H. pylori) as a cause of gastric ulcers in man (Marshall and Warren, 1984), no significant relationship has been demonstrated between Helicobacter spp. infection and gastric ulceration in dogs. In man, peptic ulcer formation as a result of excessive gastrin production and hyperacidity caused by a gastrinoma is known as the Zollinger-Ellison syndrome. This is a condition often characterized by the triad of hypergastrinaemia, hypertrophic or atrophic gastritis and GI ulceration (Hayden and Henson, 1997; Simpson and Dykes, 1997). Few cases of a canine homologue of Zollinger-Ellison syndrome associated with gastric ulcer formation are reported (Fukushima et al., 2004; Gal et al., 2011). The gastrin production by these tumours directly stimulates secretion of hydrochloric acid from the parietal cells, as well as indirectly stimulating acid production by releasing histamine from fundic enterochromaffin-like cells, thereby compromising the protective mechanisms and reducing the epithelial cell turnover of the gastric mucosa (Hughes, 2006). 3 - Chronic Gastritis Chronic gastritis is defined clinically as intermittent vomiting with duration of more than 1 – 2 weeks (Dowdle et al., 2003). The diagnosis is based on the microscopic examination of gastric biopsy samples and is subclassified according to the histopathological changes and aetiology. For the histologic diagnosis of gastritis, an increase in mucosal leukocytes accompanied by other evidence of inflammation such as hyperaemia, oedema and lesions in the adjacent structural components of the mucosa (e.g., epithelial injury or reparative fibrosis) must be identified. Canine gastritis is commonly categorized according to the nature of the predominant cellular infiltrate (e.g., eosinophilic, lymphocytic, plasmacytic, granulomatous, lymphoid follicular), the presence of architectural abnormalities (e.g., atrophy, hypertrophy, fibrosis, oedema, ulceration, metaplasia), and their subjective severity (e.g., mild, moderate, severe) (Day et al., 2008; Wilcock, 2013). 3.1 - Lymphoplasmacytic gastritis Lymphoplasmacytic gastritis, with or without concomitant lymphoid follicular hyperplasia, remains the most common form of canine gastritis (CC Brown, 2007). It 49 45 General Introduction CHAPTER 1 may occur in isolation or together with enteritis and colitis, as part of the inflammatory bowel disease (IBD) syndrome. Chronic gastritis is seen predominantly in middle aged to older animals, although young animals may also be affected. There is no sex nor breed predisposition (Sullivan and Yool, 1998). Histologically, lymphoplasmacytic inflammation is characterized by infiltration of these cells into the lamina propria (Fig. 2A) and is often accompanied by distinct increases in the number of intraepithelial lymphocytes within both surface and deep epithelial structures (Day et al., 2008). The cellular infiltrate varies widely in severity and it may be accompanied by mucosal atrophy or fibrosis, and less commonly hyperplasia (Simpson, 2013). In accordance with WSAVA guidelines (Day et al., 2008), the mucosa of the normal gastric body may have 0.5-13 (mean: 4.2) lamina propria lymphocytes and 0-5.83 (mean: 1.59) lamina propria plasma cells, while in the antrum, 0.5-13 (mean: 4.2) lamina propria lymphocytes and 0.5-15.5 (mean: 6.8) lamina propria plasma cells are considered normal. Another distinctive feature of this particular gastric inflammation is hyperplasia of the gastric lymphoid aggregates (Fig. 2B), which may sometimes be severe (Day et al., 2008). This form of canine gastritis may sometimes be associated with the presence of Helicobacter spp. organisms but there is no evidence supporting a causative role between this infection and gastric pathology. In dogs with idiopathic lymphoplasmacytic gastritis, gastric atrophy correlates with the expression of mRNA for interleukin (IL)-1β and IL-10 and the presence of neutrophils. Furthermore, the histological severity of lymphoplasmacytic gastritis is correlated with atrophy, infiltration with lymphocytes and macrophages, and expression of genes encoding IL-10 and interferon (IFN)-γ (Wiinberg et al., 2005). 3.2 - Eosinophilic gastritis Eosinophilic gastritis is a less frequent condition of the canine stomach (Lidbury et al., 2009), often believed to be a manifestation of a generalized GI tract hypersensitivity reaction (eosinophilic gastroenteritis) (Neiger, 2008). Dogs of < 5 years of age are most commonly affected; there is no sex predisposition, and German shepherd dogs and rottweilers are more predisposed (van der Gaag, 1988). However, eosinophilic gastritis has also been identified in dogs as an isolated condition of the stomach alone (Hayden and Fleischman, 1977). Macroscopically, eosinophilic gastritis may present as hypertrophy of the rugal folds with mucosal ulceration (Neiger, 2008) as well as thickening of the gastric wall, mucosal necrosis and haemorrhagic foci. 50 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 The diagnosis of eosinophilic gastritis requires an increased number of mucosal eosinophils and that eosinophils are the dominant inflammatory cell type. Other causes of eosinophilic infiltration of the gastric mucosa (e.g. parasitic disease) must be excluded. According to the WSAVA guidelines (Day et al., 2008) , the mucosa of the normal gastric body and antrum can have 0-2 (mean: 0.5) and 0-6 (mean: 2.7) eosinophils per 10,000 µm2, respectively. Eosinophils counts above these levels would suggest eosinophilic gastritis (Figure 2C). The eosinophilic infiltration is mainly limited to the gastric mucosa and rarely extends into the muscularis mucosa or serosa (Sattasathuchana and Steiner, 2014). The cause of canine eosinophilic gastritis remains unknown, but factors such as genetic predisposition and diet are likely to be involved in its pathogenesis (Sattasathuchana and Steiner, 2014). Important differentials include gastric parasitic infections by Physaloptera spp. (Neiger, 2008), Gnathostoma spp. (Maleewong et al., 1992), Cryptosporidium spp. (Ellis et al., 2010) and Heterobilharzia americana (Rodriguez et al., 2014) and eosinophilic infiltration accompanying mast cell tumours (Ozaki et al., 2002). Scirrhous eosinophilic gastritis is a rare variant of eosinophilic gastritis (Hayden and Fleischman, 1977) in which the stomach is enlarged and has a greatly thickened wall. Microscopically, there is marked infiltration of eosinophils, dense bands of fibrous tissue (granulation tissue and mature collagen) replacing most of the muscular layers and thickening the submucosa and serosa. The eosinophils are distributed in a linear-like to diffuse fashion, often forming perivascular aggregates, primarily within the muscular layers and serosa. Associated degeneration of the gastric arteries is evident ranging from fibrinoid necrosis to panarteritis. Gastric inflammation does not extend into the intestinal tract. Although the cause of this condition was not determined, its basis was probably immunological. 3.3 - Granulomatous gastritis Granulomas may be present in the canine gastric mucosa in a variety of infectious diseases and as a reaction to endogenous substances and foreign objects, such as sewing needles (Pratt et al., 2014). Endoscopic appearance ranges from non-specific minor changes to thickened mucosal folds with outlet obstruction. Histologically, the granulomatous lesions consist of the typical nodular, circumscribed collections of macrophages admixed with variable numbers of lymphocytes, eosinophils, multinucleated giant cells and neutrophils, with or without central necrosis and a peripheral lymphoid cuff (Ectors et al., 1993). Parasites that target the canine stomach are uncommon, but spirocercosis caused by the nematode S. lupi is one example. After ingestion, the larvae of this organism penetrate the gastric mucosa and provoke a severe mixed inflammatory/neoplastic-like response. These nodular foci are usually referred to as granulomas; however, some authors have 51 45 General Introduction CHAPTER 1 suggested that this designation is inappropriate (van der Merwe et al., 2008). Initially the larvae are surrounded by highly vascularized loose connective tissue, which contains fibrinrich fluid, neutrophils and foci of necrosis. Later, this tissue is composed mostly of actively dividing fibroblasts with an embryonic appearance, sometimes resembling a sarcoma (Bailey, 1963). Another nematode that may infest the stomach of dogs is Gnathostoma spinigerum. Groups of up to 10 adults can be lodged in nodules in the submucosa, which may protrude into the gastric lumen. The wall of these nodules is comprised of granulation tissue and reactive fibrous stroma and in the centre the parasites are embedded in a purulent and haemorrhagic exudate. Focal granulomas may centre on eggs trapped in the connective tissue (Maleewong et al., 1992). Another trematode parasite, endemic to the Gulf coast and south Atlantic region of the USA, Heterobilharzia americana (H. americana) was recently associated with gastric granulomatous inflammation circumscribing trapped eggs in one of 32 infected dogs (Rodriguez et al., 2014). Histoplasmosis is a relatively common systemic fungal disease of the dog caused by Histoplasma capsulatum. The infection is probably acquired via inhalation or ingestion of infective conidia. These are phagocytosed by macrophages and can be disseminated via the bloodstream or lymphatics to any organ system. The disease can be subclinical or cause clinical granulomatous disease affecting, among other organs, the canine GI tract (Bromel and Sykes, 2005; Lin Blache et al., 2011). Young, large-breed, male dogs in the US Gulf States may develop mucosal and submucosal suppurating granulomas in the stomach and elsewhere in the GI tract, caused by the fungus Pythium insidiosum (Grooters and Gee, 2002). Additionally, cryptococcosis resulting in a granulomatous gastritis mimicking carcinoma has been reported in a Doberman pinscher as a cause of gastric outlet obstruction (van der Gaag et al., 1991). 4 – Gastric mucosal fibrosis and atrophy Atrophy of the gastric mucosa has been traditionally defined as “the loss of appropriate glands” (Rugge et al., 2002) presumably following a chronic inflammatory process, since the mucosa becomes inactive and fibrosis replaces the normal glandular structures. Endoscopically, the gastric mucosa appears discoloured and thin, with prominent blood vessels (Sullivan and Yool, 1998). In human mucosal atrophy, especially in severe cases, the gastric rugae can disappear from the fundus or body, while in the antrum, mucosal thinning can be grossly unapparent (Dixon et al., 1996). In man, an inherited form 52 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 of atrophic gastritis typically confined to the gastric body and fundus is termed autoimmune chronic atrophic gastritis and is characterized by an immune response directed toward parietal cells and intrinsic factor (Neumann et al., 2013) .This will result in the destruction of parietal cells, which leads to profound hypochlorhydria (and elevated gastrin levels), and in inadequate production of intrinsic factor that may leads to vitamin B12 malabsorption and pernicious anemia. In turn, the gastrin trophic effect exert on enterochromaffin-like cells is hypothesized to be one mechanism counteracting in the malignant transformation of these cells into carcinoid tumours (Neumann et al., 2013). Microscopically, chronic atrophic gastritis is the condition that combines mononuclear infiltration, glandular atrophy (Fig. 2D), regenerative glandular nesting and mucosal fibrosis. Lymphoid follicle hyperplasia may be present. There is a reduction in overall mucosal thickness and in most cases, an obvious reduction in the number of parietal cells with a corresponding increase in the proportion of mucus-producing epithelial cells within the gland (Wilcock, 2013). In dogs, atrophic gastritis is often associated with a marked cellular infiltrate (Rousseau, 2005). However, evidence that the pathogenesis of fibrosis is associated with previous inflammatory conditions is still lacking. Atrophic gastritis has been infrequently found in dogs and there is no proof of its clinical significance (Simpson, 2013). In one study of experimentally-induced intestinal metaplasia (IM) in the stomach of dogs, Wang and colleagues described expression of Bcl2 in precedent lesions of chronic atrophic gastritis, suggesting that this protein may be involved in the initial process of genetic instability and that disturbances in the regulation of apoptosis may constitute an initial step in the malignant transformation of the normal gastric mucosa (Wang, 2000). Atrophy of the gastric mucosa that may progress to adenocarcinoma has been reported in Norwegian Lundehund dogs (Berghoff et al., 2007; Kolbjørnsen et al., 1994) and in this breed a reduction in parietal cells and hyperplasia of neuroendocrine cells has been associated with gastric adenocarcinoma (Qvigstad et al., 2008). 5 – Helicobacter spp. infection The gastric mucosa of dogs is often colonized by Helicobacter spp. other than H. pylori. Non-H. pylori Helicobacters (NHPH) are present in 67-86% of clinically healthy dogs, in 61-100% of animals presenting with chronic vomiting (Geyer et al., 1993; Hermanns et al., 1995; Hwang et al., 2002; Lecoindre P., 1995; Polanco et al., 2011) and in 100% of laboratory dogs and dogs from local shelters (Eaton et al., 1996; Henry et al., 1987; 53 45 General Introduction CHAPTER 1 Simpson et al., 1999). The predominant gastric Helicobacter spp. in dogs are H. felis, H. bizzozeronii and H. heilmannii sensu stricto (s.s.), while H. salomonis is less often detected and the prevalence of H. cynogastricus and H. baculiformis has not yet been studied (Baele et al., 2009; Haesebrouck et al., 2009; Haesebrouck et al., 2011; Van den Bulck et al., 2006). However, H. pylori has occasionally been recognized in the canine stomach (Buczolits et al., 2003; Chung et al., 2014; Ekman et al., 2013; Taulescu et al., 2009). Mixed infections with different species can also occur (Ekman et al., 2013; Van den Bulck et al., 2005) and the number of organisms present can be variable. While many studies have reported that the fundus and body have higher bacterial density and a higher probability of finding Helicobacter spp. (Anacleto et al., 2011; Simpson et al., 1999; Yamasaki et al., 1998) others have found no significant differences between the density of NHPH in the fundus, body and antrum (Buczolits et al., 2003; Cattoli et al., 1999; Gombač et al., 2010; Prachasilpchai et al., 2007; Recordati et al., 2009) of the canine stomach. The discrepancies in these results can be attributed to the different laboratory diagnostic methodologies used by the various research groups. Canine NHPH colonize the superficial mucus layer and gastric glands and may also be observed intracellularly (Fig. 3). Vacuolation and necrosis of parietal cells has also been observed in the gastric mucosa of dogs naturally infected by Helicobacter spp. (Fig. 3B) (Happonen et al., 1996; Hermanns et al., 1995; Takemura et al., 2013; Yamasaki et al., 1998). In contrast, in man, H. pylori does not affect the parietal cells or the deep gastric glands, being located more superficially and primarily affecting the antrum. Furthermore, the characteristic human H. pylori surface epithelium and foveolar neutrophil infiltration and “pit abscess” formation (Fig. 3D) (Dixon et al., 1996) is not usually seen with NHPH infection in the canine stomach. Presumably, in the acute phase, the infection briefly triggers trivial initial signs and goes unnoticed, only being detected later as an occasional finding or due to the persistence or deterioration of the animal’s clinical signs. In dogs, the inflammation is generally mononuclear in nature and ranges from mild to moderate in severity. Thus, gastritis associated with NHPH is mostly less active, compared with H. pylori-associated gastritis. Although H. pylori is well accepted as an important human pathogen and is believed to be the primary cause of chronic gastritis, gastric and duodenal ulceration, and even gastric carcinoma and mucosal-associated lymphoid tissue (MALT) lymphoma, the pathogenic significance of gastric Helicobacter species in dogs is debatable. Histological findings, such as mild to moderate gastritis, reduction in mucus production, glandular degeneration, interstitial oedema, fibrosis of lamina propria, hyperplasia, vacuolation, and necrosis of parietal cells were described in dogs naturally infected by Helicobacter species (Happonen et al., 1996; Hermanns et al., 1995; Takemura et al., 2013; Yamasaki et al., 1998). 54 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 Peyrol et al. (1998) found no pathogenic significance for H. bizzozeronii in the canine stomach (Peyrol et al., 1998). Simpson et al. (1999) found no significant difference in clinical symptoms, inflammation and gastric function between control and experimentally H. felis infected dogs. (Simpson et al., 1999). Wiinberg et al. (2005) have quantitatively examined mucosal inflammatory and immune responses in dogs with gastritis and assessed the relationship of these responses to infection with Helicobacter spp. (Wiinberg et al., 2005). They concluded that gastric infection by NHPH in dogs was associated with increased expression of transforming growth factor (TGF)-β and fibrosis. However, no significant associations between Helicobacter spp. infection and proinflammatory cytokine expression, severity of gastritis or differences in the pathogenicity of the different Helicobacter spp. were found. Another investigation documented no significant change in epithelial proliferation or E-cadherin expression in the gastric mucosa of dogs infected with Helicobacter spp. (Takemura et al., 2013). Additionally, canine H. heilmannii infection was found to be associated with increased numbers of lymphoid follicles (Takemura et al., 2013). In the majority of dogs infected with NHPH, the limited inflammatory response and the absence of clinical signs suggests that these are commensal rather than pathogenic organisms, although seroconversion and hyperplasia of the lymphoid follicles indicates that significant antigenic stimulation occurs (Simpson, 2013). Nevertheless, the presence of NHPH in the canine stomach in conjunction with more severe pathologies has been documented. For instance, Helicobacter spp. organisms were found in all cases of canine gastric polyps (Taulescu et al., 2014) and Poutahidis and colleagues detected moderate numbers of large Helicobacter-like organisms in all parts of the glandular stomach of a Poodle affected by a choriocarcinoma (Poutahidis et al., 2008). To the best of our knowledge, so far there are no evidences reporting the occurrence of gastric lymphoma and concomitant presence of Helicobacter spp. in the dog. Comparative analyses between the available genome sequences revealed that canine NHPH lack all known H. pylori adhesins (Haesebrouck et al., 2009). Although both H. pylori and NHPH share virulence factors involved in epithelial cell death such as the ɣ-glutamyl transpeptidase, all NHPH lack cytotoxin-associated genes, pathogenicity islands and a functional vacuolating cytotoxin A. A recent study has characterized the glycosylation profile of the normal canine gastric mucosa, with focus on the expression of histo-blood group antigens (Amorim et al., 2014b). The canine gastric mucosa lacks type 1 Lewis antigens, but has extensive expression of type 2 Lewis structures and A antigen, both in the surface and glandular epithelium. Furthermore, to determine if this glycosylation profile was somehow related to the host-adaptation of the different Helicobacter spp., their binding capacity to canine gastric mucosa was evaluated: H. heilmannii s.s. had the highest adhesion scores in the canine antral mucosa, while the SabA-positive H. pylori strain showed the highest adhesion 55 45 General Introduction CHAPTER 1 and carcinoma, such as Barrett’s mucosa in oesophageal biopsies of baboons and people (Rubio, 2009; Rubio et al., 2009). Glassy cells have also been identified in the canine stomach (unpublished observations). Cells with the phenotype of human glassy cells were noted in the stomach of a 12-year-old male German shepherd dog, mainly in the transitional area between the oesophagus and the cardia. This is consistent with previous primates studies in which GCs were identified in different areas of the stomach (Rubio et al., 1991; Rubio et al., 2008). The canine glassy cells had large and glassy cytoplasm with nuclei displaced towards the luminal aspect of the gland. They were organized in clusters that mainly retained glandular architecture (Fig. 5B). This dog had a poorly cohesive carcinoma apparently originating in the gastric fundus and progressing towards the oesophagus. The significance of these cells in the canine gastric mucosa remains to be further elucidated. 9.3 - Mucous (pseudopyloric) metaplasia Mucous metaplasia involves metaplastic transformation of the fundic-oxyntic mucosa to a mucus cell form. Normal oxyntic mucosa has straight glands composed of tightly packed chief cells, parietal cells, endocrine cells, and mucus cells with a higher ratio of glands to foveola compared with the antrum. It is postulated that continuous inflammation and atrophy leads to progressive loss of parietal cells and eventually the oxyntic mucosal glands come to resemble the antral/pyloric glands (pseudopyloric metaplasia) (Fig. 5C). Pseudopyloric metaplasia was reported in 17 dogs that mostly had simple diffuse or atrophic forms of gastritis (van der Gaag, 1988). 9.4 - Intestinal metaplasia (IM) Originally described in the 19th century, gastric IM is defined as the transformation of the foveolar and/or the glandular epithelium of the gastric mucosa by intestinal-like epithelium with goblet cells, enterocyte-like cells and Paneth cells. The last two cell phenotypes are absent in the so-called incomplete IM (Rubio, 2007). Human IM is often encountered in chronic gastritis following H. pylori infection, which leads to gastric atrophy, progression to IM and, ultimately, GC (Correa, 1992). The variable outcome of H. pylori infection suggests that IM results from distinct adaptive responses in adverse circumstances, with the interplay between individual genetic variation and environmental exposures determining individual susceptibility (Barros et al., 2010). In 62 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 man, about 80% of gastric carcinomas appear in the context of IM thus, IM is considered to be a preneoplastic condition, resulting in an increased risk for subsequent development of cancer (Uemura et al., 2001). Advances in molecular biology revealed that intestinal transcriptional factors regulate the proliferation and differentiation of intestinal epithelial cells by the expression of the main homeobox transcription factor CDX2 (Almeida et al., 2003; Eda et al., 2002; Silberg et al., 2002). Under normal conditions, human CDX2 expression is restricted to the intestine, but it becomes expressed ectopically in human gastric IM lesions (Almeida et al., 2003; Eda et al., 2002). Two major pathways for the development of IM are proposed: (1) activation of signalling pathways normally absent from the stomach, but involved in intestinal development and differentiation, or (2) inactivation of pathways involved in establishing and maintaining the gastric phenotype. Moreover, it is hypothesized that H. pylori infection and consequent gastric inflammation provide the initial trigger for CDX2 expression in human gastric cells by inducing an altered signalling network. This will result in a certain level of CDX2 overexpression, which will persist via an autoregulatory mechanism that precipitates the subsequent cellular events (Barros et al., 2010). Although these CDX2 regulatory mechanisms are insufficient to explain the maintenance of CDX2 expression, especially after eradication of infection and resolution of inflammation, the low reversibility of IM observed in human lesions suggests that another level of complexity is present in CDX2 regulation in vivo (Barros et al., 2010). IM is well-known and extensively studied in man and has been induced experimentally in some animal models including in gerbils, rats and dogs (Hashimoto et al., 1980; Li et al., 1994; Wang, 2000; Watanabe et al., 1992). The Hashimoto group (1980) successfully induced IM in the stomach of Beagle dogs treated with N-methyl-N-nito-N-nitrosoguanidine (MNNG); however, this observation occurred after a long-term treatment, since IM was only achieved at the 128th experimental week. Wang (2000) also induced IM in Beagle dogs, but in a shorter period of time by combining long-term oral administration of a small dose of MNNG and ranitidine plus intermittent low-dose localized X-irradiation of the stomach. Similar to the changes in man, the transition from canine NGM to IM involved several progressive steps including superficial gastritis, chronic atrophic gastritis, slight focal IM and moderate or severe IM (Wang, 2000). Furthermore, there was expression of APC, Bcl2 and Kras in the lesions of canine IM, suggesting that these tumour-related proteins may play a role in the malignant transformation of IM. Additionally, no p53 expression was detected in these induced canine IM lesions. To the best of our knowledge, spontaneous development of gastric IM has never 63 45 General Introduction CHAPTER 1 been documented in normal or inflamed canine stomach but has been described in association with canine hyperplastic gastric polyps (Taulescu et al., 2014). Additionally, in all cases of canine gastric adenocarcinoma, CDX2 expression has been recorded in foci of metaplastic change which included numerous goblet cells (Fig. 5D) (Doster et al., 2011). Eventually, under certain pathological circumstances some changes may occur in the canine GI microenvironment that probably lead to IM and similarly to that seen in man, the upregulation of CDX2 may also be involved in this pathway (unpublished observations). 10 – Gastric dysplasia 10.1 - Epithelial dysplasia The diagnosis of gastric dysplasia in animals is based on concepts described in human medicine, but is hampered by factors including (1) lack of a uniformly recognized classification, (2) significant interobserver variability in microscopic diagnosis, (3) absence of well-defined dysplastic features associated with particular entities of gastric carcinoma, and (4) a limited understanding of the clinical significance of a diagnosis of dysplasia (Lauwers and Riddell, 1999). Microscopically, gastric epithelial dysplasia is characterized by a set of histological alterations based on cytological and architectural abnormalities. The cytological changes include cytoplasmic mucin depletion; cellular crowding and pleomorphism; nuclear hyperchromatism, pleomorphism and stratification; increased nuclear:cytoplasmic ratio, and increased mitotic activity. The architectural changes include glandular disorganization or budding, with irregular branching and dilatation and intraluminal folding. However, the unequivocal distinction between regenerative changes and dysplasia in gastric tissues remains difficult for veterinary pathologists. In accordance with the Vienna classification, human dysplastic changes may be low-grade or high-grade (Schlemper et al., 2000). Low-grade dysplasia is characterized by preservation of the glandular architecture, sometimes with the presence of pseudovilli, cystically dilated or slightly irregular glands with discrete intraluminal papillary projections or a serrated appearance. Glands are composed of tall and crowded cells, with or without mucus vacuoles. The nuclei are discretely pleomorphic, elongated, stratified and are located in the lower half of the cytoplasm. Mitotic index is low. High-grade epithelial dysplasia is characterized by marked distortion of the glandular architecture with crowded, irregular and ramified glands and frequent papillary intraluminal projections lined by stratified epithelium, 64 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 exhibiting pleomorphic nuclei that may overlap. There is marked mitotic activity and loss of normal cell polarity (Fig. 5E). 10.2 - Globoid Dysplasia “Tubule neck dysplasia”, “non-metaplastic dysplasia” or “globoid dysplasia” (GD) is a subtle alteration occasionally found in human gastric non-metaplastic mucosa (GhandurMnaymneh et al., 1988) and which is believed to be a precursor lesion of signet ring carcinoma (Ghandur-Mnaymneh et al., 1988; Misdraji and Lauwers, 2002). In GD, the tortuous tubules have crowding of the neck region by enlarged clear cells lining the foveolae. The most distinctive morphological characteristic of GD is the presence of abundant vacuolated cytoplasm with excessive amounts of mucin, which compress the eccentric and hyperchromatic nuclei, giving an appearance similar to signet ring cells (GhandurMnaymneh et al., 1988; Misdraji and Lauwers, 2002). An immunohistochemical analysis of six cases of human gastric GD showed that the expression profile was MUC5AC (+), Ki-67 (+), p53 (+), p27 (+), p16 (+), MUC6 (-), Na+K+ ATPase (-) and E-cadherin expression with reduced intensity (Lino-Silva et al., 2013). GD has not been documented in the dog, but similar changes were noted in the antrum of a 12-year-old male crossbred Poodle (unpublished observations). This dog had generalized and diffuse foveolar hyperplasia with tufting of the superficial epithelium consistent with globoid change. The alteration was characterized by distinctive and increased retention of mucin in the supranuclear cytoplasm without nuclear atypia or disruption of the epithelial structures (Figure5F). High numbers of NHPH were also present in the stomach of this dog. No signs of neoplastic transformation were identified. However, similar lesions were reported in the adjacent non-neoplastic mucosa of patients diagnosed with hereditary diffuse gastric cancer (Carneiro et al., 2004). Further studies are warranted in order to understand the implications of this histological alteration in the canine gastric mucosa. 11 – Gastric epithelial neoplasia 11.1 - Epidemiological features of canine gastric carcinoma (GC) Gastric cancer accounts for < 1% of all reported neoplasms in dogs (Arnesen K, 2001; Crow, 1985; Patnaik et al., 1977; Sullivan et al., 1987). Carcinoma is the most frequent gastric tumour, comprising 50 –90% of all canine gastric malignancies and usually results in death (Patnaik et al., 1977; Sautter and Hanlon, 1975; Swann and Holt, 2002). 65 45 General Introduction CHAPTER 1 Most canine GCs are located in the lesser curvature and pylorus, often progressing to involve most of the stomach body (Fonda et al., 1989; Patnaik et al., 1977; Sautter and Hanlon, 1975; Scanziani et al., 1991; Swann and Holt, 2002). The reported age range of dogs with GC is 3 to 16 years (mean: 7.5 years) (Fonda et al., 1989; Patnaik et al., 1977; Scanziani et al., 1991; Sullivan et al., 1987; Swann and Holt, 2002) and, as in man, a higher incidence in males is reported (Carrasco et al., 2011; Fonda et al., 1989; Patnaik et al., 1977; Scanziani et al., 1991; Swann and Holt, 2002). Breed predisposition to GC in Belgian Shepherd dogs (Tervuren and Groenendael) (Fonda et al., 1989; Lubbes et al., 2009; Sautter and Hanlon, 1975; Scanziani et al., 1991; Seim-Wikse et al., 2013), Rough Collies (Seim-Wikse et al., 2013; Sullivan et al., 1987), Staffordshire Terrier (Sullivan et al., 1987), Chow Chows (Bilek and Hirt, 2007; Penninck et al., 1998) and standard Poodles (Seim-Wikse et al., 2013) are reported. In the author’s institutions, sled dogs (e.g. Alaskan malamute, Siberian husky, Pyrenean Mountain dog) and Japanese Akitas are often affected (unpublished data); however, in determining any canine breed predisposition, the popularity of the breeds in the geographical location must be considered. The incidence of GC is lower in dogs than in man (Crow, 1985; Fonda et al., 1989), but in recent years this disease appears to be diagnosed more frequently. This likely reflects the use of more accurate diagnostic techniques such as endoscopy. Despite this, canine GCs are often diagnosed at an advanced stage, resulting in a poor prognosis and limited treatment options. A median survival time of 35 days has been described (Swann and Holt, 2002) and in about 70-90% of cases, the tumours have already metastasized at the time of diagnosis or death (Scanziani et al., 1991; Sullivan et al., 1987; Swann and Holt, 2002). Metastatic sites include the gastric lymph nodes, omentum, liver, duodenum, pancreas, spleen, oesophagus, adrenal glands, and lungs (Head et al., 2008). Most canine GCs produce localized or diffuse thickening and/or ulceration of the gastric wall, serosal pallor and occasionally, a substantial decrease in the number of rugae on imaging and gastroscopy (Fig. 6A) (Guilford, 1996). 11.2 - Classification of gastric cancer A. Early and advanced gastric carcinoma Early gastric carcinoma (EGC) is defined as an invasive carcinoma confined to the mucosa and/or submucosa, with or without lymph node metastases, irrespective of the tumour size. In man, most EGCs are small (2-5 cm in size), located in the lesser curvature and seen endoscopically as polypoid (pedunculated or sessile), slightly elevated, flat, 66 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 slightly depressed or excavated lesions. Histologically, the most common forms of EGC are well differentiated, mostly with tubular and papillary architecture. Despite advances in veterinary diagnosis and treatment, the early stages of GC are often asymptomatic and the late onset of clinical signs means that canine cases carry a poor prognosis in comparison with human patients, in which the prognosis of this condition is often excellent (Uedo et al., 2006). In contrast, a recent publication described the successful treatment of EGC in a Shih-Tzu dog (Lee et al., 2014). The rarity of this lesion in veterinary medicine inhibits more detailed investigation and classification. In human medicine, the Paris classification (2005), which focuses on the endoscopical categorization of superficial lesions in GI tract, remains a clinically important tool that helps to decide if neoadjuvant (pre-operative) therapy is necessary. Advanced gastric carcinoma (AGC), which invades into the tunica muscularis or beyond, carries a worse prognosis. These lesions can be grossly exophytic, ulcerated, infiltrative or combined. The Borrmann’s classification is usually employed for the macroscopic evaluation of human AGC. Histologically, these tumours often exhibit marked architectural and cytological heterogeneity. Due to the advanced stage of disease at the time of diagnosis, the great majority of canine GCs would be included in this stage. B. Histological classification of gastric carcinoma According to the WHO classification for domestic animals (Head, 2003), gastric carcinomas are divided into three categories based on the histopathological features including adenocarcinoma, squamous cell carcinoma and undifferentiated carcinoma. Adenocarcinomas are subdivided into papillary, tubular, mucinous and signet ring cell types, depending on the predominant histological picture and the principal cell type of the tumour (Head and Ring, 2003). The human Lauren classification for GC is based on the architectural and cytological features of the tumour and has been applied successfully in previous investigations in the dog (Fonda et al., 1989; Janke et al., 2010; Patnaik et al., 1978). Generally, papillary and tubular variants are classified into intestinal, expanding, or differentiated type, while mucinous and signet ring cell variants are categorized into diffuse, infiltrative, or undifferentiated type (Lauren, 1965). In dogs, tubular adenocarcinoma is considered the most common histological subtype (Wilcock, 2013) (Figure 6B). Macroscopically, it tends to form polypoid masses and histologically, demonstrates irregularly distended, fused or branching tubules of various sizes sometimes with intraluminal mucus and nuclear and inflammatory debris. Papillary 67 45 General Introduction CHAPTER 1 adenocarcinoma is another variant that, histologically, is characterized by epithelial projections supported by a central fibrovascular core. Mucinous adenocarcinoma is defined as a gastric adenocarcinoma with a substantial amount of extracellular mucin (≥ 50% of tumour volume) (Figure 6C). The neoplastic cells can form glandular structures or irregular cell clusters, with occasional scattered signet ring cells floating in the extracellular mucin lakes. Signet ring cell adenocarcinoma of the stomach is characterized by diffuse infiltration of signet ring tumour cells in the gastric wall. Histologically, this lesion consists of isolated or small groups of malignant cells containing intracytoplasmic mucin with eccentric nuclei (Fig. 6D), often accompanied by marked desmoplasia with an infiltrative pattern. In some cases, as in human GCs, the canine signet ring tumours appear to form a linitis plasticatype tumour by spreading intramurally, and not usually involving the mucosa (unpublished observations). The 2010 WHO classification of human GCs includes another histological variant termed ‘other poorly-cohesive carcinomas’. These are often composed of a mixture of nonsignet ring cells, morphologically resembling histiocytes, lymphocytes, and plasma cells. Those tumour cells can form irregular microtrebaculae or lace-like abortive glands, often accompanied by marked desmoplasia in the gastric wall and with a grossly depressed or ulcerated surface. Although the WHO classification for domestic animals (Head, 2003) does not recognize this specific histological type, we have observed some canine gastric neoplastic lesions that fit this description (Figure 6E). In addition to the above major histological patterns of gastric cancers, the 2010 WHO human classification recognizes other variants, based on the presence or absence of poorly differentiated components, such as the mixed carcinoma. This entity consists of a well differentiated adenocarcinoma mixed with < 50% of poorly differentiated components. Equivalent tumours are recognized in the dog (unpublished observations). Other uncommon histological variants of GC are rare in the dog. There is one report of neoplasia displaying features consistent with primary gastric choriocarcinoma in a 9-yearold, male Poodle (Poutahidis et al., 2008). The microscopic examination of the area of pyloric thickening revealed extensive transmural infiltration by a highly malignant neoplasm with two distinct components: signet ring cells with eccentrically placed nuclei and clear cytoplasm; and highly pleomorphic cells plus bizarre multinucleate giant cells resembling, respectively, cytotrophoblasts and syncytiotrophoblasts (Poutahidis et al., 2008). Carcinoids are also rare neoplasms (Brown et al., 2007) arising from neuroendocrine cells that share particular gross, histological, ultrastructural, histochemical and 68 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 immunohistochemical features. The first case of this type of lesion arising in the stomach of a 13-year-old female whippet was reported by Albers et al. (Albers et al., 1998). Round to polyhedral neoplastic cells were observed as individual cells, small nests, short palisading cords or as acini, along the mucosa and infiltrating the antral stomach wall. The neoplastic cells were positive by Grimelius stain (argyrophilic) and immunohistochemically they expressed chromogranin, low and high molecular weight cytokeratins, neuron-specific enolase (NSE) and synaptophysin. The presence of intracytoplasmic neurosecretory granules was further confirmed by electron microscopy. Qvigstad et al. (2008) proposed that the Norwegian Lundehund has an increased risk of developing chronic inflammation of the fundic mucosa, leading to atrophy and probably hypoacidity, and that these changes may progress to development of GCs, most likely of neuroendocrine origin (enterochromaffin-like cells) (Qvigstad et al., 2008). Gastrinoma is a rare malignant neuroendocrine neoplasia that results in autonomous gastrin secretion (Gal et al., 2011). However, in contrast to the predominant origin of human gastrinomas from the gastric antrum and duodenum (Pipeleers-Marichal et al., 1990), canine gastrinomas are supposed to arise from non-beta pancreatic islet cells (Simpson and Dykes, 1997). This neoplasm stimulates hypersecretion of gastric acid, resulting in severe gastric and duodenal ulceration and gastric mucosal hypertrophy, and also decreases the tone of the pyloric sphincter, allowing duodenal-gastric reflux of bile. 11.3 - Application of Molecular Pathology in Canine Gastric Carcinoma In human medicine, the understanding of the molecular events and pathways in GC have led to advances in prevention, early diagnosis, tumour classification and therapeutic intervention. An accumulation of genetic and molecular abnormalities occurring during gastric carcinogenesis has been described in detail, including activation of oncogenes, overexpression of growth factors/receptors, inactivation of tumour suppression genes, involvement of DNA repair genes and cell adhesion molecules (Yasui et al., 2006), loss of heterogeneity and point mutations of tumour suppressor genes, and silencing of tumour suppressors by CpG island methylation (Kitaura et al., 1999). In veterinary pathology the first steps towards molecular investigations are being taken. The reduced sampling and the difficulty in validating and optimizing the molecular biology techniques in animal tissues and samples could underlie this delay. Nevertheless, some studies have occurred in this area and often replicate results obtained in human pathology, since strong similarities have been observed between both species with regard to the clinical presentation and histopathological features of GC. 69 45 General Introduction CHAPTER 1 The Sialyl Lewis x–modified core 2 branched O-glycans (C2-O-sLex) on human leukocytes mediate much higher-affinity adhesion to selectins on activated vascular endothelium than does sialyl Lewis x on other structures. Janke et al. (2010) tested 16 canine gastric carcinomas and found an overexpression of C2-O-sLe(x) in 56% of the tumours, when compared with normal gastric mucosa. The authors hypothesized that these carbohydrates are highly expressed in more malignant types of canine gastric carcinomas; promote adhesion to selectins, attachment of cancer cells to the vascular endothelium and therefore, metastasis and a poorer prognosis . The type I growth factor receptor family consists of the prototype EGFR and the related members, c-erbB-2, c-erbB-3 and c-erbB-4 (also known as HER-1, HER-2, HER-3 and HER-4, respectively). The membrane receptors that have a role in function have been shown to be important in the development of neoplasia (Wiseman et al., 2005). The molecule HER-3 differs from the other members of this family because it has little or no tyrosine kinase activity. In man, blockade of HER-3 activity decreased downstream signalling of the phosphoinositide 3-kinase/protein kinase B pathway and the cell cycle, resulting in the death of tumour cells (Kunii et al., 2008). Previous investigations have suggested a close relationship between cytoplasmic/membrane HER-3 expression and tumour prognosis for human GC (Hayashi et al., 2008). Doster and colleagues (2011) determined CDX2 and HER-3 expression in five gastric mucinous adenocarcinomas. In all cases, strong cytoplasmic HER-3 labelling was recorded in > 10% of tumour cells and most nuclei of the neoplastic cells showed strong CDX2 expression. However, in this study, CDX2 and HER-3 expression could not be correlated with prognosis due to the small population size and the lack of a complete clinical history and post-surgical follow-up (Doster et al., 2011). A recent study in dogs focused on others members of this family, EGFR and its analogue HER-2, and KRAS, which is a small protein encoded by the KRAS gene that mediates the transduction of signals between EGFR receptors and the nucleus. In human oncology it has been shown that the KRAS gene can suffer mutations, most of which involve codons 12, 13 and 61 (exons 2 and 3) identified as a predictor of resistance to anti-EGFR drugs (Jimeno et al., 2008). Terragni et al. (2014), evaluated EGFR/HER2 immunoexpression and KRAS mutational status retrospectively in five adenomas and 14 carcinomas (five intestinal-type and nine diffuse-type carcinomas). Overexpression of EGFR and HER-2 was observed in 42.1% and 57.9% of the cases, respectively, regardless of tumour location and biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type than in the diffuse-type. Furthermore, the KRAS gene was wild type in 18 cases, while one infiltrative mucinous carcinoma of the gastric fundus with regional lymph node metastases harboured a point mutation at codon 12. This mutation is among the most frequently detected in man and leads to substitution of a glycine by an arginine, thus leading to a geometric alteration of the protein (Terragni et al., 2014). 70 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 This results in a lack of GTP hydrolysis which keeps KRAS in a permanently activated state (Eberhard et al., 2005). Genetic and/or epigenetic alterations in E-cadherin-encoding gene (CDH1) or alterations in its protein expression, often result in tissue disorder, cellular de-differentiation, increased invasiveness of tumour cells and ultimately in metastasis (Carneiro et al., 2012). In man, CDH1 is regarded as a classical tumour suppressor gene in gastric carcinogenesis, being involved in the initiation and progression of both sporadic and hereditary forms. We are currently evaluating the potential role of E-cadherin (E-cad) and its encoding-gene (CDH1) in canine GCs. Our preliminary results show that the majority of canine CGs have abnormal E-cad expression in comparison with the normal gastric mucosa. The different patterns of E-cad immunoexpression observed in canine GCs are similar to those already described in human GCs and the existence of CDH1 somatic alterations in such lesions may provide useful information for the clinical management and prognosis of these animals (Amorim et al., 2014a). In another investigation, the expression of gastrin was investigated immunohistochemically in gastric biopsy samples from 64 dogs with gastric carcinoma. Only 8% of the canine carcinomas demonstrated expression of gastrin which is considered less common than in man. The authors concluded that this may not be a reliable prognostic marker in the dog (Seim-Wikse et al., 2014). 12 – Conclusions This review has examined the spectrum and classification of lesions of the canine stomach and compared these with the equivalent human disorders. The use of standardized reporting systems for grading the histological changes in canine gastric biopsy samples is required in order to standardize the interpretation of lesions by veterinary pathologists. Some such schemes, such as that proposed by the WSAVA GI Standardization Group for gastric inflammation (Day et al., 2008) have been introduced, but more refined classification schemes are required for canine gastric cancer in which histological and immunohistochemical features, together with molecular pathogenesis, might be considered. Such schemes should not be developed in isolation and must be related to the biological behaviour and response to therapy of the tumours considered. Canine gastric lesions often show great histological similarities to their human 71 45 General Introduction CHAPTER 1 Figure 4 - Gross and histological findings of canine hypertrophic gastritis and gastric polyps. A). Marked thickening of the gastric mucosa in the pyloric region. Bar=1cm; B) The body region showing diffuse hypertrophic gastritis with cerebriform appearance of the rugal folds (formalin fixed tissue). Bar=1cm; C) Marked foveolar and gastric gland hyperplasia and dilation, and mucus (pseudopyloric) metaplasia of the fundic glands. Bar=200µm. Inset shows the superficial lamina propria with severe inflammatory infiltration by mononuclear cells (Bar=50 µm) (HE staining); D) Multiple inflammatory polyps in the pyloric region (arrow). Bar=1cm; E) Inflammatory gastric polyp with moderate foveolar hyperplasia and marked infiltration of the lamina propria with lymphocytes and plasma cells; inset shows a detail of the infiltrate. Bar=200µm, respectively 20µm (HE staining); F) Adenomatous polyp composed of tubular and/or villous structures lined by dysplastic epithelium; inset shows the hyperplastic epithelium with tubulopapillary architecture. Bar=200µm, respectively 50µm (HE staining). 78 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 Figure 5 - Histological aspects of different metaplastic and dysplastic changes of canine gastric mucosa. A) Osseous metaplasia (heterotopic bone formation) consisting of osteoid and fully mature bone associated with a gastric carcinoma (black asterisks). H&E. Bar=20µm; B) Glands from the cardia showing glassy cell change, characterized by cells with a eosinophilic cytoplasm with ground-glass appearance and luminal, small and eccentric nuclei. HE. Bar=20µm; C) The fundic region revealing diffuse and marked replacement of parietal and chief cells by mucous cells (mucous metaplasia) (arrow). H&E. Bar=50µm; D) CDX2 immunohistochemical reactivity of Goblet 79 45 General Introduction CHAPTER 1 cells (arrow) in a gastric tubulopapillary adenocarcinoma. Immunoperoxidase-diaminobenzidine stain with Mayer’s haematoxylin counterstain. Bar=50µm; E) High-grade epithelial dysplasia of fundic mucosa characterized by highly distorted glandular architecture, irregular and dilated glands covered by a stratified epithelium with crowded and pleomorphic nuclei. H&E. Bar=20µm; F) The foveolar epithelium from pyloric region presenting diffuse globoid dysplasia. Cells have an abundant and vacuolated cytoplasm, with excessive amounts of mucin, which compress the eccentric and hyperchromatic nuclei, resembling signet ring-cells (arrows). HE. Bar=50µm. 80 45 General Introduction CHAPTER 1 45 General Introduction CHAPTER 1 Figure 6 - Morphological appearances of gastric epithelial neoplasia in dogs. A) Diffuse thickening and ulceration of the gastric wall located in the lesser curvature. Bar=1cm. Photomicrographs revealing several histological variants of canine gastric carcinomas: B) Tubulopapillary type. HE. Bar=50µm; C) Mucinous type. HE. Bar=50µm; D) Poorly cohesive carcinoma. HE. Bar=20µm; E) Signet ring cell carcinoma. HE. 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Acta Pathol Microbiol Scand, 64, 31-49. 87 45 Aim and Objectives CHAPTER 1 45 Aim and Objectives CHAPTER 1 -Aim and Objectives General aim: To characterize the epidemiological, pathological and molecular features of the spontaneous gastric infection with different NHPH in canine stomach and to evaluate the relevance of particular glycan structures in Helicobacter spp. adhesion to canine gastric mucosa. Specific aims: Paper I - To examine the spectrum, classification, pathophysiology, macroscopic and histopathological findings of the most common canine gastric lesions and compare these with the equivalent human disorders. Paper II In humans, the normal gastric mucosa glycosylation profile is characterized by the expression of neutral glycans, including the expression of type 1 Lewis antigens, Lea and Leb, in the superficial foveolar epithelium and type 2 Lewis antigens, Lex and Ley, in the glands. Sialylated antigens are rarely found in health conditions. In chapter 2, we intend to: will characterize the glycosylation profile of the normal canine gastric mucosa. - To characterize the glycosylation profile of canine gastric mucosa, focusing on the expression of the histo-blood group antigens, in order to understand their relevance in NHPH adhesion. - To evaluate the adhesion ability of different Helicobacter species to the canine gastric mucosa. The in vitro binding capacity of FITC-labelled H. pylori and NHPH to the canine gastric mucosa will be assessed in cases representative of the canine glycosylation pattern. 94 45 Aim and Objectives CHAPTER 1 95 Paper III Several works have discussed the prevalence of Helicobacter spp. in dogs, but few determine the specific species present in the canine stomach. The identification of NHPH at the species level has become possible through the use of PCR, allowing the determination of the prevalence of these various species in human and canine stomach biopsies. The accurate identification of Helicobacter spp. is essential in order to determine the prevalence and clinical significance of all taxa. In the chapter 3 we intend: - To determine the prevalence of Helicobacter spp. in the canine stomach; - To determine the distribution of Helicobacter spp. in the different portions of canine gastric mucosa and correlate it with possible histopathological alterations; - To determine the specific Helicobacter species involved in canine gastric infection. Paper IV - To characterise the molecular features of particular canine gastric lesions. In humans, and in contrast to the situation verified in the colon, most gastric polyps are considered non-neoplastic lesions. In dogs, gastric polyps are uncommon lesions thus, the molecular properties and biological significance of these canine lesions remains a poorly explored area. In chapter 4, we intended to analyse immunohistochemically a series of spontaneous canine gastric polyps regarding the presence of Helicobacter spp., cellular proliferative activity, potential phenotypic alterations and COX-2 and p53 expression. Additionally, the expression of these molecules in normal gastric mucosa will be investigated to evaluate the significance of their loss or overexpression in the gastric lesions. - Helicobacter spp. adhesion to canine gastric mucosa A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. binding to canine gastric mucosa with defined gastric glycophenotype Irina Amorim, Daniela P. Freitas, Ana Magalhães, Fátima Faria, Célia Lopes, Augusto M. Faustino, Annemieke Smet, Freddy Haesebrouck, Celso A Reis, Fátima Gärtner Helicobacter 2014; 19: 249–259 Helicobacter spp. adhesion to canine gastric mucosa CHAPTER 2 Helicobacter spp. adhesion to canine gastric mucosa CHAPTER 2 A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. Binding to Canine Gastric Mucosa with Defined Gastric Glycophenotype Irina Amorim,* ,† Daniela P. Freitas, † Ana Magalh~ aes, † F� atima Faria,* C� elia Lopes,* Augusto M. Faustino,* Annemieke Smet, ‡ Freddy Haesebrouck, ‡ Celso A. Reis †,§,¶ and F� atima G€ artner* ,† *Department of Pathology and Molecular Immunology of the Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Rua Jorge Viterbo Ferreira nr. 228, Porto 4050-313, Portugal, † Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, Porto 4200-465, Portugal, ‡ Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke B-9820, Belgium, § Department of Molecular Biology of the Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Rua Jorge Viterbo Ferreira nr. 228, Porto 4050-313, Portugal, ¶ Department of Pathology and Oncology, Faculty of Medicine of Porto, Alameda Prof. Hern^ ani Monteiro, Porto 4200-319, Portugal Keywords Canine gastric mucosa, bacterial adhesion, Helicobacter pylori, histo blood group antigens, non-Helicobacter pylori helicobacters. Reprint requests to:F � atima G€ artner, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, Porto 4200-465, Portugal. E-mail: [email protected] Abstract Background: The gastric mucosa of dogs is often colonized by non-Helicobacter pylori helicobacters (NHPH), while H. pylori is the predominant gastric Helicobacter species in humans. The colonization of the human gastric mucosa by H. pylori is highly dependent on the recognition of host glycan receptors. Our goal was to define the canine gastric mucosa glycophenotype and to evaluate the capacity of different gastric Helicobacter species to adhere to the canine gastric mucosa. Materials and Methods: The glycosylation profile in body and antral compartments of the canine gastric mucosa, with focus on the expression of histo-blood group antigens was evaluated. The in vitro binding capacity of FITC-labeled H. pylori and NHPH to the canine gastric mucosa was assessed in cases representative of the canine glycosylation pattern. Results: The canine gastric mucosa lacks expression of type 1 Lewis antigens and presents a broad expression of type 2 structures and A antigen, both in the surface and glandular epithelium. Regarding the canine antral mucosa, H. heilmannii s.s. presented the highest adhesion score whereas in the body region the SabA-positive H. pylori strain was the strain that adhered more. Conclusions: The canine gastric mucosa showed a glycosylation profile different from the human gastric mucosa suggesting that alternative glycan receptors may be involved in Helicobacter spp. binding. Helicobacter pylori and NHPH strains differ in their ability to adhere to canine gastric mucosa. Among the NHPH, H. heilmannii s.s. presented the highest adhesion capacity in agreement with its reported colonization of the canine stomach. The gastric mucosa of dogs is often colonized by helicobacters different from Helicobacter pylori (H. pylori). These non-H. pylori helicobacters (NHPH) are present in 67–86% of clinically healthy dogs, in 61–100% of animals presenting chronic vomiting [1–5] and in about 100% of laboratory dogs and dogs from local shelters [6–8]. The predominant gastric Helicobacter species in dogs are H. felis,H. bizzozeronii, and H. heilmannii sensu stricto (s.s.), while H. salomonis is less often detected and the prevalence of H. cynogastricus and H. baculiformis has not yet been studied [9–12]. Mixed infections with different species can also occur [13]. The pathogenic significance of gastric NHPH in dogs is poorly understood and remains controversial; therefore veterinarians are facing the dilemma of either treating or ignoring spiral organisms observed in canine gastric biopsies. However, these NHPH are of zoonotic significance and have been associated with gastritis, peptic ulcers and mucosa associated lymphoid tissue lymphomas in human patients [11]. Dogs may constitute a source of ©2014 John Wiley & Sons Ltd, Helicobacter 19: 249–259 249 Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12125 101 Helicobacter spp. adhesion to canine gastric mucosa CHAPTER 2 infection for their owners, although the prevalence of these canineand feline-associated helicobacters in humans is much lower than that of H. pylori. On the other hand, H. pylori has occasionally been identified in the canine stomach [14,15]. Gastric Helicobacter species have a host species preference, but may occasionally cross this host species barrier. The molecular mechanisms underlying this partial host-adaptation are not known, but may be related to differences in glycosylation profile of the gastric mucosa. The gastrointestinal tract glycosylation profile determines the colonization capability of various infectious agents. Bacterial binding occurs through recognition of specific glycan receptors expressed by the host epithelial cells [16–18]. The biosynthesis of glycan chains is controlled by enzymatic activity of several glycosyltransferases, which are expressed in a cell, tissue and speciesspecific manner (for review see [19]). The expression of the terminal Lewis glycan structures that are recognized as receptors for H. pylori in human gastric tissue depends on the enzymatic activity of specific fucosyltransferases and sialyltransferases [20] and are schematically represented in Fig. 1A, B. Two backbone structures are the precursors for the Lewis antigens biosynthesis: the type 1 Galb1-3GlcNAc, and the type 2 Galb1-4GlcNAc chains. Addition of a fucose to terminal galactose on type 1 structures leads to H-type 1 structure, which can be further modified with a fucose on the GlcNAc residue resulting in Lewis b (Le b ). Alternatively, the type 1 backbone may be fucosylated on the GlcNAc residue leading to Lewis a (Le a ) structure. Furthermore, the action of a sialyltransferase toward type 1 chains can lead to the biosynthesis of the sialyl-Le a (sLe a ) antigen. The biosynthesis of type 2 based Lewis antigens depends on the addition of the same glycan units but with different linkages, originating the isomers Lewis x (Le x ), Lewis y (Le y ) and sialyl-Le x (sLe x ) antigens (Fig. 1B) (as reviewed by [20]). The H. pylori blood group antigen-binding adhesin (BabA) recognizes both H-type 1 and Le b fucosylated antigens [16] expressed on the surface of the gastric mucosa of secretor individuals [21,22]. The secretor status is determined by the activity of the fucosyltransferase 2 enzyme (FUT2) [23] and the capacity to synthesize H-type 1 and Le b antigens in body secretions. The secretor and Lewis status in humans are associated with the adhesion and infection of H. pylori strains expressing the BabA adhesin [21,24,25]. In addition, a second H. pylori adhesin, the sialic acid-binding adhesin (SabA) has been described, which recognizes the sialylated antigens sLe a and sLe x [17]. These sialylated structures are absent in normal human gastric mucosa but are induced upon H. pylori infection and gastric mucosa inflammation [17,26,27]. The canine secretory alloantigen alloantibody system (CSA) is closely related to the human ABH-Le system being equivalent to the major human ABO exocrine glycophenotype. The CSA was first described in 1966 by Zweibaum and colleagues [28]. The genetic polymorphisms of the CSA were later analysed [29] and the structure of canine polymorphic antigens was further characterized in four phenotypes: A, X, Y, and AY [30] (Fig. 1C). However, limited information is available about the glycosylation profile of the canine gastric mucosa. Here we describe, for the first time, the glycophenotype observed in the canine gastric mucosa, with focus on the expression of Lewis glycan antigens. Furthermore, A B C Figure 1 Schematic representation of Lewis antigens. Type 1 chains (panel A) are characterized by the Galb1,3 linkage, while type 2 chains (panel B) display a Galb1,4 linkage. (Panel A) Addition of a fucose to terminal galactose on type 1 structures leads to H-type 1 structure, which can be further modified with a fucose on the GlcNAc residue resulting in Le b antigen. The type 1 backbone may be just fucosylated on the GlcNAc residue leading to Le a antigen biosynthesis. The action of a sialyltransferase towards the type 1 chain can lead to the biosynthesis of the sLe a antigen. (Panel B) the biosynthesis of type 2 based Lewis antigens depends on the addition of the same glycan units but with different linkages, originating the isomers Le x , Le y and sLe x antigens. Addition of a fucose to terminal galactose on type 2 structures leads to H-type 2, which can be further modified with a fucose on the GlcNAc residue resulting in Ley antigen. The type 2 backbone may be just fucosylated on the GlcNAc residue leading to Le x antigen biosynthesis. The action of a sialyltransferase toward the type 2 chain can lead to the biosynthesis of the sLex antigen. (Panel C) Schematic representation of canine secretory alloantiagens (CSA) polymorphisms (adapted from Oriol et al. [30]). ©2014 John Wiley & Sons Ltd, Helicobacter 19: 249–259250 Helicobacter Adhesion to Canine Gastric Mucosa Amorim et al. 102 Helicobacter spp. adhesion to canine gastric mucosa CHAPTER 2 Helicobacter spp. adhesion to canine gastric mucosa CHAPTER 2 the binding capacity of H. pylori and NHPH to canine gastric mucosa was evaluated. This study provides valuable information about the canine gastric glycosylation profile and about the Helicobacter capacity to adhere to the canine gastric mucosa, contributing to the understanding of the host colonization spectrum of the different Helicobacter species. Materials and Methods Tissue Samples and Histology Gastric samples were obtained during necropsy procedures from dogs that died from non-infectious causes (eight representative sections of the body and six of the antrum of the stomach). Tissues were fixed in 10% buffered formalin and paraffin-embedded. Serial sections 3 lm thick were made, one being stained with haematoxylin and eosin (HE) for histopathology and the others were used for the histochemical and immunohistochemical studies. Sections stained with HE were examined by three pathologists and were considered as normal gastric tissues, according to the criteria proposed by Prachasilpchain et al. [31]. Additionally, the presence of Helicobacter spp. was excluded based on the negative results of modified Giemsa stain and of anti-H. pylori immunohistochemistry, using a polyclonal antibody (RBK012; Zytomed, Berlin, Germany), which shows immunoreactivity with a wide range of bacteria belonging to the Helicobacter genus. Immunohistochemistry For the immunohistochemical study, sections were deparaffinized, hydrated and antigen retrieval was performed in a pressure cooker in 10 mmol/L sodium citrate buffer, pH 6.0, for 2 minutes. Slides were cooled for 10 minutes at room temperature and rinsed twice in triphosphate buffered saline (TBS) for 5 minutes. The Novolink TM Max-Polymer detection system (Novocastra, Newcastle, UK) was used for visualization, according to the manufacturer0s instructions. After blocking endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 minutes, sections were incubated, overnight at 4 °C, with the monoclonal antibodies specific for the carbohydrate antigens (Table 1). Sections were rinsed with TBS between each step of the procedure. Color was developed for up to 7 minutes at room temperature with 3,30-diamino-benzidine (DAB; Sigma, St. Louis, MO, USA) and sections were then lightly counterstained with haematoxylin, dehydrated, and mounted. Sections of human gastric mucosa with intestinal metaplasia were obtained from the pathology department archive of Hospital Santo Antonio –Porto and were used as positive controls. Negative controls were performed by replacing the primary antibody with an antibody of the same immunoglobulin isotype. Lectin Staining The biotinylated lectin Dolichus biflorus agglutinin (DBA), for detection of terminal alpha linked GalNAc, and the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) solution were purchased from Vector Laboratories (Burlingame, CA, USA). After deparaffination and rehydration, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes at room temperature. After washing twice with phosphate buffered saline (PBS) for 5 minutes, sections were incubated with 10% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature and then in the biotinylated lectin solution (1 : 100 in PBS) for 1 hour, at room temperature. The slides were subsequently washed in PBS and immersed in ABC solution for 30 minutes at room temperature, according to manufacturer0s recommendations. After washing twice with PBS for 5 minutes, slides were immersed in a freshly prepared solution of DAB containing 0.02% hydrogen peroxide for 7 minutes at room temperature to visualize the lectin binding sites, counterstained with haematoxylin, dehydrated and mounted. Sections of canine normal intestine were used as positive controls. Negative controls were performed by replacing the lectin by PBS. Evaluation of Gastric Tissue Glycosylation Profile A microscopic evaluation was performed by analyzing the entire section of the gastric tissue. Positive immunoreactivity was recorded as a distinct brown labeling of cytoplasm and/or membrane of epithelial cells. Extracellular staining of the superficial mucus was Table 1 Antibodies used in immunohistochemistry Monoclonal antibody Antigen Supplier/ References Dilution CA3F4 Le a Young et al. [55] 1 : 5 BG6 Le b Signet © (Dedham, MA, USA) 1 : 50 CA19-9 Sialyl-Le a Santa Cruz © (Santa Cruz, CA, USA) 1 : 500 SH1 Le x Fukushi et al. 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J Biol Chem 1984;259:4681–5. 57 Abe K, McKibbin JM, Hakamori S. The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant). J Biol Chem 1983;258:11793–7. Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Alignment of the peptide sequences of the predicted galactoside 3(4)-L-fucosyltransferase-like from Canis lupus familiaris and the Homo sapiens a1,3 and a1,3/1,4-fucosyltransferase FUT3. ©2014 John Wiley & Sons Ltd, Helicobacter 19: 249–259 259 Amorim et al. Helicobacter Adhesion to Canine Gastric Mucosa 111 - Epidemiological characterization of Helicobacter spp. in canine gastric mucosa Epidemiological characterization of the Helicobacter spp. present in canine gastric mucosa Irina Amorim, Annemieke Smet, Odete Alves, Silvia Teixeira, Ana Laura Saraiva, Freddy Haesebrouck, Celso Reis, Fátima Gärtner (In preparation) Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 Epidemiological characterization of the Helicobacter spp. present in canine gastric mucosa Irina Amorim1,2, Annemieke Smet3, Odete Alves1, Silvia Teixeira1, Ana Laura Saraiva1, Freddy Haesebrouck3, Celso Reis1,2,4, Fátima Gärtner1,2 1 Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Rua Jorge Viterbo Ferreira nr. 228, 4050-313 Porto, Portugal. 2 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal. 3 Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke B-9820, Belgium. 4 Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200319 Porto, Portugal. Key-words: Canine gastric mucosa; dogs; non-Helicobacter pylori helicobacters (NHPH); histochemistry; immunohistochemistry (IHC); polymerase chain reaction (PCR); stomach. Abstract To determine the prevalence of Helicobacter species and the gastric histopathological findings, gastric mucosa samples of 69 dogs were evaluated. Helicobacter species were detected in 59 dogs (85.5%) by at least one detection method. Histological, histochemical and immunohistochemical evaluations revealed Helicobacter spp. in 45 (65.2%), 52 (75.4%) and in 57 (82.6%) dogs, respectively. Bacteria were detected by PCR analysis in 33 (47.8%) dogs. H. heilmannii-like organisms were the most commonly found being identified in 22 animals (66.7%) and predominantly in the antral gastric region. H. salomonis was the second more prevalent species (51.5%) although it was mainly found in association with other than NHPH and in the body gastric region. H. bizzozeronni and H. felis were the less frequent species and H. cynogastricus and H. baculiformis were not detected at all. It was concluded that, despite the high incidence and worldwide distribution of NHPH, geographic variations partially account for the prevalence of a specific Helicobacter species in the dog stomach. 117 Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 Introduction The genus Helicobacter is composed of at least 32 species (Haesebrouck et al., 2009). Among these, Helicobacter pylori (H. pylori) is consider an important pathogen whose natural host is man but its presence in the canine stomach has been rarely reported (Buczolits et al., 2003; Chung et al., 2014; Ekman et al., 2013). A large number of non-Helicobacter pylori Helicobacter (NHPH) species have been also recognized in humans and in several animals. Previously, NHPHs were generally referred to as “H. heilmannii” which was further subdivided in two taxa, types 1 and 2 (Haesebrouck et al., 2009). Microorganisms referred to as H. heilmannii type 1 are identical to H. suis, a species colonizing the stomachs of pigs. The former H. heilmannii type 2 represents a group of species known to colonize the gastric mucosa of dogs: H. felis, H. bizzozeronii, H. salomonis, H. cynogastricus, H. baculiformis (Haesebrouck et al. 2009) and a bacterium which was, in 2004, given the provisional name ‘‘Candidatus H. heilmannii’’ because, at that time, it could not be cultured in vitro (O’Rourke et al., 2004). In 2011, a description of H. heilmannii as a novel species was defined (Smet et al., 2012). The name H. heilmannii sensu stricto (s.s.) was proposed to refer to the novel Helicobacter species and the term H. heilmannii sensu lato (s.l.) to refer to the whole NHPH group (Haesebrouck et al., 2011). NHPHs are present in 67–86% of clinically healthy dogs, in 61–100% of animals presenting chronic vomiting (Geyer et al., 1993; Hermanns et al., 1995; Hwang et al., 2002; Lecoindre P., 1995; Polanco et al., 2011) and in about 100% of laboratory Beagle dogs and dogs from local shelters (Eaton et al., 1996; Henry et al., 1987; Simpson et al., 1999). The predominant gastric Helicobacter spp. in dogs are H. felis, H. bizzozeronii and H. heilmannii sensu stricto (s.s.), while H. salomonis is less often detected and the prevalence of H. cynogastricus and H. baculiformis has not yet been studied (Baele et al., 2009; Haesebrouck et al., 2009; van den Bulck et al., 2005; Van den Bulck et al., 2006). The diagnostic methods used for Helicobacter spp. can be non-invasive and invasive (Prachasilpchai et al., 2007). Non-invasive methods like serology or detection of bacterial DNA and antigens in stools do not require a gastric biopsy or anaesthesia. The invasive methods, like bacterial cultures, histopathology, smears, electron microscopy or polymerase chain reaction (PCR), require a gastric biopsy, which is frequently obtained through endoscopy under anaesthesia or during necropsies. Typically Helicobacter spp. organisms are not easily visualized with the haematoxylin and eosin (HE) stain and so, their direct observation in biopsied specimens is highlighted by the use of special stains, such as the modified Giemsa (MG) stain. More elaborate and sensitive detection methods such as immunohistochemistry (IHC) or polymerase chain reaction (PCR) are research tools rarely used in a diagnostic setting. 118 Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 Several investigations have discussed the prevalence of Helicobacter spp. in dogs (Buczolits et al., 2003; Geyer et al., 1993; Henry et al., 1987; Hermanns et al., 1995; Polanco et al., 2011) but few determine the specific species present in the canine stomach (Chung et al., 2014; Jalava et al., 1998; Priestnall et al., 2004; van den Bulck et al., 2005). The accurate identification of the Helicobacter spp. is essential in order to determine the prevalence and clinical significance of all taxa. The aim of this study was to determine the prevalence and to identify the Helicobacter species present in distinct stomach regions of the canine stomach (body and antrum) using histological, histochemistry, immunohistochemistry and molecular diagnostic techniques. The degree of colonization was characterized and correlated with the respective histopathological changes in canine gastric mucosa. Materials and Methods Sample collection The gastric tissues were obtained from 69 dogs (45 male and 24 female, ranging in age from 3 months to 15 years). The samples were randomly selected from the archives of the Laboratory of Veterinary Pathology, ICBAS-UP (Portugal) where they were received between 2010 and 2013. Twenty samples were collected during endoscopic procedures, five during surgery and 44 during necropsy examinations. When available, the detailed description of each case was recorded and included information such as breed, sex, age and clinical signs. Only the gastric samples in good condition of preservation were included in this study. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin wax. Three consecutive sections 3 µm thick were made, one being stained with HE, other with a MG stain and the third was used for the immunohistochemical study. Sample evaluation Histopathological parameters such as alterations in cellularity, fibrosis of the lamina propria and gland atrophy were analysed according to the World Small Animal Veterinary Association (WSAVA) guidelines (Day et al., 2008). The degree of morphological features and inflammatory changes was graded as normal, mild, moderate or marked by using the available WSAVA gastrointestinal standardization visual analogue (Day et al., 2008). 119 Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 others, in the colonization process. Regarding the antrum region, the in vitro binding assays are in accordance with the results herein obtained and support the high prevalence of H. heilmannii-like organisms in this specific location (Amorim et al., 2014). Given these results and the proportion of dogs showing the H. heilmannii-like and H. salomonis bacterial combination, it seems plausible that direct competition between at least these two species may occur in order to achieve the successful colonization of the gastric niche in the dog. Furthermore, the high percentage of mixed infections comprising H. salomonis may suggest that the colonization capacity of this specific species in the gastric context may be enhanced when associated with other NHPH. However, further studies are needed in order to clarify these hypotheses. Taken together, all these results suggest that, despite the high incidence and worldwide distribution of NHPH, geographic variations amongst other factors, partially account for the prevalence of a specific Helicobacter species in the dog stomach. Acknowledgments I. Amorim (SFRH/BD/76237/2011) acknowledges FCT, the Portuguese Foundation for Science and Technology, for financial support. The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. Table 1 - List of primers used for qPCR 126 Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 PCR positiveresults with specific species identification, regardless gastric location (percent&number) (n=33) Positive results for Helicobacter spp. (percent& number) (n=27) Negative results (percent & number) (n=9) Hh (n=10) Hf (n=2) Hb (n=2) Hs (n=3) Hf+Hb (n=1) Hh+Hs (n=11) Hh+Hf (n=1) Hb+Hs (n=1) Hf+Hb+Hs (n=2) Histopathology grading (Day et al., 2008) Normal 3.0 (1/33) 0 0 3.0 (1/33) 0 0 0 0 0 7.4 (2/27) 11.1 (1/9) Mild gastritis 15.2 (5/33) 3.0 (1/33) 3.0 (1/33) 6.1 (2/33) 0 18.2 (6/33) 3.0 (1/33) 3.0 (1/33) 6.1 (2/33) 44.4 (12/27) 33.3 (3/9) Moderate gastritis 12.1 (4/33) 3.0 (1/33) 3.0 (1/33) 0 3.0 (1/33) 9.1 (3/33) 0 0 0 48.1 (13/27) 44.4 (4/9) Marked gastritis 0 0 0 0 0 6.1 (2/33) 0 0 0 0 11.1 (1/9) Epithelial injury Mild 27.3 (9/33) 3.0 (1/33) 0 9.1 (3/33) 3.0 (1/33) 30.3 (10/33) 0 3.0 1/3(3) 6.1 (2/33) 77.8 (21/27) 33.3 (3/9) Moderate 3.0 (1/33) 3.0 (1/33) 6.1 (2/33) 0 0 0 3.0 (1/33) 0 0 14.8 (4/27) 11.1 (1/9) Fibrosis/mucosal atrophy Mild 18.2 (6/33) 6.1 (2/33) 6.1 (2/33) 6.1 (2/33) 0 18.2 (6/33) 0 0 3.0 (1/33) 55.6 (15/27) 33.3 (3/9) Moderate 3.0 (1/33) 0 0 0 3.0 (1/33) 0 0 0 0 3.7 (1/27) 11.1 (1/9) Intraepithelial lymphocytes Mild 15.2 (5/33) 3.0 (1/33) 6.1 (2/33) 6.1 (2/33) 0 15.2 (5/33) 0 0 6.1 (2/33) 29.6 (8/27) 11.1 (1/9) Moderate 3.0 (1/33) 3.0 (1/33) 0 0 3.0 (1/33) 9.1 (3/33) 0 0 0 14.8 (4/27) 0 Lymphofollicular hyperplasia Mild 6.1 (2/33) 0 0 0 3.0 (1/33) 0 0 0 0 29.6 (8/27) 11.1 (1/9) Bacterial density (based on the IHC results) NA +0 0 0 0 0 0 0 0 0 14.8 (4/27) ++ 3.0 (1/33) 0 0 0 3.0 (1/33) 0 0 0 0 25.9 (7/27) +++ 24.2 (8/33) 6.1 (2/33) 6.1 (2/33) 9.1 (3/33) 0 33.3 (11/33) 3.0 (1/33) 3.0 (1/33) 6.1 (2/33) 51.9 (14/27) Table 2 - Table summarising the histopathological alterations and colonisation density observed in the canine stomach, according to the respective positive NHPH species-specific PCR results, positive genus results and negative results. 127 Legend: Hh, H. heilmannii-like; Hf, H.felis; Hb, H. bizozzeronni; Hs, H. salomonis. Bacterial density: +, few organisms; ++, moderate number of organisms; +++, large number of organisms. NA: not applicable Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 Table 3 – Detection of Helicobacter spp. in the different stomach compartments of the canine stomach recurring to different diagnostic methods. Gastric region Detection methods Positive (percent & number) HE MGS IHC* PCR Body (n=66) 62.1 (41/66) 68.2 (45/66) 84.8 (56/66) 37.9 (25/66) Antrum (n=51) 70.6 (36/51) 78.4 (40/51) 80.4 (41/51) 51.0 (41/51) * The positive results obtained with IHC did not differ significantly across each stomach region (p > 0.05). Table 4 - Specific Helicobacter species detected by PCR in the different stomach compartments of the canine stomach. Specific PCR-Helicobacter spp. positive results Gastric region (percent & number) Body (n=25) Antrum (n=26) H. heilmannii-like 24.0 (6/25) 57.7 (15/26) H. salomonis 44.0 (11/25) 7.7 (2/26) H. felis 8.0 (2/25) 3.8 (1/26) H. bizzozeronnii 4.0 (1/25) 11.5 (3/26) H. felis + H. bizzozeronnii 4.0 (1/25) 3.8 (1/26) H. heilmannii-like + H. salomonis 8.0 (2/25) 11.5 (3/26) H. heilmannii-like + H. felis 4.0 (1/25) 3.8 (1/26) H. felis + H. salomonis 4.0 (1/25) 0 128 Epidemiological characterization of Helicobacter spp. in canine gastric mucosa CHAPTER 3 References Amorim, I., Freitas, D. P., Magalhães, A., Faria, F., Lopes, C., Faustino, A. M., Smet, A., Haesebrouck, F., Reis, C. A. and Gärtner, F. (2014). A comparison of Helicobacter pylori and nonHelicobacter pylori Helicobacter spp. Binding to Canine Gastric Mucosa with Defined Gastric Glycophenotype. Helicobacter, 19, 249-259. Baele, M., Pasmans, F., Flahou, B., Chiers, K., Ducatelle, R. and Haesebrouck, F. (2009). 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