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11 Animal models and vaccine development
Animal models and vaccine development ADRIAN LEE Long before the culture of Helicobacter pylori, spiral bacteria had been observed in the stomachs of a range of different animals. In the later part of the last century such bacteria were first described in cats and dogs colonizing the gastric mucus and glands in large numbers with occasional organisms seen in parietal cells (Rappin, 1881; Bizzozero, 1892; Salomon, 1896). Further descriptions of gastric bacteria appeared in the early part of this century culminating in the extensive study by Doenges in 1939 in which he described these bacteria in monkeys and gave the first well documented description of such bacteria in humans. OTHER HELICQBACTER MORPHOLOGY
SPECIES;
CHARACTERISTICS
AND
With Warren and Marshall’s achievement in isolating H. pylori, it was natural that there were attempts to culture gastric bacteria from other animals (Marshall et al, 1984). The rationale for these investigations was that these bacteria presumably had common characteristics that allowed them to inhabit the gastric mucosa, thus comparative studies might reveal information useful in understanding factors involved in colonization and the pathogenesis of Helicobacter infection of the stomach. The importance of these bacteria increased when it became apparent that H. pylori appeared to have a remarkable host specificity and would not colonize the animal species usually used in development of animal models of human disease. As these bacteria were isolated, their physiological characteristics defined and their DNA sequenced, it became apparent that these organisms were very closely related. They were all placed in the same genus, Helicobacter and given different species names (Goodwin et al, 1989; Bronsdon et al, 1991; Paster et al, 1991b; Eaton et al, 1993). The criterion commonly used to justify positioning these animal isolates was the sequencing of part of their ribosomal RNA, the so called 16s rRNA gene. 16s rRNA is a component of the ribosome, a structure essential for protein synthesis. As production of proteins is so fundamental to life, the critical function of this molecule has stayed the same over the millennia of evolution and so the sequence has remained the same. However, there are known regions of the molecule which have randomly altered over time. The degree of similarity of the 16s BailliLre’s Clinical GastroenterologyVol. 9, No. 3, September 1995 ISBN 0-7020-1961-5
61.5 Copyright 0 1995, by Baillikre Tindall All rights of reproduction in any form reserved
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rRNA sequence is taken to correlate with ancestorship (Woese, 1987). Bacteria that have very similar sequences are considered close relatives and if there is greater than a 90% similarity they can be legitimately placed in the same genus. This is what has happened to the helicobacters which can be regarded as ancestors from a common organism that evolved to inhabit the primordial stomach many years ago. As animal species evolved, so did these bacteria taking on a remarkable diversity of morphologies as can be seen below. However they can all be considered in the one group of highly evolved bacteria exquisitely adapted to the gastric milieu of their animal host. The human helicobacter is IX pylori. Their relatives are described below. Helicobacter mustelae The first animal helicobacter to be cultured was by Fox, who in 1985, isolated a small slightly curved rod shaped bacterium from the stomach of an adult ferret (Fox et al, 1986). These organisms shown in Figures la and lb are 3 pm x 0.5 pm in size with an unusual configuration of lateral and terminal flagella.
Figure 1. Other gastric helicobacter species. (A) Negative stain of Helicobacter musteke showing the unusual lateral and terminal flagella configuration (bar = 5 pm). (B) ferret gastric tissue showing Helicobacier musfelue in close association and adhering to epithelial cells (bar = 1 pm).
Helicobacter
felis
The cat and dog stomach are packed with spiral/helical shaped bacteria, indeed in early morphological studies, three different morphological types were described in the one animal (Lockard and Boler, 1970). The first of these organisms to be isolated was a very tightly spiralled bacterium with five to seven coils (Lee et al, 1988a). This organism had a very striking
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ultrastructure and was seen to be entwined with distinctive periplasmic fibrils and with large tufts of polar flagella (Figures lc & d). It is presumably due to these structures that the bacteria move with a rapid ‘boring’ type motility allowing them to cope with the viscous nature of gastric mucus, just as been suggested for H. pylori (Hazel1 et al, 1986). The name given to this organism was Helicobacterfelis as it was first isolated from a cat. The same bacterium has been subsequently isolated from dog stomachs. There have been two reports of H. felis infection in humans both associated with an acute neutrophilic gastritis (Wegmann et al, 1991; Lavelle et al, 1994).
Figure 1 (contd.). (C) negative stain of Helicobacter felis showing the characteristic helical shape. Periplasmic fibres (arrow) and tufts of polar flagella (bar = 0.5 pm). (D) gastric tissue from a lemur showing a Helicobacterfelis-like organism (bar = 1 pm).
‘Helicobacter
heizmannii
( ‘Gastrospirillum
hominis’) and friends
The dominant morphological bacterium seen in cats and dogs was not H. felis. The bacteria had a similar tight spiral morphology but the characteristic periplasmic fibrils were not present (Figure le). This same morphology was the most commonly seen in any animal, being reported in pigs, primates, and cheetahs (Curry et al, 1987; Queiroz et al, 1990; Eaton et al, 1991a). Interest in these organisms increased following the short report of Dent et al (1987) who observed a bacterium with a similar morphology in 3/1300 human biopsies from patients with histological gastritis, Based on serological results, it was suggested that the human patients had probably acquired this organism from their pets (Lee et al, 1988b). Subsequently this organism was tentatively named ‘Gastrospirillum hominis’ (McNulty et al,
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Figure 1 (contd.). (E) gastric tissue from a Mandrill heilmannii-like organisms (bar = 0.5 pm).
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monkey
showing
large numbers
of Helicobucter
1989). There have now been more than 200 cases of infection with this bacterium reported and it is considered to contribute to about 1% of human gastritis (Lee et al, 1994). No one has had success at culturing this bacterium from either humans or animals. In 1896, Salomon had published a bizarre paper in which he fed the gastric mucosa from various animal species to rodents and showed that their gastric mucosa rapidly became colonized with huge numbers of a bacterium which, from his drawings, appeared to have the morphology of ‘Gustrospirillum (Salomon, 1896). Based on this paper, the homogenate of a gastric biopsy from a patient with ‘G. hominis’ gastritis was fed to mice, and it was found that the organism colonized the mouse stomach in large numbers (Dick et al, 1989; Lee et al, 1989). Subsequently, similar bacteria from a wide range of animals including lemurs, macaques, mandrill monkeys and a bobcat have been isolated using this in vivo culture method, but in vitro culture has never been successful (J. O’Rourke, unpublished observations). Using molecular techniques, the 16s rRNA gene of some of these bacteria have been amplified from infected mouse stomachs allowing the whole gene to be sequenced. This has shown, not surprisingly, that these bacteria belong to the genus Helicobacter (Solnick et al, 1993). The name ‘Helicobacter heilmannii’ has been tentatively assigned to one of the human isolates. Whether each bacterium from all of the different animal species will be given a separate name remains to be determined. It is sufficient for the gastroenterologist to appreciate that this broad group of morphologically similar bacteria are the most common helicobacter and most animals are infected with them. Where the organisms infecting their patients comes from depends on the type of animals that person has come in contact with. Given the number of infected cases and the small number of humans infected, transmission must be difficult and depends on very close contact with the animal. Presumably most come from pet cats and dogs unless your patient happens to have a pet cheetah! (See below).
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acinonyx
The most exotic helicobacter yet isolated comes from the cheetah. Animals in a closed colony in Ohio were found to be seriously ill and presented with chronic vomiting (Eaton et al, 199 1b). Histopathology revealed the cheetahs had a severe lymphoplasmacytic gastritis characterized by variable numbers of neutrophils, gland abscesses, lymphoid follicles and epithelial erosions. Although bacteria with the morphology of ‘H. heilmannii’ were seen in the gastric mucosa, the organism cultured had a morphology much closer to H. pylori except it was smaller. These S-shaped bacteria are 0.3 pm x 2 pm in size with 2-5 polar sheathed flagella and share many biochemical properties with H. pylori. The bacterium has been named Helicobacter acinonyx, after the taxonomic name for the cheetah (i.e. Acinonyxjubilatus) (Eaton et al, 1991a). The gastric versus the non-gastric
helicobacters
As time goes by, many other helicobacters will probably be cultured from animal stomachs. These may be of interest in what they tell us about gastric colonization and pathogenesis. However, recently there have been a plethora of new helicobacters reported that have no relevance to our understanding of gastroduodenal pathology and will only serve to clutter the Helicobacter databases. These are the non-gastric helicobacters and it is important that gastroenterologists appreciate what these bacteria are so they need not chase the literature on these organisms. Soon after the sequencing of H. pylori and H. mustelae, and based on short sequences that were seen to be specific for the genus Helicobacter, specific gene probes were produced and used to screen large numbers of intestinal bacteria (Paster et al, 199 1a). A number of bacteria were shown to react with the specific probe. Indeed some which were previously in the genus Campylobacter have been renamed, for example Helicobacter cinaedi and Helicobacter fennelliae (Vandamme et al, 1991). In other cases new species have been identified, for example Helicobacter canis (Stanley et al, 1993). These non-gastric bacteria do not colonize the stomach. They lack the enzyme urease which is certainly a unifying property of the gastric helicobacters and appears necessary for these bacteria to colonize the acid laden stomach. The only exception is Helicobacter muridarum, which has a urease enzyme (Lee et al, 1992b) and in special circumstances does inhabit gastric mucus (Lee et al, 1993a).
NATURAL AND EXPERIMENTALLY IN ANIMALS; ULTRASTRUCTURAL, MACROSCOPIC CHANGES
INDUCED INFECTION MICROSCOPIC AND
For any microbial infection, it is important to have appropriate animal models in order to better understand pathogenesis, test new therapies and to develop vaccines. There is no perfect model of human H. pylori infection, rather we have to rely on a number of differing situations which either
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utilize H. pylori infection in animals, follow the consequence of natural helicobacter infection in animals, or involve infection of unnatural animal hosts with some of the non- H. pylori gastric helicobacters described above. The natural and experimental infections of animals reported to date are described below. For each animal the potential use of the model is evaluated to serve as a guide for potential researchers into H. pylori-associated disease. Primates
Many species of primates are colonized with H. heilmannii: (Fox and Lee, 1993). The most extensive studies have been those of Dubois et al (1991) in which they found 14 out of 29 rhesus monkeys to be colonized. The bacteria were found to localize in the mucus covering the surface epithelial cells and deep into the gastric pits, however there were no signs of gastritis. Ultrastructural study of the parietal cells revealed bacteria in the cytoplasm of healthy and injured cells and this correlated with an increased basal acid output (BAO) and peak postload acid output (PAO) in these animals. In 1988, bacteria resembling H. pylori were isolated from a colony of laboratory housed Rhesus monkeys (Baskerville and Newell, 1988). Since then many others have found bacteria almost identical to H. pylori in a range of primates (Bronsdon and Schoenknecht, 1988; Euler et al, 1990; Dubois et al, 1991). A very similar organism called Helicobacter nemenstrina has also been described (Bronsdon et al, 1991). It may be that these bacteria are in some cases human strains that have been transmitted to the primates by their H. pylori infected keepers. Alternately, given the close evolutionary linkage between the non-human primate and the human, one could predict similar bacteria could be present. In the primates naturally infected with these H. pylori-like bacteria, a more significant inflammation was seen than with H. heilmannii: (Euler et al, 1990). Consistent with this have been studies where non-human primates have been infected with human isolates of H. pylori. In Japanese monkeys there was an initial polymorphonuclear and mononuclear cell infiltration after infection with H. pylori with antral erosions and erythema observed macroscopically. This persisted for up to six months, however the severity of the gastritis decreased over time (Shut0 et al, 1993). Elements of acute and chronic gastritis were also detected in chimpanzees infected with H. pylori (Hazel1 et al, 1992). The expense of primate research restricts the usefulness of the model, although in the development of new vaccines it will be essential to eventually involve a primate-model. For other studies alternative models are required. Pigs and piglets
Although in early studies, H. pylori did not colonize traditional animal models such as mice, rats, guinea pigs and rabbits, two groups indepen-
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dently showed that gnotobiotic piglets derived by caesarean section and raised in germ-free isolators did become infected with human isolates of H. pylori (Krakowka et al, 1987; Lambert et al, 1987). The bacteria colonized the antral mucosa in a manner very similar to that of humans. Helicobacter pylori was seen in large numbers in the mucus and appeared to firmly adhere to the gastric mucosa. The inflammation induced, resembled that seen in H. pylori infected human children rather than adults (Bertram et al, 1991). The cellular infiltration was primarily mononuclear with large lymphoid aggregates with nodular protrusions seen on gross examination of the gastric mucosa. A problem with the gnotobiotic piglet as an experimental animal is cost and the fact that the piglets become pigs and can no longer be housed in the isolators. However, the model has proved extremely useful in studies on H. pylori colonization, and the definitive work showing the importance of motility and urease was performed with this model, using H. pylori variants and mutants (Eaton et al, 1989; Eaton et al, 1991~). Other workers have used barrier raised specific pathogen free pigs and shown that H. pylori will colonize for long periods of time and have developed a 13C-urea breath test for diagnosis of infection (Engstrand et al, 1990). Ferrets
There are a number of shared characteristics between the H. mustelae infected ferret and the H. pylori infected human. Ferrets present varying degrees of gastritis ranging from superficial gastritis in the oxyntic mucosa to a diffuse antral gastritis with elements of chronic atrophic gastritis. The severity of gastritis has been found to increase with age and in some instances ulceration has been noted at the pyloroduodenum junction (Fox and Lee, 1993). The ultrastructure of the H. mustelae colonized gastric mucosa reveals that like H. pylori, this bacterium adheres firmly to the gastric mucosa (O’Rourke et al, 1992). Indeed the majority of the organisms were seen close to the mucosa with many cases of actual endocytosis of the organism into the epithelial cells (Figure lb). This has been observed with H. pylori in vitro but is not commonly seen in vivo. The adhesion of H. mustelae seen in the ferret is the only other example of helicobacter adhesion. Also, the ferret is the only other animal where peptic ulceration is commonly seen. Although not common enough to be used as an experimental model of helicobacter-associated ulceration it does give weight to the importance of the ferret as a natural model of human H. pyloriassociated disease. Another use for the ferret-model has been as a screen for antimicrobial therapies. For example, experiments have shown that H. mustelae can be eradicated from the majority of colonized ferrets tested using a combination of bismuth subcitrate, amoxycillin and metronidazole, whereas monotherapies such as bismuth or chloramphenicol were unsuccessful (Otto et al, 1990; McColm et al, 1993).
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Dogs In pre-H. pylori days the pathology presumably induced by natural helicobacter infection was described by Henry et al (1987), who looked at a number of laboratory reared Beagles. The bacteria were most commonly detected at the fundic-pyloric junction and the cardia which corresponded with the presence of large lymphoid follicles. From this association he postulated that the presence of these bacteria may result in the induction of lymphoreticular hyperplasia. Koch’s postulates for a helicobacter causing this pathology was confirmed experimentally when living cultures of H. felis were fed to germ free Beagle puppies (Lee et al, 1992a). In the infected animals, large lymphoid follicles were found throughout the gastric mucosa with the largest and most extensive follicles seen in the fundus and body. No such follicles were detected in the control animals. Throughout the subglandular region of the stomach, a diffuse infiltrate of lymphocytes, eosinophils and plasma cells were also seen. At the same time as these experiments, H. pylori was fed to another litter of the germ free Beagle pups (Radin et al, 1990). Limited colonization was observed in the dogs and they presented a chronic active gastritis characterized by focal to diffuse lymphoplasmacytic cellular infiltrate in addition to occasional lymphoid follicles in the antrum. Gnotobiotic dog-work is expensive and normal dogs have heavy infection with their own helicobacters, and so there would appear to be limited use of a dog-model. However, as veterinarians become familiar with the concept of human H. pyEori-associated gastric disease they are starting to look for parallels with canine disease (Geyer et al, 1993). As has been the case with H. pylori and non-ulcer dyspepsia the role of the bacterium as a causal agent is not proven, although logic has it that some animals probably suffer from their helicobacters. Cats The consequence of natural helicobacter infection in cats would appear similar to dogs. In a study looking at the post mortem studies of 55 cats and kittens, an associated pathology was observed (Otto et al, 1994). Gastritis was recorded that varied from a mild infiltrate of inflammatory cells to a diffuse mixed subglandular leukocytic infiltrate with lymphoid nodules that displaced entire gastric glands. Mixtures of lymphocytes, plasma cells and occasional polymorphonuclear leukocytes were also detected in focal cellular aggregates. The severity of the histopathology was found to correlate with the density of bacterial colonization. There have been no experimental studies with cats, although a remarkable recent finding would suggest that this will soon change. Handt et al (1994) has isolated bacteria from domestic cats which were shown by morphology, biochemical properties and 16s rRNA sequence analysis to be H. pylori. Histopathology revealed the cats had gastritis characterized by a mild to severe diffuse lymphofollicular infiltrate with occasional neutrophils and eosinophils in the subglandular and glandular mucosae.
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Mice
Mice do not have a natural gastric helicobacter, although some strains of conventional mice are colonized with H. muridarum in their lower bowel (Lee et al, 1992b), although as seen above, these animals can easily become colonized with other animal helicobacter species. The most important mouse model has utilized H. felis (Dick et al, 1989). When administered orally, H. felis colonizes the mouse stomach in large numbers where it will remain for the life of the animal. The pathology resulting from infection varies depending on the animal strain used, highlighting the importance of host factors. When given to germ-free Swiss mice an active chronic gastritis is seen (Lee et al, 1990). Initially there is an acute inflammatory response composed primarily of eosinophils and neutrophils, the latter increases with time resulting in the formation of small micro-abscesses. By 8 weeks post-infection there is a chronic infection characterized by increasing numbers of mononuclear cells and the appearance of large lymphoid follicles with micro-abscesses still present in the pyloric mucosa. The H. felis infected germ-free mouse has been followed for up to 50 weeks in which, after an initial active chronic inflammation was observed, a chronic gastritis persisted for the length of the experiment (Fox et al, 1993). This was the first convenient animal model of helicobacter infection where the active component of the gastritis, that is characteristic of H. pylori infection in adult humans, was recorded (Figures 2a-c). Long-term
Figure 2. Animal models of helicobacter infection (H&E stains). Germfree mouse colonized with Helicobncter felis for 4 weeks showing an increase in the cell infiltrate. (A) (magn x 50) with the migration of polymorphonuclear leukocytes from the lamina propria into the cytoplasm of the glandular epithelial cells. (B) (magn x 100) higher magnification of the migration of the polymorphonuclear leukocytes. (C) (magn x 100) and micro abscesses. (Reproduced from Lee et al (1990, Gastroenterology 90: 1315-1323) with permission.
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studies in a conventional colony of Swiss mice demonstrated the progression of gastritis from active/chronic gastritis to a highly destructive atrophic gastritis in which some dysplastic changes were seen (Figure 2d) (Lee et al, 1993a). This was the first experimental evidence of the progression of helicobacter induced chronic gastritis to atrophy, an observation that is consistent with the Correa hypothesis (1984) of the progressive steps in the development of gastric adenocarcinoma. It has been suggested that the long term H. $&-infected mouse may be a very important model in which to study helicobacter induced gastric carcinogenesis.
Figure 2 (contd.). Conventional mice colonized with Helicobacter felis for 18 months showing extensive infiltration of inflammatory cells and obvious loss of oxyntic glands. (D) (magn x 125). Reproduced from Lee et al (1993a, Zentrulbluttfiir Bakteriologie 280: 38-50) with permission.
Mice from a specific pathogen free colony of BALB/c mice were followed long-term after infection with H. felis, and for up to 18 months, very little inflammation was seen. However, post-18 months lymphoid follicles started to appear only in infected animals. By 26 months, dramatic lesions exactly analogous to low grade B-cell lymphomas were observed in 25% of the infected animals (Enno et al, 1995). The rodent has much potential as an animal model, even though mice do not develop ulcers there are a number of H. pylori-associated pathologies that are mimicked. However, there are differences with respect to H. pylori that have to be noted. The bacterium is restricted to the antrum and cardia with very little colonization seen in the body of the stomach. There is no adherence to the gastric mucosa and the bacteria travel much deeper into the gastric glands than normally occurs with H. pylori. Despite these differences the susceptibility of the H. &Z&infected mice to antimicrobial agents is very similar to that found in the H. pylori infected human.
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Consequently, monotherapy will not eradicate H. felis whereas triple therapy is very effective. Given the unreliability of in vitro testing of newer antimicrobials as a predictor on in vivo efficacy, it has been proposed that the H. felis-mouse model is a very convenient and economical screen for potential new anti-H. pylori agents (Dick-Hegedus and Lee, 1991). The other major use of this model has been in immunization studies and this will be discussed later. Even though early studies in mice showed that mice could not be infected with H. pylori, recent work suggests that this may be an attainable goal (Karita et al, 1991). Japanese workers claim to be able to infect BALB/c nude mice with fresh human isolates of H. pylori. Counts appear to be low and no pictures of colonization have been published. The critical factor seems to be the freshness of the isolate used. The development of a H. pylori-mouse model is a very worthwhile goal and more developmental work is required. Rats Rats can be infected with helicobacters such as H. felis or H. heilmannii. The inflammation reported has been limited but this probably relates to the strain of rat used (Fox et al, 1991). Due to the convenience of mice over rats and no obvious advantage at this stage, there has been little further work done on a rat-model. TOWARDS
A VACCINE
As is seen above there are a number of animal models available for the study of H. pylori-associated disease. Now that this bacterium is firmly established as a major bacterial pathogen infecting more than half the world’s population, possibly threatening the health and indeed lives of millions of people, the time is ripe for the development of a strategy that has proved so successful with many of the world’s other infectious diseases, namely immunization. The latter part of this chapter shows that it is the development of animal models that now makes it possible to develop an anti-H. pylori vaccine. The possibility Helicobacter pylori is a remarkably well adapted pathogen that colonizes its host for life. This is despite the induction of an immune response so large that serological assays can be accurately used to predict active infection (Lee et al, 1993b). There are other chronic diseases where the organism survives in the host for life, but they are usually sequestered away (e.g. tuberculosis, herpes). In contrast, H. pylori can be found in huge numbers swimming in the gastric mucus or firmly adhered to the epithelial surface despite the infiltration of large numbers of immunocompetent cells and active phagocytes.
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With such highly evolved evasion strategies it could be asked whether immunization is an attainable goal. Is it possible to beat nature? The early workers responded, by suggesting that there is a difference between an immune response necessary to prevent an infection and the response needed to remove an already established one. The first evidence The first data that suggested the goal of effective immunization was attainable was the result of the fortunate teaming together of Stephen Czinn, a paediatric gastroenterologist with an interest in H. pylori and a mucosal immunologist, John Nedrud. Czinn and Nedrud immunized both mice and ferrets by oral administration of a sonicate of H. pylori together with the adjuvant cholera toxin (CT) (Czinn and Nedrud, 1991). As predicted, they achieved a highly significantly raised level of anti-H. pylori IgA in gastric secretions. They concluded with the comment that these results suggested that it is possible to develop an oral immunization protocol for prevention of H. pylori infection and associated gastritis. However, they were not in a position to easily test their hypothesis as mice could not be colonized with H. pylori and the ferrets were all infected with H. mustelae. By the time the Czinn and Nedrud work had been reported, we had developed the H. felis mouse model and thus were in a position to test their hypothesis. Groups of mice were given oral doses of H. felis sonicate alone or with CT on days 1, 3, 6, 30 and 54. Three weeks later, after these animals and appropriate control groups were challenged with large numbers of viable H. felis. A highly significant degree of protection was seen with the vaccine comprising the H. felis sonicate and CT (Chen et al, 1993). At about the same time Czinn and Nedrud found the same result in a series of germ-free mice (Czinn et al, 1993). They also demonstrated that a monoclonal IgA antibody against H. felis was protective. The enhanced response noted in the actively-immunized protected mice was later found to correlate with increased levels of specific anti-H. felis-IgA antibody in gastric and intestinal secretions (Chen et al, 1993). Composition
of vaccines
Since these early experiments were published, many companies have commenced the highly competitive race to develop the first human vaccine. All these groups have used the H. felis-mouse model. Based on mostly published abstracts, we know much more about the possibilities for the composition of the eventual vaccine (Blanchard et al, 1993; Corthesy-Theulaz et al, 1993; Ferrer0 et al, 1993; Thomas et al, 1993). The specificity of the Czinn and Nedrud monoclonal antibody was found to be against the H. felis urease (Blanchard et al, 1993). Given the known importance of this molecule, it was not surprising urease would be a good candidate for a protective antigen, although it should be noted that the monoclonal antibody did not inhibit urease activity. Following the
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sequencing of the urease genes of H. pylori and the elegant work of Labigne and her team in developing methods for the genetic manipulation of this organism, a vaccine was constructed that used recombinant H. pylori urease with CT (Cussac et al, 1992; Ferrer0 et al, 1993; Corthesy-Theulaz et al, 1993). This vaccine protected against challenge with viable H. felis. This was consistent with studies that showed immunization with whole cell sonicates of H. pylori protected against H. felis. This was a relief to the workers in the field as it meant that they were indeed not developing a vaccine to protect mice against H. felis, but could work on the H. pylori vaccine! At present the most likely vaccine candidate is urease. How this vaccine will be formulated is unknown and this is another area of intense activity. Cholera toxin is clearly an unacceptable adjuvant as it is too toxic to humans. An alternative adjuvant is needed. Recently experiments with the non-toxic B-subunit of cholera toxin (CTB) has shown protection (Lee and Chen, 1994). The CTB used was a commercial preparation which is known to have minute amounts of holotoxin contamination. Thus the CTB effect could be due to the contaminating CT. None the less these results are encouraging and suggest a non-toxic, effective vaccine formulation will be found. Therapeutic vaccinationa new dimension in Helicobacter management Using the H. felis model some results have recently been published that add a new dimension to the potential of H. pylori immunization. Success with our mouse-model encouraged us to determine whether it is possible to eliminate existing gastric infection by immunization. Specific pathogen free (SPF) BALB/c mice were infected with a living culture of H. felis. Four weeks after infection, 80 animals were immunized orogastrically on days 1, 15, 17, and 20 with H. felis whole cell sonicate (4 x 1 mg) and cholera toxin (4 x 10 pg). Eighty infected mice were retained as controls. One week, 1 month, 2 months and 3 months after completion of immunization, groups of immunized and non-immunized mice were euthanased and their H. felis status determined. Results of this experiment are shown in Table 1. A high proportion of previously infected animals had no detectable organisms in their stomachs for up to three months post-immunization. Table
1. Therapeutic
Vaccine Experiment 1 Nil H. feZis sonicate H. felis sonicate H. felis sonicate H. felis sonicate * Fisher’s
+ + f +
CT CT CT CT
immunization
Time of assay post-immunization
Number animals infected
1 1 1 2 3
IO/76 2120 3/20 6120 l/17
exact test (two tail).
week, 1,2, 3 month week month months months
f&s
of Helicobacter
infected
mice.
of % Not infected
Significance*
9% 90% 85% 70% 94%
F1 P z? p.
< < < <
0.0001 0.0001 0.0001 0.0001
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According to the current definitions of anti-Helicobacter chemotherapy, i.e. clearance of bacteria for more than a month after the cessation of therapy; therapeutic immunization appeared to have eradicated the infection (Doidge et al, 1994). In a second experiment a significant result was achieved by administration of a H. pylori sonicate plus cholera toxin. These results showed, for the first time, that it was possible to eradicate active Helicobacter infection of the gastric mucosa by administration of an oral vaccine. This raised the intriguing possibility that therapeutic immunization might be a viable option in the management of Helicobacter-associated disease. If immunization as a therapy for peptic ulcers was combined with short-term acid suppression, the possibility of reinfection may also be eliminated. In those countries where H. pylori infection rates are very high and infection occurs at an early age, large scale oral immunization of sections of the community would not only protect the young from the deleterious consequences of long-term H. pylori infection, but could also cure existing disease. On reflection the success of therapeutic immunization was predictable. Lifelong helicobacter infection is the result of a balance between host and parasite that maintains an equilibrium which is normally of no major consequence to the host. The bacterium can persist because it has evolved to circumvent the normal immune response. The host control mechanisms ensure that a comprehensive local immunity is not induced, as the normal mechanisms of oral tolerance are in action. Subverting oral tolerance against the helicobacter antigens by the administration of the cholera toxin adjuvant results in an enhanced local response, that is much greater than the response that the resident helicobacter evolved to evade. Thus, the balance swings in favour of the host and the bacterium is eliminated. A commentary by Cohen (1994) recently, drew attention to the development of vaccine therapies for a number of other chronic diseases where a permanent carrier status has been established, for example herpes virus infection, leprosy, leishmaniasis, tuberculosis and Hepatitis B. In the veterinary field, immunization has been used as a therapy of chronic Campylobacter fetus infection in bulls for many years (Bouters et al, 1973). Conclusion
The development of animal models of helicobacter infection provides us with a number of powerful tools to investigate many facets of infection by these fascinating helical pathogens. There is no one best model despite the vigorous defences of those of us in the field. However, perhaps many of them will be used in what is now possibly the most exciting development in the fight against human H. pylori infection, namely the development of a safe and effective vaccine that can be used for the treatment and prevention of these diseases. This opens up the potential for the complete eradication of this bacterium from many populations, thus consigning the major topic of this current volume to the history books and the status of a medical curiosity.
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SUMMARY Following the demonstration of Helicobacter pylori as a major gastroduodenal pathogen there was a need to develop animal models in order to investigate mechanisms of pathogenesis and to be able to test new treatment strategies. Helicobacter pylori will only colonize a limited number of hosts including non-human primates, germ-free or barrier raised piglets, germ-free dogs and recently laboratory raised cats. Although these models have proved useful there is a need for more convenient small animal models. The ferret infected with its natural gastric organism, Helicobacter mustelae, is the only other animal to show peptic ulceration and has been successfully used to investigate gastritis and antimicrobial agents. The other commonly used animal model is the laboratory mouse or rat infected with either Helicobacter felis or Helicobacter heilmannii, bacteria that normally colonize cat or dog gastric mucosae. Active/chronic gastritis, gastric atrophy, and lymphoma-like lesions have been shown to develop in H. felis infected mice. The most recent and exciting use of an animal model has been the use of the H. felis mouse model in the development of human vaccines against H. pylori. Mice can be protected against infection with large doses of viable H. feZis by oral immunization using sonicates of H. f&s or H. pylori or recombinant H. pylori urease together with cholera toxin or cholera toxinB subunit as the mucosal adjuvant. More importantly it has been shown that immunization of already infected animals results in eradication of infection. This raises the intriguing possibility that therapeutic immunization might be a viable option in the management of Helicobacter-associated disease. If immunization as a therapy of peptic ulcers was combined with short-term acid suppression, the possibility of reinfection may also be eliminated. In those countries where H. pylori infection rates are very high and infection occurs at an early age, large scale oral immunization of sections of the community would not only protect the young from the deleterious consequences of long-term H. pylori infection but could also cure existing disease. Acknowledgments The help of Jani O’Rourke in the preparation of this manuscript is gratefully acknowledged as is research support from the National Health and Medical Research Council of Australia, CSL Ltd, Parkville, Australia and Astra Hassle, Molndal, Sweden.
REFERENCES Baskerville A & Newell DG (1988) Naturally occurring chronic gastritis and C. pylori infection in the rhesus monkey: a potential model for gastritis in man. Gut29: 465-472. Bertram TA, Murrav PD. Morgan DR et al (1991) Gastritis associated with infection bv Helicobkter pyiori in hum&: geographical differences, Scandinavian Journal of Gas& enterology 26: 1-8. Bizzozero G (1892) Ueber die schlauchfoermigen drusen des magendarmkanals und die beziehungen ihres epithels zu dem oberfachenepithel der schleimhaut. Archivfiir Mikrobiologie und Anatomia 42: 82-152.
A. LEE Blanchard T, Czinn S & Nedrud J (1993) Identification of Helicobacter urea.se as a potential vaccine antigen for protection against Helicobacter infection. Acta Gastro-Enterologica Belgica 56 (supplement): 53. Bouters R, Dekeyser J, Vandeplassche M et al (1973) Vibrofetus infection in bulls: curative and preventative vaccination. British Vetermary Journal 129: 52-57. Bronsdon MA & Schoenknecht FD (1988) Campylobacter pylon’ isolated from the stomach of the monkey, Macaca nemestrina. Journal of Clinical Microbiology 26: 1725-1728. Bronsdon MA, Goodwin CS, Sly LI et al (1991) Helicobacter nemestrinae sp. nav., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina). International Journal of Systematic Bacteriology 41: 148-153. Chen MH, Lee A, Hazel1 S et al (1993) Immunization against gastric infection with Helicobacter species-first step in the prophylaxis of gastric cancer? Zentralblatt fur BakteriologieInternational Journal of Medical Microbiology 280: 155-165. Cohen J (1994) Vaccines get a new twist. Science 264: 503-505. Correa P (1984) Chronic gastritis is a cancer precursor. Scandinavian Journal of Gastroenterology 19 (supplement 104): 131-136. Corthesy-Theulaz I, Haas R, Davin C et al (1993) Helicobacterpylori urease elicits protection against Helicobacterfelis infection in mice. Acta Gastro-Enterologica Belgica 56 (supplement): 34. Curry A. Jones DM & Eldridge J (1987) Spiral organisms in the baboon stomach. Lancet ii: 634-635. Cussac V, Ferrer0 RL & cbigne A ‘(1992) Eipression of Helicobacter pylon’ urease genes in Escherichia coli grown under nitrogen limiting conditions. Journal of Bacteriology 174: 2466-2473. Czinn SJ & Nedrud JG (1991) Oral immunization against Helicobacter pylon‘. Infection and Immunity 59: 2359-2363. Czinn SJ, Cai A & Nedrud JG (1993) Protection of germ-free mice from infection by Helicobacter felis after active oral or passive IgA immunization. Vaccine 11: 637-642. Dent JC, McNulty CAM, Uff JC et al (1987) Spiral organisms in the gastric antrum. Lancet ii: 96. Dick E, Lee A, Watson G & O’Rourke J (1989) Use of the mouse for the isolation and investigation of stomach-associated, spiral-helical shaped bacteria from man and other animals. Journal of Medical Microbiology 29: 55-62. Dick-Hegedus E & Lee A (199 1) Use of a mouse model to examine anti-Helicobacter pylon’ agents. Scandinavian Journal of Gastroenterology 26: 909-915. Doenges JL (1939) Spirochaetes in the gastric glands of Macacus rhesus and humans without definite history of related disease. Archives of Pathology 27: 469-477. Doidge C, Gust I, Lee Aet al (1994) Therapeutic immunization against Helicobacter infection. Lancet 343: 914-915. Dubois A, Tarnawski A, Newell DG et al (1991) Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys. Are gastritis and hyperchlorhydria infectious diseases? Gastroenterology 100: 884-891. Eaton KA, Morgan DR & Krakowka S (1989) Campylobacterpylori virulence factors in gnotobiotic piglets. Infection and Immunity 57: 1119-1125. Eaton KA, Radin MJ, Fox JG et al (1991a) Helicobacter a&onyx, a new species of Helicobacter isolated from cheetahs with gastritis. Microbial Ecology in Health and Disease 4 (supplement): s104. Eaton KA, Radin MJ, Kramer L et al (199lb) Gastric spiral bacilli in captive cheetahs. Scandinavian Journal of Gastroenterology 26: 38-42. Eaton KA, Brooks CL, Morgan DR & Krakowka S (1991c) Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylon’ in gnotobiotic piglets. Infection and Immunity 59: 247@2475. Eaton KA, Dewhirst FE, Radin MJ et al (1993) Helicobacter acinonyx sp.nov., isolated from cheetahs with gastritis. International Journal of Systematic Bacteriology 43: 99-106. Engstrand L, Gustavsson S, Jorgensen A et al (1990) Inoculation of barrier-born pigs with Helicobacterpylori: a useful animal model for gastritis type B. Infection and Immunity 58: 1763-1768. Enno A, O’Rourke JL, Howlett CR et al (1995) Maltoma-like lesions in the murine gastric mucosa after long-term infection with Helicobacterfelis. A mouse model of Helicobacter pylon’-induced gastric lymphoma. American Journal of Pathology 147: 217-222. Euler AR, Zurenko GE, Moe JB et al (1990) Evaluation of two monkey species (Macaca mulatta and Macaca fascicularis) as possible models for human Helicobacter pylon’ disease. Journal of Clinical Microbiology 28: 2285-2290.
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Ferrer0 RL, Thiberge JM & Labigne A (1993) Expression of recombinant urease polypeptides from Helicobacter spp. candidate antigens in the development of an anti-Heielicobacterpylon’ vaccine. Acta Gastro-Enterologica Belgica 56 (supplement): 53. Fox JG & Lee A (1993) Gastric Helicobacter infection in animals: natural and experimental infections. In Goodwin CS & Worsley BW (eds) Helicobacter pylori: Biology and Clinical Practice, pp 407-430. Boca Raton: CRC Press. Fox JG, Edrise BM, Cabot EB et al (1986) Cumpylobactcr-like organisms isolated from gastric mucosa of ferrets. American Journal of Veterinary Research 47: 236-239. Fox JG, Lee A, Otto G et al (1991) Helicobacterfelis gastritis in gnotobiotic rats: an animal model of Helicobacter pylori gastritis. Infection and Immunity 59: 785-791. Fox JG, Blanc0 M, Murphy JC et al (1993) Local and systemic immune responses in murine Helicobacterfelis active chronic gastritis. Infection and Immunity 61: 2309-2315. Geyer C, Colbatzky F, Lechner J & Hermanns W (1993) Occurrence of spiral-shaped bacteria in gastric biopsies of dogs and cats. Veterinary Record 133: 18-19. Goodwin CS, Armstrong JA, Chilvers T et al (1989) Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacterpylori gen. nov. and Helicobacter mustelae comb. nov. respectively. International Journal of Systematic Bacteriology 39: 397-405. Handt LK, Fox JG, Dewhirst FE et al (1994) Helicobacter pylori isolated from the domestic cat: public health implications. Infection and Immunity 62: (in press). Hazel1 SL, Lee A, Brady L & Hennessy W (1986) Cumpylobucterpyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. Journal oflnfectious Diseases 153: 658-663. Hazel1 SL, Eichberg JW, Lee DR et al (1992) Selection of the chimpanzee over the baboon as a model for Helicobacter pylori infection. Gastroenterology 103: 848-854. Henry GA, Long PH, Bums JL & Charbonneau DL (1987) Gastric spirillosis in beagles. American Journal of Veterinary Research 48: 831-836. Karita M, Kouchiyama T, Okita K & Nakazawa T (1991) New small animal model for human gastric Helicobucter pylori infection: Success in both nude and euthymic mice. American Journal of Gastroenterology 86: 1569-1603. Krakowka S, Morgan DR, Kraft WG & Leunk RD (1987) Establishment of gastric Cumpylobacter pylon’ infection in the neonatal gnotobiotic piglet. Infection and Immunity 55: 2789-2796. Lambert JR, Borromeo M, Pickard KJ et al (1987) Colonization of gnotobiotic piglets with Campylobacter pyloridis-an animal model? Journal of Infectious Diseases 155: 1344. Lavelle JP, Landar S, Mitros TH & Contelin JL (1994) Acute gastritis associated with spiral organisms from cats. Digestive Diseases and Science 39: 744-750. Lee A & Chen M (1994) Successful immunization against gastric infection with Helicobacter species: Use of a cholera toxin B-subunit whole cell vaccine. Infection and Immuniiy 62: 3594-3597. Lee A, Hazel1 SL, O’Rourke J & Kouprach S (1988a) Isolation of a spiral-shaped bacterium from the cat stomach. Infection and Immunity 56: 2843-2850. Lee A, Dent J, Hazel1 S & McNulty C (1988b) Origin of spiral organisms in human gastric antmm. Lancet i: 300-301. Lee A, Eckstein RP, Fevre DI et al (1989) Non-Campylobacterpylori spiral organisms in the gastric antrum. Australian and New Zealand Journal of Medicine 19: 156-158. Lee A, Fox JG, Otto G & Murphy J (1990) A small animal model of human Helicobacterpylori active chronic gastritis. Gastroenterology 99: 1315-1323. Lee A, Krakowka S, Fox JG et al (1992a) Role of Helicobacter felis in chronic canine gastritis. Veterinary Pathology 29: 487494. Lee A, Phillips MW, O’Rourke JL et al (1992b) Helicobacter muridarum sp. nov., a microaerophilic helical bacterium with a novel ultrastructure isolated from the intestinal mucosa of rodents. International Journal of Systematic Bacteriology 42: 27-36. Lee A, Chen MH, Coltro N et al (1993a) Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection. Zentralblattfur Bakteriologie-International Journal of Medical Microbiology 280: 38-50. Lee A, Fox J & Hazel1 S (1993b) Pathogenicity of Helicobacterpylori-a perspective. Infection and Immunity 61: 1601-1610. Lee A, O’Rourke JL & Kellow JE (1995) Gastrospirillum hominis (Helicobacter heilmannii) and other gastric infections in humans. In Blaser MJ et al (eds) Infections of the Gastrointestinal Tract. New York: Raven Press (in press).
A. LEE Lockard VG & Baler RK (1970) Ultrastructure of a spiral microorganism in the gastric mucosa of dogs. American Journal of Veterinary Research 31: 1453-1462. Marshall BJ, Royce H, Annear DI et al (1984) Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbiology Letters 25: 83-88. McColm AA, Bagshaw JA & Omalley CF (1993) Development of a 14C-urea breath test in ferrets colonized with Helicobacter mustelae-ffects of treatment with bismuth, antibiotics, and urease inhibitors. Gut34: 181-186. McNulty CAM, Dent JC, Curry A et al (1989) New spiral bacterium in gastric mucosa. Journal of Clinical Pathology 42: 585-591. O’Rourke JL, Lee A & Fox JG (1992) An ultrastructural study of Helicobacter mustelae and evidence of a specific association with gastric mucosa. Journal of Medical Microbiology 36: 42&427. Otto G, Fox JG, Wu PY & Taylor NS (1990) Eradication of Helicobacter mustelae from the ferret stomach: an animal model of Helicobacter (Campylobacter) pylori chemotherapy. Antimicrobial Agents and Chemotherapy 34: 1232-1236. Otto G, Hazel1 SH, Fox JG et al (1994) Animal and public health implications of gastric colonization of cats by helicobacter-like organisms. Journal of Clinical Microbiology 32: 1043-1049. Paster BJ, Dewhirst FlE, Seymour C et al (1991a) Helicobacter species isolated from bird and swine faeces. Microbial Ecology in Health and Disease 4 (supplement): S107. Paster BJ, Lee A, Fox JG et al (1991b) Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria. International Journal of Systematic Bacteriology 41: 31-38. Queiroz DM, Rocha GA, Mendes EN et al (1990) A spiral micro-organism in the stomach of pigs. Veterinaxy Microbiology 24: 199-204. Radin MJ, Eaton KA, Krakowka S et al (1990) Helicobacter pylori gastric infection in gnotobiotic beagle dogs. Infection and Immunity 58: 2606-2612. Rappin JP (188 1) Contra 1’Ctude de bacterium de la bouche a l’Ct& normal. Quoted by. In Breed RS et al (eds) BergeyS Manual of Determinative Bacteriology, 6th edn, pp 68. Baltimore: Williams & Wilkins. Salomon H (1896) Uber das spirillum des saugetiermagens und sein verhalten zu den belegzellen. Zentralblattfcr Bakteriologie-International Journal of Medical Microbiology 19: 433-441. Shuto R, Fujioka T, Kubota T & Nasu M (1993) Experimental gastritis induced by Helicobacterpylori in Japanese monkeys. Infection and Immunity 61: 933-939. Solnick JV, O’Rourke J, Lee A et al (1993) An uncultured gastric spiral organism is a newly identified helicobacter in humans. Journal of Infectious Diseases 168: 379-385. Stanley J, Linton D, Burnens AP et al (1993) Helicobacter canis sp.nov., a new species from dogsan integrated study of phenotype and genotype. Journal of General Microbiology 139: 2495-2504. Thomas WD, Lee CK, Lofsky Aet al (1993) Effect of oral imunization with Helicobacterpylori antigens on colonization by H. felis in mice. Acta Gastro-Enterologica Belgica 56 (supplement): 54. Vandamme P, Falsen E, Rossau R et al (1991) Revision of Campylobacter, Helicobacter; and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov. International Journal of Systematic Bacteriology 41: 88-103. Wegmann W, Aschwanden M, Schaub N et al (1991) Gastrospirillum-hominis-assoziierte gastritiseine zoonose? Schweizerische Medizinische Wochenschrift 121: 245-254. Woese CR (1987) Bacterial evolution. Microbiology Reviews 51: 221-271.
SPECIES;
CHARACTERISTICS
AND
With Warren and Marshall’s achievement in isolating H. pylori, it was natural that there were attempts to culture gastric bacteria from other animals (Marshall et al, 1984). The rationale for these investigations was that these bacteria presumably had common characteristics that allowed them to inhabit the gastric mucosa, thus comparative studies might reveal information useful in understanding factors involved in colonization and the pathogenesis of Helicobacter infection of the stomach. The importance of these bacteria increased when it became apparent that H. pylori appeared to have a remarkable host specificity and would not colonize the animal species usually used in development of animal models of human disease. As these bacteria were isolated, their physiological characteristics defined and their DNA sequenced, it became apparent that these organisms were very closely related. They were all placed in the same genus, Helicobacter and given different species names (Goodwin et al, 1989; Bronsdon et al, 1991; Paster et al, 1991b; Eaton et al, 1993). The criterion commonly used to justify positioning these animal isolates was the sequencing of part of their ribosomal RNA, the so called 16s rRNA gene. 16s rRNA is a component of the ribosome, a structure essential for protein synthesis. As production of proteins is so fundamental to life, the critical function of this molecule has stayed the same over the millennia of evolution and so the sequence has remained the same. However, there are known regions of the molecule which have randomly altered over time. The degree of similarity of the 16s BailliLre’s Clinical GastroenterologyVol. 9, No. 3, September 1995 ISBN 0-7020-1961-5
61.5 Copyright 0 1995, by Baillikre Tindall All rights of reproduction in any form reserved
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rRNA sequence is taken to correlate with ancestorship (Woese, 1987). Bacteria that have very similar sequences are considered close relatives and if there is greater than a 90% similarity they can be legitimately placed in the same genus. This is what has happened to the helicobacters which can be regarded as ancestors from a common organism that evolved to inhabit the primordial stomach many years ago. As animal species evolved, so did these bacteria taking on a remarkable diversity of morphologies as can be seen below. However they can all be considered in the one group of highly evolved bacteria exquisitely adapted to the gastric milieu of their animal host. The human helicobacter is IX pylori. Their relatives are described below. Helicobacter mustelae The first animal helicobacter to be cultured was by Fox, who in 1985, isolated a small slightly curved rod shaped bacterium from the stomach of an adult ferret (Fox et al, 1986). These organisms shown in Figures la and lb are 3 pm x 0.5 pm in size with an unusual configuration of lateral and terminal flagella.
Figure 1. Other gastric helicobacter species. (A) Negative stain of Helicobacter musteke showing the unusual lateral and terminal flagella configuration (bar = 5 pm). (B) ferret gastric tissue showing Helicobacier musfelue in close association and adhering to epithelial cells (bar = 1 pm).
Helicobacter
felis
The cat and dog stomach are packed with spiral/helical shaped bacteria, indeed in early morphological studies, three different morphological types were described in the one animal (Lockard and Boler, 1970). The first of these organisms to be isolated was a very tightly spiralled bacterium with five to seven coils (Lee et al, 1988a). This organism had a very striking
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ultrastructure and was seen to be entwined with distinctive periplasmic fibrils and with large tufts of polar flagella (Figures lc & d). It is presumably due to these structures that the bacteria move with a rapid ‘boring’ type motility allowing them to cope with the viscous nature of gastric mucus, just as been suggested for H. pylori (Hazel1 et al, 1986). The name given to this organism was Helicobacterfelis as it was first isolated from a cat. The same bacterium has been subsequently isolated from dog stomachs. There have been two reports of H. felis infection in humans both associated with an acute neutrophilic gastritis (Wegmann et al, 1991; Lavelle et al, 1994).
Figure 1 (contd.). (C) negative stain of Helicobacter felis showing the characteristic helical shape. Periplasmic fibres (arrow) and tufts of polar flagella (bar = 0.5 pm). (D) gastric tissue from a lemur showing a Helicobacterfelis-like organism (bar = 1 pm).
‘Helicobacter
heizmannii
( ‘Gastrospirillum
hominis’) and friends
The dominant morphological bacterium seen in cats and dogs was not H. felis. The bacteria had a similar tight spiral morphology but the characteristic periplasmic fibrils were not present (Figure le). This same morphology was the most commonly seen in any animal, being reported in pigs, primates, and cheetahs (Curry et al, 1987; Queiroz et al, 1990; Eaton et al, 1991a). Interest in these organisms increased following the short report of Dent et al (1987) who observed a bacterium with a similar morphology in 3/1300 human biopsies from patients with histological gastritis, Based on serological results, it was suggested that the human patients had probably acquired this organism from their pets (Lee et al, 1988b). Subsequently this organism was tentatively named ‘Gastrospirillum hominis’ (McNulty et al,
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Figure 1 (contd.). (E) gastric tissue from a Mandrill heilmannii-like organisms (bar = 0.5 pm).
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monkey
showing
large numbers
of Helicobucter
1989). There have now been more than 200 cases of infection with this bacterium reported and it is considered to contribute to about 1% of human gastritis (Lee et al, 1994). No one has had success at culturing this bacterium from either humans or animals. In 1896, Salomon had published a bizarre paper in which he fed the gastric mucosa from various animal species to rodents and showed that their gastric mucosa rapidly became colonized with huge numbers of a bacterium which, from his drawings, appeared to have the morphology of ‘Gustrospirillum (Salomon, 1896). Based on this paper, the homogenate of a gastric biopsy from a patient with ‘G. hominis’ gastritis was fed to mice, and it was found that the organism colonized the mouse stomach in large numbers (Dick et al, 1989; Lee et al, 1989). Subsequently, similar bacteria from a wide range of animals including lemurs, macaques, mandrill monkeys and a bobcat have been isolated using this in vivo culture method, but in vitro culture has never been successful (J. O’Rourke, unpublished observations). Using molecular techniques, the 16s rRNA gene of some of these bacteria have been amplified from infected mouse stomachs allowing the whole gene to be sequenced. This has shown, not surprisingly, that these bacteria belong to the genus Helicobacter (Solnick et al, 1993). The name ‘Helicobacter heilmannii’ has been tentatively assigned to one of the human isolates. Whether each bacterium from all of the different animal species will be given a separate name remains to be determined. It is sufficient for the gastroenterologist to appreciate that this broad group of morphologically similar bacteria are the most common helicobacter and most animals are infected with them. Where the organisms infecting their patients comes from depends on the type of animals that person has come in contact with. Given the number of infected cases and the small number of humans infected, transmission must be difficult and depends on very close contact with the animal. Presumably most come from pet cats and dogs unless your patient happens to have a pet cheetah! (See below).
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acinonyx
The most exotic helicobacter yet isolated comes from the cheetah. Animals in a closed colony in Ohio were found to be seriously ill and presented with chronic vomiting (Eaton et al, 199 1b). Histopathology revealed the cheetahs had a severe lymphoplasmacytic gastritis characterized by variable numbers of neutrophils, gland abscesses, lymphoid follicles and epithelial erosions. Although bacteria with the morphology of ‘H. heilmannii’ were seen in the gastric mucosa, the organism cultured had a morphology much closer to H. pylori except it was smaller. These S-shaped bacteria are 0.3 pm x 2 pm in size with 2-5 polar sheathed flagella and share many biochemical properties with H. pylori. The bacterium has been named Helicobacter acinonyx, after the taxonomic name for the cheetah (i.e. Acinonyxjubilatus) (Eaton et al, 1991a). The gastric versus the non-gastric
helicobacters
As time goes by, many other helicobacters will probably be cultured from animal stomachs. These may be of interest in what they tell us about gastric colonization and pathogenesis. However, recently there have been a plethora of new helicobacters reported that have no relevance to our understanding of gastroduodenal pathology and will only serve to clutter the Helicobacter databases. These are the non-gastric helicobacters and it is important that gastroenterologists appreciate what these bacteria are so they need not chase the literature on these organisms. Soon after the sequencing of H. pylori and H. mustelae, and based on short sequences that were seen to be specific for the genus Helicobacter, specific gene probes were produced and used to screen large numbers of intestinal bacteria (Paster et al, 199 1a). A number of bacteria were shown to react with the specific probe. Indeed some which were previously in the genus Campylobacter have been renamed, for example Helicobacter cinaedi and Helicobacter fennelliae (Vandamme et al, 1991). In other cases new species have been identified, for example Helicobacter canis (Stanley et al, 1993). These non-gastric bacteria do not colonize the stomach. They lack the enzyme urease which is certainly a unifying property of the gastric helicobacters and appears necessary for these bacteria to colonize the acid laden stomach. The only exception is Helicobacter muridarum, which has a urease enzyme (Lee et al, 1992b) and in special circumstances does inhabit gastric mucus (Lee et al, 1993a).
NATURAL AND EXPERIMENTALLY IN ANIMALS; ULTRASTRUCTURAL, MACROSCOPIC CHANGES
INDUCED INFECTION MICROSCOPIC AND
For any microbial infection, it is important to have appropriate animal models in order to better understand pathogenesis, test new therapies and to develop vaccines. There is no perfect model of human H. pylori infection, rather we have to rely on a number of differing situations which either
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utilize H. pylori infection in animals, follow the consequence of natural helicobacter infection in animals, or involve infection of unnatural animal hosts with some of the non- H. pylori gastric helicobacters described above. The natural and experimental infections of animals reported to date are described below. For each animal the potential use of the model is evaluated to serve as a guide for potential researchers into H. pylori-associated disease. Primates
Many species of primates are colonized with H. heilmannii: (Fox and Lee, 1993). The most extensive studies have been those of Dubois et al (1991) in which they found 14 out of 29 rhesus monkeys to be colonized. The bacteria were found to localize in the mucus covering the surface epithelial cells and deep into the gastric pits, however there were no signs of gastritis. Ultrastructural study of the parietal cells revealed bacteria in the cytoplasm of healthy and injured cells and this correlated with an increased basal acid output (BAO) and peak postload acid output (PAO) in these animals. In 1988, bacteria resembling H. pylori were isolated from a colony of laboratory housed Rhesus monkeys (Baskerville and Newell, 1988). Since then many others have found bacteria almost identical to H. pylori in a range of primates (Bronsdon and Schoenknecht, 1988; Euler et al, 1990; Dubois et al, 1991). A very similar organism called Helicobacter nemenstrina has also been described (Bronsdon et al, 1991). It may be that these bacteria are in some cases human strains that have been transmitted to the primates by their H. pylori infected keepers. Alternately, given the close evolutionary linkage between the non-human primate and the human, one could predict similar bacteria could be present. In the primates naturally infected with these H. pylori-like bacteria, a more significant inflammation was seen than with H. heilmannii: (Euler et al, 1990). Consistent with this have been studies where non-human primates have been infected with human isolates of H. pylori. In Japanese monkeys there was an initial polymorphonuclear and mononuclear cell infiltration after infection with H. pylori with antral erosions and erythema observed macroscopically. This persisted for up to six months, however the severity of the gastritis decreased over time (Shut0 et al, 1993). Elements of acute and chronic gastritis were also detected in chimpanzees infected with H. pylori (Hazel1 et al, 1992). The expense of primate research restricts the usefulness of the model, although in the development of new vaccines it will be essential to eventually involve a primate-model. For other studies alternative models are required. Pigs and piglets
Although in early studies, H. pylori did not colonize traditional animal models such as mice, rats, guinea pigs and rabbits, two groups indepen-
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dently showed that gnotobiotic piglets derived by caesarean section and raised in germ-free isolators did become infected with human isolates of H. pylori (Krakowka et al, 1987; Lambert et al, 1987). The bacteria colonized the antral mucosa in a manner very similar to that of humans. Helicobacter pylori was seen in large numbers in the mucus and appeared to firmly adhere to the gastric mucosa. The inflammation induced, resembled that seen in H. pylori infected human children rather than adults (Bertram et al, 1991). The cellular infiltration was primarily mononuclear with large lymphoid aggregates with nodular protrusions seen on gross examination of the gastric mucosa. A problem with the gnotobiotic piglet as an experimental animal is cost and the fact that the piglets become pigs and can no longer be housed in the isolators. However, the model has proved extremely useful in studies on H. pylori colonization, and the definitive work showing the importance of motility and urease was performed with this model, using H. pylori variants and mutants (Eaton et al, 1989; Eaton et al, 1991~). Other workers have used barrier raised specific pathogen free pigs and shown that H. pylori will colonize for long periods of time and have developed a 13C-urea breath test for diagnosis of infection (Engstrand et al, 1990). Ferrets
There are a number of shared characteristics between the H. mustelae infected ferret and the H. pylori infected human. Ferrets present varying degrees of gastritis ranging from superficial gastritis in the oxyntic mucosa to a diffuse antral gastritis with elements of chronic atrophic gastritis. The severity of gastritis has been found to increase with age and in some instances ulceration has been noted at the pyloroduodenum junction (Fox and Lee, 1993). The ultrastructure of the H. mustelae colonized gastric mucosa reveals that like H. pylori, this bacterium adheres firmly to the gastric mucosa (O’Rourke et al, 1992). Indeed the majority of the organisms were seen close to the mucosa with many cases of actual endocytosis of the organism into the epithelial cells (Figure lb). This has been observed with H. pylori in vitro but is not commonly seen in vivo. The adhesion of H. mustelae seen in the ferret is the only other example of helicobacter adhesion. Also, the ferret is the only other animal where peptic ulceration is commonly seen. Although not common enough to be used as an experimental model of helicobacter-associated ulceration it does give weight to the importance of the ferret as a natural model of human H. pyloriassociated disease. Another use for the ferret-model has been as a screen for antimicrobial therapies. For example, experiments have shown that H. mustelae can be eradicated from the majority of colonized ferrets tested using a combination of bismuth subcitrate, amoxycillin and metronidazole, whereas monotherapies such as bismuth or chloramphenicol were unsuccessful (Otto et al, 1990; McColm et al, 1993).
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Dogs In pre-H. pylori days the pathology presumably induced by natural helicobacter infection was described by Henry et al (1987), who looked at a number of laboratory reared Beagles. The bacteria were most commonly detected at the fundic-pyloric junction and the cardia which corresponded with the presence of large lymphoid follicles. From this association he postulated that the presence of these bacteria may result in the induction of lymphoreticular hyperplasia. Koch’s postulates for a helicobacter causing this pathology was confirmed experimentally when living cultures of H. felis were fed to germ free Beagle puppies (Lee et al, 1992a). In the infected animals, large lymphoid follicles were found throughout the gastric mucosa with the largest and most extensive follicles seen in the fundus and body. No such follicles were detected in the control animals. Throughout the subglandular region of the stomach, a diffuse infiltrate of lymphocytes, eosinophils and plasma cells were also seen. At the same time as these experiments, H. pylori was fed to another litter of the germ free Beagle pups (Radin et al, 1990). Limited colonization was observed in the dogs and they presented a chronic active gastritis characterized by focal to diffuse lymphoplasmacytic cellular infiltrate in addition to occasional lymphoid follicles in the antrum. Gnotobiotic dog-work is expensive and normal dogs have heavy infection with their own helicobacters, and so there would appear to be limited use of a dog-model. However, as veterinarians become familiar with the concept of human H. pyEori-associated gastric disease they are starting to look for parallels with canine disease (Geyer et al, 1993). As has been the case with H. pylori and non-ulcer dyspepsia the role of the bacterium as a causal agent is not proven, although logic has it that some animals probably suffer from their helicobacters. Cats The consequence of natural helicobacter infection in cats would appear similar to dogs. In a study looking at the post mortem studies of 55 cats and kittens, an associated pathology was observed (Otto et al, 1994). Gastritis was recorded that varied from a mild infiltrate of inflammatory cells to a diffuse mixed subglandular leukocytic infiltrate with lymphoid nodules that displaced entire gastric glands. Mixtures of lymphocytes, plasma cells and occasional polymorphonuclear leukocytes were also detected in focal cellular aggregates. The severity of the histopathology was found to correlate with the density of bacterial colonization. There have been no experimental studies with cats, although a remarkable recent finding would suggest that this will soon change. Handt et al (1994) has isolated bacteria from domestic cats which were shown by morphology, biochemical properties and 16s rRNA sequence analysis to be H. pylori. Histopathology revealed the cats had gastritis characterized by a mild to severe diffuse lymphofollicular infiltrate with occasional neutrophils and eosinophils in the subglandular and glandular mucosae.
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Mice
Mice do not have a natural gastric helicobacter, although some strains of conventional mice are colonized with H. muridarum in their lower bowel (Lee et al, 1992b), although as seen above, these animals can easily become colonized with other animal helicobacter species. The most important mouse model has utilized H. felis (Dick et al, 1989). When administered orally, H. felis colonizes the mouse stomach in large numbers where it will remain for the life of the animal. The pathology resulting from infection varies depending on the animal strain used, highlighting the importance of host factors. When given to germ-free Swiss mice an active chronic gastritis is seen (Lee et al, 1990). Initially there is an acute inflammatory response composed primarily of eosinophils and neutrophils, the latter increases with time resulting in the formation of small micro-abscesses. By 8 weeks post-infection there is a chronic infection characterized by increasing numbers of mononuclear cells and the appearance of large lymphoid follicles with micro-abscesses still present in the pyloric mucosa. The H. felis infected germ-free mouse has been followed for up to 50 weeks in which, after an initial active chronic inflammation was observed, a chronic gastritis persisted for the length of the experiment (Fox et al, 1993). This was the first convenient animal model of helicobacter infection where the active component of the gastritis, that is characteristic of H. pylori infection in adult humans, was recorded (Figures 2a-c). Long-term
Figure 2. Animal models of helicobacter infection (H&E stains). Germfree mouse colonized with Helicobncter felis for 4 weeks showing an increase in the cell infiltrate. (A) (magn x 50) with the migration of polymorphonuclear leukocytes from the lamina propria into the cytoplasm of the glandular epithelial cells. (B) (magn x 100) higher magnification of the migration of the polymorphonuclear leukocytes. (C) (magn x 100) and micro abscesses. (Reproduced from Lee et al (1990, Gastroenterology 90: 1315-1323) with permission.
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studies in a conventional colony of Swiss mice demonstrated the progression of gastritis from active/chronic gastritis to a highly destructive atrophic gastritis in which some dysplastic changes were seen (Figure 2d) (Lee et al, 1993a). This was the first experimental evidence of the progression of helicobacter induced chronic gastritis to atrophy, an observation that is consistent with the Correa hypothesis (1984) of the progressive steps in the development of gastric adenocarcinoma. It has been suggested that the long term H. $&-infected mouse may be a very important model in which to study helicobacter induced gastric carcinogenesis.
Figure 2 (contd.). Conventional mice colonized with Helicobacter felis for 18 months showing extensive infiltration of inflammatory cells and obvious loss of oxyntic glands. (D) (magn x 125). Reproduced from Lee et al (1993a, Zentrulbluttfiir Bakteriologie 280: 38-50) with permission.
Mice from a specific pathogen free colony of BALB/c mice were followed long-term after infection with H. felis, and for up to 18 months, very little inflammation was seen. However, post-18 months lymphoid follicles started to appear only in infected animals. By 26 months, dramatic lesions exactly analogous to low grade B-cell lymphomas were observed in 25% of the infected animals (Enno et al, 1995). The rodent has much potential as an animal model, even though mice do not develop ulcers there are a number of H. pylori-associated pathologies that are mimicked. However, there are differences with respect to H. pylori that have to be noted. The bacterium is restricted to the antrum and cardia with very little colonization seen in the body of the stomach. There is no adherence to the gastric mucosa and the bacteria travel much deeper into the gastric glands than normally occurs with H. pylori. Despite these differences the susceptibility of the H. &Z&infected mice to antimicrobial agents is very similar to that found in the H. pylori infected human.
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Consequently, monotherapy will not eradicate H. felis whereas triple therapy is very effective. Given the unreliability of in vitro testing of newer antimicrobials as a predictor on in vivo efficacy, it has been proposed that the H. felis-mouse model is a very convenient and economical screen for potential new anti-H. pylori agents (Dick-Hegedus and Lee, 1991). The other major use of this model has been in immunization studies and this will be discussed later. Even though early studies in mice showed that mice could not be infected with H. pylori, recent work suggests that this may be an attainable goal (Karita et al, 1991). Japanese workers claim to be able to infect BALB/c nude mice with fresh human isolates of H. pylori. Counts appear to be low and no pictures of colonization have been published. The critical factor seems to be the freshness of the isolate used. The development of a H. pylori-mouse model is a very worthwhile goal and more developmental work is required. Rats Rats can be infected with helicobacters such as H. felis or H. heilmannii. The inflammation reported has been limited but this probably relates to the strain of rat used (Fox et al, 1991). Due to the convenience of mice over rats and no obvious advantage at this stage, there has been little further work done on a rat-model. TOWARDS
A VACCINE
As is seen above there are a number of animal models available for the study of H. pylori-associated disease. Now that this bacterium is firmly established as a major bacterial pathogen infecting more than half the world’s population, possibly threatening the health and indeed lives of millions of people, the time is ripe for the development of a strategy that has proved so successful with many of the world’s other infectious diseases, namely immunization. The latter part of this chapter shows that it is the development of animal models that now makes it possible to develop an anti-H. pylori vaccine. The possibility Helicobacter pylori is a remarkably well adapted pathogen that colonizes its host for life. This is despite the induction of an immune response so large that serological assays can be accurately used to predict active infection (Lee et al, 1993b). There are other chronic diseases where the organism survives in the host for life, but they are usually sequestered away (e.g. tuberculosis, herpes). In contrast, H. pylori can be found in huge numbers swimming in the gastric mucus or firmly adhered to the epithelial surface despite the infiltration of large numbers of immunocompetent cells and active phagocytes.
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With such highly evolved evasion strategies it could be asked whether immunization is an attainable goal. Is it possible to beat nature? The early workers responded, by suggesting that there is a difference between an immune response necessary to prevent an infection and the response needed to remove an already established one. The first evidence The first data that suggested the goal of effective immunization was attainable was the result of the fortunate teaming together of Stephen Czinn, a paediatric gastroenterologist with an interest in H. pylori and a mucosal immunologist, John Nedrud. Czinn and Nedrud immunized both mice and ferrets by oral administration of a sonicate of H. pylori together with the adjuvant cholera toxin (CT) (Czinn and Nedrud, 1991). As predicted, they achieved a highly significantly raised level of anti-H. pylori IgA in gastric secretions. They concluded with the comment that these results suggested that it is possible to develop an oral immunization protocol for prevention of H. pylori infection and associated gastritis. However, they were not in a position to easily test their hypothesis as mice could not be colonized with H. pylori and the ferrets were all infected with H. mustelae. By the time the Czinn and Nedrud work had been reported, we had developed the H. felis mouse model and thus were in a position to test their hypothesis. Groups of mice were given oral doses of H. felis sonicate alone or with CT on days 1, 3, 6, 30 and 54. Three weeks later, after these animals and appropriate control groups were challenged with large numbers of viable H. felis. A highly significant degree of protection was seen with the vaccine comprising the H. felis sonicate and CT (Chen et al, 1993). At about the same time Czinn and Nedrud found the same result in a series of germ-free mice (Czinn et al, 1993). They also demonstrated that a monoclonal IgA antibody against H. felis was protective. The enhanced response noted in the actively-immunized protected mice was later found to correlate with increased levels of specific anti-H. felis-IgA antibody in gastric and intestinal secretions (Chen et al, 1993). Composition
of vaccines
Since these early experiments were published, many companies have commenced the highly competitive race to develop the first human vaccine. All these groups have used the H. felis-mouse model. Based on mostly published abstracts, we know much more about the possibilities for the composition of the eventual vaccine (Blanchard et al, 1993; Corthesy-Theulaz et al, 1993; Ferrer0 et al, 1993; Thomas et al, 1993). The specificity of the Czinn and Nedrud monoclonal antibody was found to be against the H. felis urease (Blanchard et al, 1993). Given the known importance of this molecule, it was not surprising urease would be a good candidate for a protective antigen, although it should be noted that the monoclonal antibody did not inhibit urease activity. Following the
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sequencing of the urease genes of H. pylori and the elegant work of Labigne and her team in developing methods for the genetic manipulation of this organism, a vaccine was constructed that used recombinant H. pylori urease with CT (Cussac et al, 1992; Ferrer0 et al, 1993; Corthesy-Theulaz et al, 1993). This vaccine protected against challenge with viable H. felis. This was consistent with studies that showed immunization with whole cell sonicates of H. pylori protected against H. felis. This was a relief to the workers in the field as it meant that they were indeed not developing a vaccine to protect mice against H. felis, but could work on the H. pylori vaccine! At present the most likely vaccine candidate is urease. How this vaccine will be formulated is unknown and this is another area of intense activity. Cholera toxin is clearly an unacceptable adjuvant as it is too toxic to humans. An alternative adjuvant is needed. Recently experiments with the non-toxic B-subunit of cholera toxin (CTB) has shown protection (Lee and Chen, 1994). The CTB used was a commercial preparation which is known to have minute amounts of holotoxin contamination. Thus the CTB effect could be due to the contaminating CT. None the less these results are encouraging and suggest a non-toxic, effective vaccine formulation will be found. Therapeutic vaccinationa new dimension in Helicobacter management Using the H. felis model some results have recently been published that add a new dimension to the potential of H. pylori immunization. Success with our mouse-model encouraged us to determine whether it is possible to eliminate existing gastric infection by immunization. Specific pathogen free (SPF) BALB/c mice were infected with a living culture of H. felis. Four weeks after infection, 80 animals were immunized orogastrically on days 1, 15, 17, and 20 with H. felis whole cell sonicate (4 x 1 mg) and cholera toxin (4 x 10 pg). Eighty infected mice were retained as controls. One week, 1 month, 2 months and 3 months after completion of immunization, groups of immunized and non-immunized mice were euthanased and their H. felis status determined. Results of this experiment are shown in Table 1. A high proportion of previously infected animals had no detectable organisms in their stomachs for up to three months post-immunization. Table
1. Therapeutic
Vaccine Experiment 1 Nil H. feZis sonicate H. felis sonicate H. felis sonicate H. felis sonicate * Fisher’s
+ + f +
CT CT CT CT
immunization
Time of assay post-immunization
Number animals infected
1 1 1 2 3
IO/76 2120 3/20 6120 l/17
exact test (two tail).
week, 1,2, 3 month week month months months
f&s
of Helicobacter
infected
mice.
of % Not infected
Significance*
9% 90% 85% 70% 94%
F1 P z? p.
< < < <
0.0001 0.0001 0.0001 0.0001
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According to the current definitions of anti-Helicobacter chemotherapy, i.e. clearance of bacteria for more than a month after the cessation of therapy; therapeutic immunization appeared to have eradicated the infection (Doidge et al, 1994). In a second experiment a significant result was achieved by administration of a H. pylori sonicate plus cholera toxin. These results showed, for the first time, that it was possible to eradicate active Helicobacter infection of the gastric mucosa by administration of an oral vaccine. This raised the intriguing possibility that therapeutic immunization might be a viable option in the management of Helicobacter-associated disease. If immunization as a therapy for peptic ulcers was combined with short-term acid suppression, the possibility of reinfection may also be eliminated. In those countries where H. pylori infection rates are very high and infection occurs at an early age, large scale oral immunization of sections of the community would not only protect the young from the deleterious consequences of long-term H. pylori infection, but could also cure existing disease. On reflection the success of therapeutic immunization was predictable. Lifelong helicobacter infection is the result of a balance between host and parasite that maintains an equilibrium which is normally of no major consequence to the host. The bacterium can persist because it has evolved to circumvent the normal immune response. The host control mechanisms ensure that a comprehensive local immunity is not induced, as the normal mechanisms of oral tolerance are in action. Subverting oral tolerance against the helicobacter antigens by the administration of the cholera toxin adjuvant results in an enhanced local response, that is much greater than the response that the resident helicobacter evolved to evade. Thus, the balance swings in favour of the host and the bacterium is eliminated. A commentary by Cohen (1994) recently, drew attention to the development of vaccine therapies for a number of other chronic diseases where a permanent carrier status has been established, for example herpes virus infection, leprosy, leishmaniasis, tuberculosis and Hepatitis B. In the veterinary field, immunization has been used as a therapy of chronic Campylobacter fetus infection in bulls for many years (Bouters et al, 1973). Conclusion
The development of animal models of helicobacter infection provides us with a number of powerful tools to investigate many facets of infection by these fascinating helical pathogens. There is no one best model despite the vigorous defences of those of us in the field. However, perhaps many of them will be used in what is now possibly the most exciting development in the fight against human H. pylori infection, namely the development of a safe and effective vaccine that can be used for the treatment and prevention of these diseases. This opens up the potential for the complete eradication of this bacterium from many populations, thus consigning the major topic of this current volume to the history books and the status of a medical curiosity.
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SUMMARY Following the demonstration of Helicobacter pylori as a major gastroduodenal pathogen there was a need to develop animal models in order to investigate mechanisms of pathogenesis and to be able to test new treatment strategies. Helicobacter pylori will only colonize a limited number of hosts including non-human primates, germ-free or barrier raised piglets, germ-free dogs and recently laboratory raised cats. Although these models have proved useful there is a need for more convenient small animal models. The ferret infected with its natural gastric organism, Helicobacter mustelae, is the only other animal to show peptic ulceration and has been successfully used to investigate gastritis and antimicrobial agents. The other commonly used animal model is the laboratory mouse or rat infected with either Helicobacter felis or Helicobacter heilmannii, bacteria that normally colonize cat or dog gastric mucosae. Active/chronic gastritis, gastric atrophy, and lymphoma-like lesions have been shown to develop in H. felis infected mice. The most recent and exciting use of an animal model has been the use of the H. felis mouse model in the development of human vaccines against H. pylori. Mice can be protected against infection with large doses of viable H. feZis by oral immunization using sonicates of H. f&s or H. pylori or recombinant H. pylori urease together with cholera toxin or cholera toxinB subunit as the mucosal adjuvant. More importantly it has been shown that immunization of already infected animals results in eradication of infection. This raises the intriguing possibility that therapeutic immunization might be a viable option in the management of Helicobacter-associated disease. If immunization as a therapy of peptic ulcers was combined with short-term acid suppression, the possibility of reinfection may also be eliminated. In those countries where H. pylori infection rates are very high and infection occurs at an early age, large scale oral immunization of sections of the community would not only protect the young from the deleterious consequences of long-term H. pylori infection but could also cure existing disease. Acknowledgments The help of Jani O’Rourke in the preparation of this manuscript is gratefully acknowledged as is research support from the National Health and Medical Research Council of Australia, CSL Ltd, Parkville, Australia and Astra Hassle, Molndal, Sweden.
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