MODEL

Abstract: An animal model for experimental infection with Helicobacter pylori which displays the acute symptoms of Helicobacter pylori infection. The model can be prepared by injecting an animal, such as a xenobiotic dog, with a dose of bacteria previously adapted to the mouse. The invention also relates to the use of the animal model for the development of vaccines, chemotherapeutics and diagnostics.

Description: Model Technical field The present invention relates to an animal infected with Helicobacter pylori (H. pylori) capable of inducing acute symptoms and of maintaining a chronic infection of H. pylori, useful as a model for H. pylori infection. The model is especially useful for the development of vaccines and chemotherapeutics against H. pylori and diagnostics. Background of the Invention H. pylori was isolated in 1982 by B. Marshall and J. Warren using microaerophilic conditions that had been developed to grow Campylobacter jejuni. H. pylori bacteria are S-shaped, gram negative bacilli 2-3.5 μm in length and 0.5-1 μm in width (4). H. pylori is responsible for the most common infection in the world. In developing countries 80% of the population is infected by the bacterium at the age of 20, while in developed countries H. pylori infection increases with age from <20% in 30-year old people to >50% in 60-year olds (2, 4) The infection is transmitted by either the oro-fecal or the oro-oral route (4). Infection occurs during the first years of life and persists forever. Once established, the infection is chronic, possibly permanent. Risk factors for infection are crowding, poor hygiene and host-specific genetic factors. Chronic infection of the human gastroduodenal mucosae by H. pylori is frequently associated with chronic gastritis, peptic ulcer, and increases the risk of occurrence of gastric malignancies such as adenocarcinoma and low grade B cell lymphoma (4, 43, 44). Most of the infections remain asymptomatic, whereas symptomatic, severe diseases correlate epidemiologically with the infection by a subset of H. pylori strains, called Type I (5-7, 13, 55). This subset of strains is endowed with increased virulence due to the expression of a biologically active toxin (VacA), which is cytopathic to gastric epithelial cells in vitro and in vivo (17, 21 , 52), and also to the acquisition of a pathogenicity island (PAI), called cag, which contains a set of genes encoding several virulence factors (8) which are responsible for the induction of the synthesis of the neutrophil chemotactic cytokine IL-8 by the gastric epithelial cells (8). Colonization of the mucosa of the stomach by H. pylori is today recognized as the major cause of acute and chronic gastroduodenal pathologies in humans (4, 6). The recognition of the infectious nature of the illness is having a major impact in the treatment of the disease that is shifting from the treatment of symptoms by anti-H2 blockers to the eradication of the bacterial infection by antibiotic regimen. In spite of the unquestionable successes that will be achieved with antibiotic treatment, it should be remembered that this success will inevitably lead to the occurrence of resistant strains that in the long term will make antibiotics ineffective. This suggests that vaccination, which classically is the most effective way to prevent and control infectious diseases in a large population, could be used to prevent infection and possibly also to treat the disease. Vaccine development requires understanding of a number of critical steps that are not yet fully studied. These are: 1 ) identification of the factors important for virulence, 2) large-scale production and characterisation of the virulence factors, 3) development of appropriate animal models to test the virulence and immunogenicity of the molecules identified, and 4) development of an antigen delivery system capable of inducing the type of immunity that is necessary to confer protection against infection and disease. The increasing importance of H. pylori in the induction of a wide variety of gastric pathologies has represented a major challenge for the development of efficacious vaccine strategies. To better understand the interactions between the bacterium and the host, much effort has focused on the development of appropriate animal models of infection reproducing aspects of the natural human infection. Thus, one of the most pressing needs is the development of an animal model in which to study H. pylori infection. Several animal models of infection and disease have been developed aiming at studying the pathogenesis of infection and development of preventive and therapeutic strategies. Many of these models are highly impractical, since they employ monkeys (12) or species that are kept under gnotobiotic (that is, germ-free) conditions, for example germ-free dogs or piglets (31 , 47). Colonization of gnotobiotic piglets (31 ) has been reported using H. pylori strains isolated from patients with gastroduodenal diseases. However piglets cannot be kept under germ-free conditions for more than 2 months (47) mainly due to their nutritional needs. Successful infection of specific pathogen-free (SPF) cats has been described using a strain isolated from conventional cats (14, 20). Gnotobiotic beagle pups have also been infected with a human H. pylori isolate and kept under germ- free conditions for 30 days. However, in this model no data are available on long term infections with H. pylori (47). Other experimental animal models include athymic nu/nu (29) or germ-free mice (29). The major drawbacks of these experimental infections, however, are the sophisticated and expensive housing systems required, and, more importantly, the peculiar immunological status of the gnotobiotic or immunodeficient hosts employed. More recently, H. pylori, freshly isolated from human gastroduodenal biopsies, have been adapted to persistently colonize the gastric mucosa of xenobiotic mice (37). This model has proven particularly useful to assess the feasibility of either preventive (36-38, 46) or therapeutic (18) vaccination, as well as for the in vivo screening of anti-H. pylori antimicrobials (33), and for studying the pathogenesis of infection (48). However, to evaluate gastric infection, mice have to be sacrificed; the pathological changes induced by the chronic infection and/or the effect of therapeutic or immunizing regimens cannot, therefore, be followed up in the same individual animal. The present invention describes for the first time an animal model which can reproduce symptoms which have been clearly associated with the acute phases of infection with H. pylori (39, 40, 41 , 49) in humans. Summary of the Invention The present application is based on the discovery that H. pylori can persistently colonize the gastric mucosa of conventional xenobiotic dogs, and that this colonization causes acute symptoms, histopathological lesions and elicits specific immune responses. According to a first aspect of the present invention, there is provided a non- human, non-murine animal infected with Helicobacter pylori which exhibits one or more acute symptoms of Helicobacter pylori infection. Preferably, the Helicobacter pylori establishes chronic persistent infection in the infected animal. As used herein, the term "acute symptoms" includes chronic gastritis, peptic ulcer, and increased risk of occurrence of gastric malignancies such as adenocarcinoma and low-grade B-cell lymphoma. The animal may be any non-human, non-murine animal capable of sustaining an infection with Helicobacter pylori and exhibiting one or more acute symptoms of Helicobacter pylori infection. Preferably, the animal is a larger animal (i.e. larger than a rat, mouse or guinea pig), suitably one adapted to laboratory use, such as a cat, dog, sheep, goat, pig, monkey, or horse. Larger animal models are useful because the progression of the experimental infection can be followed in a single animal. This has the advantage of reducing the error in following disease progression associated with the analysis of a number of different, smaller animals, such as mice. Using dogs as animal models for human disease also has the advantage that the physiology of a dog is similar to that of a human. The possibility of stable infection is to be contrasted with infections which exhibit the acute symptoms of Helicobacter pylori infection. Preferably, the animal is xenobiotic dog. In the described embodiment the dog is a beagle. According to a second aspect of the present invention, there is provided a method for infecting an animal according to the first aspect of the invention with Helicobacter pylori comprising the steps of: (a) isolating Helicobacter pylori; and (b) administering a dose of the isolate of Helicobacter pylon to said animal. Preferably, the administration of Helicobacter pylori is repeated at intervals after the initial infection. Preferably, the administration of Helicobacter pylori is repeated 3 times every other day after the initial infection for one week. Preferably, prior to the administration of Helicobacter pylori, stomach acid of the animal is neutralised. Preferably, the isolate of Helicobacter pylori is passaged several times in vivo prior to administration. Preferably, the process further comprises of the step of: (c) isolating Helicobacter pylori from the animal and administering the isolate to a further animal. Preferably, the isolate of Helicobacter pylori in step (a) is isolated from a non- human animal. Preferably, the non-human animal is a mouse. In a third aspect of the present invention, there is provided an animal according to the first aspect of the invention when prepared by the method of the second aspect of the invention. According to a further aspect of the present invention, there is provided an animal for use in: (i) the study of the pathogenesis of Helicobacter py/or/-induced disease; (ii) development of vaccines; (iii) development of chemotherapeutics; or (iv) development of diagnostics. There is also provided a method for evaluating a vaccine or chemotherapeutic agent against Helicobacter pylori comprising the steps of: (a) administering to a non-human, non-murine animal an effective amount of candidate vaccine or chemotherapeutic agent; (b) administering to the non-human, non-murine animal Helicobacter pylori, in accordance with the method of the second aspect of the present invention; and (c) observing the incidence of Helicobacter pylori infection in the animal model. Finally there is provided an isolate of Helicobacter pylori obtained from an animal according to the first or third aspects of the present invention. The animal model contemplated by the present invention has the advantage that the cost of experiments is reduced. For example, many experimental mice would be required to attempt to follow the disease progression of experimental H. pylori infection compared to just one animal, for example, a dog. In the particular example of a canine model, and with respect to following H. pylori infection, the pH of a dog's stomach is as low as a human's stomach, whereas the pH of a mouse's stomach is higher. The similarity of dog physiology to human physiology has a further advantage when considering the xenobiotic nature of the dog model of one embodiment of the present invention. Gnotobiotic dog models, for instance, apparently show no human-like clinical symptoms of H. pylori infection (47). Conventional xenobiotic beagle dogs have already been utilized to reproduce experimental infections with human pathogens like Yersinia enterocolitica (24), Borrelia burgdorferi (9), and Leishmania infantum (3). It has been reported that gastroduodenal mucosae of conventional dogs can be naturally colonized by some gastrospirilla (25, 26), which may occasionally cause mild or moderate gastritis, but not by H. pylori (26). A conventional xenobiotic dog with normal intestinal flora which can be successfully infected by H. pylori would therefore be an ideal model. In one embodiment of the present invention, the feasibility of establishing H. pylori infection in conventional xenobiotic dogs, using a strain of H. pylori previously adapted to the mouse (24), has been assessed and the long term outcome of this infection has been investigated. Brief Description of the Drawings Figure 1. Histopathology and endoscopic findings in infected dogs. (A) Hematoxylin-eosin (H & E) stain of gastric mucosa at 1 week post-infection (PI) showing hyperemia (arrow) and edema (arrow head) of the mucosal lamina propria (original magnification = 400x). (B) H & E staining of antral mucosa at 1 week PI showing numerous polymorphonuclear granulocytes (arrows) infiltrating the lamina propria among the glands (original magnification = 400x). H & E stainings of antral mucosa taken at 1 week PI reveals (C) exocytosis of some polymorphonuclear cells (arrow heads) through flattened and vacuolized epithelium, and (D) several polymorphonuclear granulocytes (arrow) in the lumen (original magnifications = 1 ,000x). (E) A round hyperemic, superficial erosion of antral gastric mucosa observed by endoscopy at 4 weeks PI. (F) H & E stain of antral mucosa at 4 weeks PI showing vacuolar degeneration (arrows) of the epithelium (original magnification = 400x). (G) Evident epithelium damage in the antral mucosa at 4 week PI. Toluidine Blue stain of semi-thin section (original magnification = 200x). (H) High power magnification (1 ,000x) of the previous field showing some curved bacteria (arrow heads) in close contact with epithelial cells which are heavily vacuolized (arrows) and show loss of the apical secreting portion (open arrows). (I) endoscopical aspect of antral mucosa at 8 weeks PI showing the typical 'bumpy' appearance of the gastric wall due to foliicular gastritis. Immunohistochemical examination at 8 weeks (J), and 16 weeks (K) PI revealed the presence of some bacteria stained with the anti- VacA C1G9 monoclonal antibody (Mab) in the mucus (arrow) overlying the epithelium (original magnification = 1 ,000x). (L) H & E stain of a biopsy taken from the area shown in panel I. A characteristic lymphoplasmacytic follicle associated with chorion edema and displacement of glands, (original magnification = 100x). (M) H & E stain of antral mucosa showing a sub- epithelial follicle and gland epithelial degeneration (original magnification = 400x). Figure 2. Transmission electron microscopy. Ultrastructural aspect of antral mucosa 18 weeks PI; two typical H. pylori bacteria are visible in the glandular lumen. Close association of the bacterium and the cell membrane with a characteristic pedestal of condensed actin and myosin is shown. (Original magnification = 12,000x). Figure 3. Detection of H. pylori in gastric biopsies by PCR. H. pylori DNA amplification of the cagA sequence showing a PCR product of 298-bp in gastric biopsies of dogs collected at 4 and 12 weeks PI but not in gastric samples collected before infection. Figure 4. Antibody responses during infection. Figure 4A shows the serum IgG antibody response to a H. pylori bacterial lysate and to two purified H. pylori antigens, CagA and VacA. Each curve individuates single dogs. Figure 4B shows the IgA and IgG antibody response to H. pylori bacterial lysate in the saliva. Each curve individuates single dogs. Results are expressed as the optical densities at 405 nm in salivary samples diluted 1 :20. Figure 5. Endoscopic findings in protected and infected dogs. Shows endoscopic examination of the antral region of protected dogs (Figure 5A) and control (infected) dogs (Figure 5B) 42 days after challenge with H. pylori. Figure 6. Histological findings in protected and infected dogs. Shows histological examination (hematoxylin-eosin staining) of biopsies taken from the antral region of protected dogs (Figure 6A, magnification = 10x) and control (infected) dogs (Figure 6B, magnification = 10x; Figure 6C, magnification = 40x; Figure 6D, magnification = 100x) 42 days after challenge with H. pylori. Figure 7. Immunohistochemical findings in infected and protected dogs. Shows immunohistochemical examination of biopsies taken from the antral region of protected dogs (Figure 7A) and control (infected) dogs (Figure 7B) 42 days after challenge with H. pylori (magnification = 40x). Detailed Description of the Invention As an embodiment of the present invention, the feasibility of experimental infection of conventional dogs with H. pylori, and the long term outcome of this infection was investigated. It is shown that a mouse-adapted H. pylori Type I strain persistently colonizes the stomach of xenobiotic beagle dogs, causes acute symptoms and gastric pathology, and elicits specific immune responses. One of the most striking features of the model described herein is that the dogs presented evident clinical signs, such as vomiting and diarrhea, at the time of the onset of infection which lasted for 1-2 weeks PI. To the best of the inventors' knowledge, this is the first animal model in which acute symptoms were observed as a consequence of gastric inoculation of H. pylori. In a study carried out with 7-days-old germ-free beagle pups, Radin et al. (47) did not report any acute symptoms, with the pups remaining asymptomatic for the duration (30 days) of the experiment. There may be several explanations for the occurrence of evident clinical signs in the present study. First, a phenotypically characterized cytotoxic Type I strain which had been adapted to a non-human host by several in vivo passages was used. Second, a larger dose of bacteria (three inocula of 10 9 bacteria) than that (single bolus of 3x10 8 bacteria) employed by Radin et al. (47) was used. Third, the animals used in the present study have a normal gastrointestinal commensal microbial flora which may contribute to amplify the pathological consequences of infection. These factors possibly played a role in inducing the symptoms observed in the infected dogs. Although the documentation of the clinical consequences of early stages of acute infection in humans is scarce (39, 40, 41 , 49), it is noteworthy that in the self-infection experiments vomiting has been reported (39, 41 ), as well as an increase in intestinal peristalsis and a transient softening of feces (39). Furthermore, VacA toxic activity was also found in a proportion of diarrheal feces from children (34, 35). It is then logical to hypothesize that the acute symptoms observed in the dogs of the present study reproduce those which can be encountered during the early stages of H. pylori infection in humans. One of the advantages of the xenobiotic dog model presented herein is the ease of follow up of the infection over a long period of time, at variance with the time limitations imposed by the germ-free animal models (47). This advantage has been exploited in the present investigation, where it was possible to follow up the occurrence and progression of histopathological lesions induced by the infection for 24 weeks. Indeed, the three dogs developed early superficial gastritis, with appearance of mucosal erosions, which progressed to foliicular gastritis. It is remarkable that at early stages of infection the gastric lamina propria was heavily infiltrated by neutrophils. In other animal models, this neutrophil infiltration is usually much less pronounced (31 , 37) or has been described only at later stages of infection (32, 47). This infiltration, accompanied by symptoms like vomiting, mimic that observed in acute gastritis in infected humans (39, 41 , 49), where it has been hypothesized to be possibly mediated by some pro-inflammatory chemokines, such as IL-8, specifically induced by products of cytotoxic (Type I) H. pylori strains (1 , 11 , 28, 45, 53). With the progression of infection, there is appearance of clear signs of epithelial erosions both macroscopically and microscopically, with characteristic cellular vacuolization and loss of the apical portion of the cell. Previously, similar lesions in the gastric mucosa of H. pylori infected mice (18, 37) have been observed. One of the most striking features at later stages of infection was the presence of well structured lymphoid follicles observed in all 3 dogs, both in the corpus and in the antrum. This is a typical finding of chronic stages of infection by H. pylori, which has been extensively reported both in chronically infected patients (16, 42) and in experimentally infected animals (14, 20, 47). These organized lymphoid structures may represent the consequence of an active and persistent stimulation of the immune system at the local mucosal level, with recruitment in situ of specific T-cells (27). It has been reported that in some individuals such lymphoid structures in the gastric mucosa may precede the development of low grade B-cell lymphoma (44). During the entire follow up, H. pylori was identified in the gastric biopsies by immunohistochemistry, TEM, urease test, and PCR. Furthermore, it was also possible to culture the bacteria from the gastric biopsies and the rectal swabs. This clearly shows that the evolution of the pathology in this model is constantly associated with the presence of H. pylori. It should be stressed that viable H. pylori was recoverable at any time of observation, even at 24 weeks after infection. This finding is in agreement with the recovery of H. mustelae in the stools of experimentally infected ferrets (15). Should this be applicable to natural human infections, as suggested by some authors (30, 52) and also by the wide similarities found between the histopathological findings in our dogs and those in infected patients, it is tempting to hypothesize that infected individuals can spread infectious H. pylori in stools, and that, under certain epidemiological conditions (23), this may represent a source of dissemination to other subjects. It is however highly unlikely that oral-fecal transmission took place during the course of this experiment, since the dogs were constantly kept isolated in individual boxes. H. pylori DNA was also detected in the oropharyngeal swabs of infected dogs. However, since it was not possible to isolate viable (i.e. cultivable) forms of the bacterium, it cannot be concluded whether this might represent another possible route of transmission of infection to other individuals, as suggested by Axon (2). The experimental infection with H. pylori induced an early detectable antibody response to different antigens, including CagA and VacA, both in serum and saliva, which persisted during the entire period of follow up. The feasibility of immunizing conventional dogs with whole cell H. pylori vaccine (i.e. H. pylori cell lysate) was also investigated. It is shown that such a preparation can elicit an immunity that provides a protective effect against a later challenge with infectious H. pylori. Immunogenicity. The protective effect of a whole cell H. pylori vaccine was investigated in dogs challenged with infectious H. pylori. The details of the experimental procedures followed are set out later. As compared to time 0 and to control dogs, i.m. immunization induced very high titers of serum IgG antibodies as shown in Table 1. I.m. immunization with H. pylori lysate also induced production of antigen-specific serum IgA antibodies. Symptoms. Control dogs #2 and #3 had diarrhea during the first week after the last challenge. Control dog #2 also had vomiting. There were no symptoms of H. pylori infection in the i.m. group. Urease test on gastric (antral) biopsy and on gastric lavage. This assay, performed on antral biopsies and gastric lavage taken 10 days after the last challenge, was positive in the control group, and negative in the i.m. group as shown in Table 2 below, even 24 h later. These data were also confirmed at day 42 post-challenge. Table 2 Group dog ϋ biopsy gastric lavage Control 1 + + 2 + + 3 + + i.m. 1 2 3 Table 1 Immunogenicity of the H. pylori lysate given four times intramuscularly to Beagle dogs Serum IgG titers to: Serum IgA titers to Immunization H. pylori lysate CagA NAP H. pylori lysate Dog no. route Pre * Post-4 Pre Post-4 Pre Post-4 Pre post-4 nil (control) < 100§ 100 < 100 < 100 < 100 < 100 < 20 < 20 nil (control) < 100 100 < 100 < 100 < 100 < 100 < 20 < 20 nil (control) < 100 100 < 100 < 100 < 100 < 100 < 20 < 20 4 intramuscular < 100 > 25000 < 100 12800 < 100 > 12800 < 20 1280 5 intramuscular < 100 > 25000 < 100 12800 < 100 > 12800 < 20 320 6 intramuscular < 100 > 25000 < 100 6400 < 100 > 12800 < 20 640 Pre: serum samples taken before the immunization; Post -4: serum sample taken after four immunizations, before the challenge. §Titers are expressed as the last serum dilution giving an optical density equal or higher than 0.2. Titers < 100 (IgG) and <20 (IgA) are considered as negative. Endoscopy, histology and immunochemistry results. Protected dogs showed normal mucosa at endoscopic examination at 42 days post-challenge (Fig. 5A), with a bright and smooth surface, without signs of hyperemia or edema. Conversely, in the control (infected) dogs, the gastric mucosa was heavily red and edematous, and had a rippled surface with appearance of plicae (Fig. 5B), suggestive of the nodular (foliicular) gastritis described above. Histologically, the gastric mucosa of protected dogs conserved an intact structure, both at the surface and at the submucosa (Fig. 6A), whereas in infected dogs there was appearance of hyperemia (Fig. 6B, arrows), edema (Fig. 6B, asterisk), inflammatory cellular infiltrates (Figure 6B, arrowheads and Fig. 6C). H. pylori was also easily identified in the mucous layer (Fig. 6D). All these data were confirmed by immunohistochemistry using an anti-VacA monoclonal antibody, which heavily stained epithelial cells of infected dogs (Fig. 7B, arrows), but not those of protected dogs (Fig. 7A). Conclusions. Taken together these data show that i.m. immunization with H. pylori lysate can protect dogs against challenge with infectious H. pylori, and that the i.m. route can be considered for vaccination against this bacterium. Consequently, the presently claimed animal model is clearly useful in developing vaccines and chemotherapeutics against H. pylori and assessing their efficacy. Experimental Infection in animal model H. pylori strain. SPM326s, a streptomycin-resistant derivative of the mouse-adapted H. pylori Type I (CagA+/VacA+) strain SPM326 (37), was grown as previously described (37) and used for the infection of dogs. Animals and experimental design. Three 4-6 months-old xenobiotic beagle dogs, one male and two females (Morini s.a.s., S. Polo D'Enza, Italy), were selected on the basis of the absence of detectable serum IgG against H. pylori in Western blot (WB) analysis using total bacterial lysate as antigen (see below). The three dogs selected were housed in standard conditions and maintained on a diet of dry food (MIL, Morini s.a.s.) and tap water ad libitum. Upon arrival in our animal facilities, an additional WB analysis on sera confirmed their H. pylori status. The dogs were housed in individual cages and allowed to adapt for a month to the new environment. During the month of adaptation, two tests were carried out on fecal samples to assess the presence of intestinal parasites or common enteric pathogenic bacteria. The dogs were then challenged 3 times every other day over a one-week period with the mouse-adapted H. pylori SPM326s strain as follows: 24 h before each challenge the dogs were fasted. 2 h before bacteria inoculation, dogs received 10 mg/kg of cimetidine intramuscularly (i.m.) (Tagamet® 200; Smith Kline & French, USA). At the moment of challenge, the dogs were anesthetized with a mixture of 40 μg/kg of medetomidine chloridrate (Domitor®; Centralvet- Vetem s.p.a., Milano, Italy) and 5 mg/kg of ketamine (Ketavet®, Gellini, Latina, Italy) intravenously (i.v.); then a gastric lavage was performed with 100 ml of 0.2M NaHCO 3 sterile solution followed by oral challenge with 3 ml of a freshly prepared suspension of 10 9 CFUs in sterile saline of the H. pylori strain SPM326s, grown under microaerobic conditions (see below), prepared immediately before the inoculation procedure. At the end of the bacterial inoculation, 200 μg/kg of the anesthetic antagonist atipamezole (Antisedan®; Centralvet-Vetem s.p.a., Milano, Italy) was administered and then dogs were again treated with cimetidine and fed after 2 h. Blood samples, salivary samples, and oropharyngeal and rectal swabs were collected under sterile conditions immediately before the challenge and at 1 , 2, 4, 8, 12, 18 and 24 weeks post-challenge. At the same times, gastric endoscopies were performed using a 4.9-mm-diameter Pentax pediatric bronchoscope (Pentax Technologies, Zaventem, Belgium). Pre-challenge gastric biopsies were taken during the endoscopies using flexible pinch-biopsy forceps at the antrum, corpus, fundus, and cardias for urease testing and for microbiological, PCR, histopathological and immunohistochemical analyses. Before each endoscopy the whole instrument and the flexible forceps were soaked in 4% glutaraldehyde for 45 minutes and then rinsed in sterile saline. To avoid cross-contamination among biopsies taken at different sites, the forceps were washed with tap water and lightly flame-sterilized before the collection of each bioptic sample. The above experimental protocol was approved by the Scientific and Ethical Commitee of the University of Pisa and received official authorization (DM No. 21/97-C) from the Italian Ministry of Health (Department of Veterinary Health, Food and Nutrition). Rapid urease test. Antral biopsies were incubated for up to 24 h in 1 ml of a 10% urea solution in distilled water added with two drops of a 1 % phenol red solution (Sigma Chemical Co., St. Louis, MO, USA) in sodium phosphate buffer, pH 6.5. A positive test is indicated by change of color (from orange to dark pink) in the medium; the time necessary for the color change is recorded (Table 3). The time to positivity of this test has been shown to be proportional to the number of bacteria present at the biopsy site (22). Histopathology and Transmission Electron Microscopy. Samples for histological, immunohistochemical and ultrastructural examination were taken from the biopsies at sites adjacent to those utilized for microbiological analysis and for PCR. The samples were fixed in 10% buffered formalin and embedded in paraffin. 3 μm sections were stained with hematoxylin-eosin (H & E) and Alcian and Periodic acid Schiffs (PAS) staining using standard procedures for histopathological examination. Similar sections were also employed for immunohistochemical analyses using the Avidine-Biotine-Complex (ABC)- peroxidase technique with a monoclonal antibody (Mab) specific for H. pylori (Biogenesis Ltd, Poole, England, UK) or an anti-VacA mouse monoclonal antibody (C1G9) obtained by immunizing Balb/c mice with purified native H. pylori VacA (Burroni and Telford, unpublished observations). Biotinylated horse anti-mouse antibody was used as secondary antibody. The reaction was developed with 3-1-diaminobenzidine-chlorhydrate (DAB) (Sigma) for identification and location of bacterial antigen. For electron microscopic examination, other samples were fixed in Karnowsky, post-fixed in OsO 4 , and embedded in Epon-Araldite (Polysciences Inc., Warhngton, PA, USA). Semi- thin sections were stained with toluidine blue for evaluation of cell damage, whereas ultra-thin sections were stained with uranyl acetate and lead citrate, and then examined with a Philips EM 301 transmission electron microscope (TEM) operating at 80 KV. Bacterial growth conditions. Gastric biopsies taken from the different gastric sites, as well as the rectal and oropharyngeal swabs, were streaked onto Columbia agar plates (Gibco BRL, Paisley, UK) containing 5% horse blood supplemented with Dent's or Skirrow's antibiotic mixtures (Oxoid, Basingstoke, UK) plus 400 μg/ml streptomycin (Sigma). Plates were incubated in microaerobic conditions using an Anaerojar system with Campygen atmosphere generating system (Oxoid) for 7-12 days. Growing H. pylori colonies were identified by the typical morphology assessed by visual inspection of the plates, and then confirmed by Gram staining, positive urease test, and positive PCR amplification of the cagA gene. Polymerase chain reaction (PCR). Bacteria or gastric bioptic samples were incubated at 37°C for 30 minutes with lysozyme at 100 μg/ml in 0.1 M NaCI, 1mM EDTA, 10 mM Tris-HCI pH 8. Then SDS was added (at a final concentration of 1 % wt/vol), and the mixture was heated at 65°C. Proteinase K (25 μg/ml) was added before incubation at 50°C for 2 h. Then the mixture was extracted with phenol-cloroform. After extraction, the DNA solution was mixed with absolute ethanol at -20°C for 1 h, washed with 75% ethanol, and dried. The DNA pellet was suspended in distilled water (7). Oligonucleotides were synthesized on an Applied Biosystem synthesizer (Applied Biosystems Inc., Foster City, CA, USA) by the automated phosphoramidite coupling method and purified by standard protocols. The H. pylori CagA primers, R008 (5' TTAGAATAATCAACAAACATCACGCCAT 3') and D008 (5' AATGCTAAATTAGACAACTTGAGCGA 3'), were derived from the sequenced cagA gene (7), giving an amplified product of 298-bp. PCR mixture contained 50 mM KCI, 10 mM Tris, 200 mM deoxynucleoside triphosphates, 30 pmol of each primer, 0.1 μg of bovine serum albumin, 2.5 U of Amplitaq (Perkin-Elmer, Norwalk, Connecticut, USA), and 80 ng of H. pylori chromosomal DNA. Amplifications were performed on a PCR system 9600 thermocycler (Perkin-Elmer) with the following cycling profiles: 94°C for 30 s, 65°C for 30 s, and 72°C for 30 s for 38 cycles and then extension at 72°C for 7 minutes. Detection of anti-H. pylori antibodies. SDS-PAGE of H. pylori (strain SPM326s) and WB analysis of sera were performed according to previously published procedures (37). Briefly, dog sera were diluted 1 :200 and incubated for 2 h at room temperature. Then, horseradish peroxidase (HRP)-conjugated rabbit anti-dog IgG antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) was added at 1 :2,000 dilution for 2 h, and the reaction was developed using 4-α-chloronaphtol as substrate. Detection of antibody against H. pylori by ELISA was carried out on 96-well plates coated overnight at 4°C with SPM326s lysate (10 μg/well) or with purified native VacA or CagA (0.2 μg/well). Coated wells were blocked with PBS containing 5% non-fat milk. Twofold serial dilutions of the sera were incubated at 37°C for 2 h and then washed with PBS. Antigen specific IgG titers were determined using a 1 :4,000 dilution of HRP-conjugated rabbit anti-dog IgG polyclonal antibody for 2 h at 37°C. Antigen bound antibodies were revealed by adding o-phenylenediamine dihydrochloride (Sigma) as a substrate. For detection of H. py/or/ ' -specific IgA and IgG in the saliva, similar assays were utilized by diluting salivary samples 1 :20 and utilizing alkaline phosphatase-conjugated goat anti-lgA or -IgG antibodies. Antibody titers were determined as previously described (18). Acute symptoms following H. pylori infection. For one week after the third challenge with H. pylori, the three dogs experienced at least one episode per day of diarrhea, which also persisted, but was less frequent, during the second week. Furthermore, the dogs had at least one episode of vomiting per day during the first week PI only. These symptoms disappeared spontaneously without specific treatment and no other symptoms were observed (Table 3). All three dogs remained alert and fed normally throughout the whole study. Histopathology and endoscopic findings. Endoscopic and histological examinations of the gastric mucosa performed at time zero were normal in the three dogs (not shown). In the histological sections of biopsies taken at 1 week PI a marked hyperemia and edema of the mucosal lamina propria, particularly in the corpus and in the antrum, was observed (Fig. 1A). At this time, acute antral gastritis was evident, which was characterized histologically by the presence of numerous polymorphonuclear leukocytes infiltrating the lamina propria among the glands, the structure of which was in some cases significantly altered (Fig. 1 B). Notably, several polymorphonuclear leukocytes were interspersed in the epithelial cell layer, which was also often markedly disarrayed (Fig. 1C). Furthermore, several polymorphonuclear leukocytes were also found in the mucous layer (Fig. 1 D), suggesting that a marked neutrophil transcytosis was elicited by the infection. Upon endoscopic examination performed one and two weeks PI, a gastric hyperemia was observed in the three dogs. Edema of the mucosa and catarrh adhering to the mucosa of the antrum and corpus were present (not shown) conferring a congested aspect of gastric wall. At 2 weeks PI, histology of the gastric biopsies evidenced a marked decrease in the neutrophilic infiltration into the lamina propria and a concomitant increase of mononuclear cells. Edema and hyperemia of the lamina propria were still present (not shown). After 4 weeks, clear signs of superficial erosions were endoscopically evident in the antrum (Fig. 1 E). Histological sections showed vacuolar degeneration, loss of the apical secreting portion of the cells, piknosis and rhexis of epithelial cell nuclei (Figs. 1 F, G and H). At the 8th week PI, foliicular gastritis, with the characteristic 'bumpy' aspect of the gastric wall (Fig. 11), was easily detected by endoscopy in all three animals. Histological examination of gastric biopsies revealed the presence of small lymphoplasmacytic aggregates among the glands of the corpus and fundus; small lymphoid follicles (1.5-2 mm) appeared in the antrum (Fig. 1L). Associated with these follicles, degenerative processes in the epithelial cells, presence of scattered neutrophilic granulocytes, and an increase of exocytosis of mononuclear cells (Fig. 1 M) were observed. All these signs have been reported as being typical of chronic active gastritis in infected humans (10, 16, 19, 42, 50, 54). These macroscopic and microscopic signs remained unchanged at later times (i.e., at the 12th, 18th and 24th week PI). At this advanced stage of infection, marked modifications in the mucin composition, as detected by Alcian-PAS staining of histological section, was also observed, with a progressive depletion of PAS-positive cells in the antrum (not shown). H. pylori was identified in the biopsies immunohistochemically using H. py/or/ ' -specific monoclonal antibodies (Mabs). Figs. 1 K and J show H. pylori, stained with the C1G9 anti-VacA Mab, in the mucous layer of the antrum at 8 and 16 weeks PI, respectively. H. pylori was also observed by TEM in the lumen of antral glands, in close association with epithelial cell membranes (Fig. 2). Isolation and identification of H. pylori. The presence of H. pylori in the biopsies taken from the gastric mucosa of experimentally infected dogs was first assessed by the rapid urease test. The test, which gave negative results in the gastric biopsies taken before the oral challenge with H. pylori, became strongly and quickly positive in all three dogs starting from the first sampling, one week after infection, and remained positive at all time points during the follow up of the study (Table 3). Results of microbiological cultures are summarized in Table 3. In samples taken 1 , 2 and 4 weeks PI, H. pylori was identified by culture, although at these time points the growth of bacteria from the selective culture plates was rather scanty. From the 8th week PI onward, the growth of H. pylori from all the gastric bioptic samples was more evident from all three dogs, and 100-500 H. pylori colonies were easily recovered from each biopsy (not shown). At later times (i.e. at 18 and 24 weeks PI) in two of the three dogs, the bacterium was easily isolated from body and antrum only, whereas a marked decrease in the growth efficiency was observed in biopsies taken from cardias and fundus. Starting from the first week PI, H. pylori could also be cultured from rectal swabs of the three animals (Table 3). These microbiological findings were always confirmed by PCR performed on the bacterial colonies grown on the plates (not shown). All attempts to culture H. pylori from oropharyngeal swabs ware unsuccessful at any time during the follow up. However, the presence of H. pylori DNA was detectable by PCR amplification of the cagA gene at all times PI (not shown). PCR was carried out on antral biopsy samples taken at time zero and at each endoscopic examination using caα//4-specific primers. The presence of H. pylori was confirmed in all PI samples (Table 3). Fig. 3 shows the PCR products obtained in antral gastric biopsies taken at 4 and 12 weeks PI. Anti-H. pylori antibody response. The induction of specific H. pylori antibody response was followed up in serum samples taken before, and at different times after, infection and analyzed by WB and ELISA (Figure 4). H. pylori- specific IgG antibodies were already detectable two weeks after infection and persisted at high titer during the entire follow up of the study. Interestingly, the three dogs mounted an antibody response against VacA and CagA, which also persisted for the entire study. Furthermore, both IgG and IgA specific for H. pylori antigens appeared in the saliva of the three dogs. The titers of these antibodies increased over time, reached their peaks between weeks 12 and 15 PI, and then declined, although they were still detectable 24 weeks after infection. Table 3 Weeks post-infection 0 1 2 4 8 12 18 24 Symptoms Vomit +++ Diarrhea +++ + Rapid Urease test on antral biopsy 3 < 1 ≤ 3 < 5 < 5 < 5 < 5 < 5 Culture Gastric biopsies - + + + + + + + Rectal swabs + + + + + + + Orophar. swabs - cagA gene PCR amplification Gastric biopsies (antrum) Orophar. swabs ND C ND + + + + + a data are expressed as time (minutes) employed by the test to become positive. b negative after 24 hours c ND = not done Immunization using animal model H. pylori strains. SPM326s, a streptomycin-resistant derivative of the mouse-adapted H. pylori Type I (CagA+/VacA+) strain SPM326 (37), was grown as previously described (37) and used to challenge the dogs. The CCUG strain of H. pylori is well known in the art. Animals. Six 4-6 months-old xenobiotic beagle dogs, all female (Morini s.a.s., S. Polo D'Enza, Italy), were selected on the basis of the absence of detectable serum IgG against Helicobacter spp. in Western blot (WB) analysis using total bacterial lysate as antigen (see below). The six dogs selected were housed in standard conditions and maintained on a diet of dry food (MIL, Morini s.a.s.) and tap water ad libitum. Upon arrival in our animal facilities, an additional WB analysis on sera confirmed their H. pylori status. The dogs were housed in individual cages and allowed to adapt for a month to the new environment. During the month of adaptation, two tests were carried out on fecal samples to assess the presence of intestinal parasites or common enteric pathogenic bacteria. Preparation of H. pylori lysate. Two pellets of H. pylori CCUG strain from two 5 liter fermenters (56) were obtained. After each pellet was resuspended in 50 ml of sonication buffer (50 mM Na 2 HPO 4 .2H 2 0, 300 mM NaCI, pH 7.8), the two resuspended pellets were mixed. The OD 530nm of the combined resuspension was measured to determine bacterial concentration (= 3.2 x 10 10 CFU/ml). The resuspension was diluted with sonication buffer to bring the concentration to 2 x 10 10 CFU/ml. Before sonication, bacteria had the classical spiral form when viewed under the microscope. Sonication of the resuspended bacteria was then carried out on ice: 2 cycles of 4 minutes, and 2 cycles of 5 minutes, waiting one minute between each cycle. After sonication, all the bacteria appeared broken when viewed under the microscope. Protein concentration was then determined using the Bradford method (= 57.5 mg/ml protein). Aliquots of the cell lysate were then prepared and frozen at -80°C until use. Immunization. Three dogs were immunized on day 0 intramuscularly (i.m.) with the prepared H. pylori lysate (the equivalent of 10 10 CFU H. pylori (= 28 mg/dose)) adsorbed onto 1 mg aluminium hydroxide (Chiron Behring GmbH & Co., Marburg, Germany; Lot No. 277345) in 1 ml volume. Immunizations were repeated on days 7, 14, and 22. Serum samples were taken on day 0, 21 (post- 3) and 43 (post-4). The other three dogs, as a control group, were treated identically but saline was used in place of the H. pylori lysate. Challenge with infectious H. pylori. The dogs were then challenged on days 49, 51 and 53 with the mouse-adapted H. pylori SPM326s strain as follows: 24 h before each challenge the dogs were fasted. 2 h before bacteria inoculation, dogs received 10 mg/kg of cimetidine i.m. (Tagamet® 200; Smith Kline & French, USA). At the moment of challenge, the dogs were anesthetized with a mixture of 40 μg/kg of medetomidine chloridrate (Domitor®; Centralvet- Vetem s.p.a., Milano, Italy) and 5 mg/kg of ketamine (Ketavet®, Gellini, Latina, Italy) intravenously (i.v.); then a gastric lavage was performed with 100 ml of 0.2 M NaHCO 3 sterile solution followed by oral challenge with 3 ml of a freshly prepared suspension of 10 9 CFUs in sterile saline of the H. pylori strain SPM326s, grown under microaerobic conditions (see below), prepared immediately before the inoculation procedure. At the end of the bacterial inoculation, 200 μg/kg of the anesthetic antagonist atipamezole (Antisedan®; Centralvet-Vetem s.p.a., Milano, Italy) was administered and then dogs were again treated with cimetidine and fed after 2 h. Post-challenge follow-up. Ten and 42 days after the last challenge gastric endoscopies were performed using a 4.9-mm-diameter Pentax pediatric bronchoscope (Pentax Technologies, Zaventem, Belgium). At the same time, gastric biopsies were taken during the endoscopies using flexible pinch-biopsy forceps at the antrum, corpus fundus, and cardias for urease testing and for microbiological, histopathological and immunohistochemical analyses. Before each endoscopy the whole instrument and the flexible forceps were soaked in 4% glutaraldehyde for 45 minutes and then rinsed in sterile saline. To avoid cross-contamination among biopsies taken at different sites, the forceps were washed with tap water and lightly flame-sterilized before the collection of each bioptic sample. The above experimental protocol was approved by the Scientific and Ethical Commitee of the University of Pisa and received official authorization (DM No. 21/97-C) from the Italian Ministry of Health (Department of Veterinary Health, Food and Nutrition). Rapid urease test. Antral biopsies and liquid from gastric lavage were incubated for up to 24 h in 1 ml of a 10% urea solution in distilled water added with two drops of a 1 % phenol red solution (Sigma Chemical Co., St. Louis, MO, USA) in sodium phosphate buffer, pH 6.5. A positive test is indicated by change of color (from orange to dark pink) in the medium; the time necessary for the color change is recorded. At time 0, endoscopy was carried out on the six dogs and antral biopsies were taken for the urease test. In all six dogs, the urease test was negative at time 0. Histopathology and Transmission Electron Microscopy. Samples for histological, immunohistochemical and ultrastructural examination were taken from the biopsies at sites adjacent to those utilized for microbiological analysis. The samples were fixed in 10% buffered formalin and embedded in paraffin. 3 μm sections were stained with hematoxylin-eosin (H & E) and Alcian and Periodic acid Schiffs (PAS) staining using standard procedures for histopathological examination. Similar sections were also employed for immunohistochemical analyses using the Avidine-Biotine-Complex (ABC)- peroxidase technique with a monoclonal antibody (Mab) specific for H. pylori (Biogenesis Ltd, Poole, England, UK) or an anti-VacA mouse monoclonal antibody (C1G9) obtained by immunizing Balb/c mice with purified native H. pylori VacA (Burroni and Telford, unpublished observations). Biotinylated horse anti-mouse antibody was used as secondary antibody. The reaction was developed with 3-1-diaminobenzidine-chlorhydrate (DAB) (Sigma) for identification and location of bacterial antigen. For electron microscopic examination, other samples were fixed in Karnowsky, post-fixed in OsO 4 , and embedded in Epon-Araldite (Polysciences Inc., Warrington, PA, USA). Semi- thin sections were stained with toluidine blue for evaluation of cell damage, whereas ultra-thin sections were stained with uranyl acetate and lead citrate, and then examined with a Philips EM 301 transmission electron microscope (TEM) operating at 80 KV. Detection of anti-H. pylori antibodies. SDS-PAGE of H. pylori (strain SPM326s) and WB analysis of sera were performed according to previously published procedures (37). Briefly, dog sera were diluted 1 :200 and incubated for 2 h at room temperature. Then, horseradish peroxidase (HRP)-conjugated rabbit anti-dog IgG antibody (Nordic Immunological Laboratories, Tilburg, The Netherlands) was added at 1 :2,000 dilution for 2 h, and the reaction was developed using 4-α-chloronaphtol as substrate. 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Claims: Claims: 1. A non-human, non-murine animal infected with Helicobacter pylori which exhibits one or more acute symptoms of Helicobacter pylori infection. 2. An animal according to claim 1 , in which Helicobacter pylori establishes chronic persistent infection. 3. An animal according to claim 1 or claim 2, wherein said animal is a xenobiotic dog. 4. A method for infecting an animal according to any one of claims 1 to 3 with Helicobacter pylori comprising the steps of: (a) isolating Helicobacter pylori; and (b) administering a dose of the isolate of Helicobacter pylori to said animal. 5. A method according to claim 4, wherein the administration of Helicobacter pylori is repeated at intervals after the initial infection. 6. A method according to claim 4 or claim 5, wherein the administration of Helicobacter pylori is repeated 3 times every other day after the initial infection for one week. 7. A method according to any one of claims 4 to 6, wherein, prior to the administration of Helicobacter pylori, stomach acid of the animal is neutralised. 8. A method according to any one of claims 4 to 7, wherein the isolate of Helicobacter pylori is passaged several times in vivo prior to administration. 9. A method according to any one of claims 4 to 8, further comprising the step of: 5 (c) isolating Helicobacter pylori from the animal and administering the isolate to a further animal. 10. A method according to any one of claims 4 to 9, wherein the isolate of 10 Helicobacter pylori in step (a) is isolated from a non-human animal. 11. A method according to claim 10, wherein the non-human animal is a mouse. 15 12. An animal according to any one of claims 1 to 3 or a method according to any one of claims 4 to 11 , wherein said animal is a dog, preferably a beagle dog. 13. An animal according to any one of claims 1 to 3 or claim 12 when 20 prepared by the method of any one of claims 4 to 11. 14. An animal according to any one of claims 1 to 3, 12 or 13 for use in: (i) the study of the pathogenesis of Helicobacter pylori-Induced disease; 25 (ii) development of vaccines; (iii) development of chemotherapeutics; or 30 (iv) development of diagnostics. 15. A method for evaluating a vaccine or chemotherapeutic agent against Helicobacter pylori comprising the steps of: (a) administering to a non-human, non-murine animal an effective amount of candidate vaccine or chemotherapeutic agent; (b) administering to the non-human, non-murine animal Helicobacter pylori, in accordance with the method of any one of claims 4 to 11 ; and (c) observing the incidence of Helicobacter pylori infection in the animal model. 16. An isolate of Helicobacter pylori obtained from an animal according to any one of claims 1 to 3, 12 or 13.

Inventors: DEL GIUDICE GIUSEPPE, RAPPUOLI RINO, DEL GIUDICE, GIUSEPPE, RAPPUOLI, RINO
Applicants: CHIRON SPA, DEL GIUDICE GIUSEPPE, RAPPUOLI RINO, CHIRON S.P.A., DEL GIUDICE, GIUSEPPE, RAPPUOLI, RINO
Application year: 1999
Main classification: A01K67
IPC codes: A01K67, G01N33