Laboratory Animal Science Vol 48, No 6 Copyright 1998 December 1998 by the American Association for Laboratory Animal Science

Animal Models of Helicobacter Infection

Andre Dubois Our next speaker will be Dr. Andre Dubois, who will speak human disease led to interest in animal diseases, not the about animal models of Helicobacter infection. Dr. Dubois. opposite, and I am convinced that this interest will benefit Dr. Dubois: Thank you, Dr. Frenkel, and thank you to veterinary science. the organizers of this meeting for having invited me. It is a I have divided my presentation into two parts. In the privilege to be presenting these data today. first part, I will discuss natural infection in various ani- The Helicobacter was named only about 10 years mals, and in the second part, I will more briefly present ago to group a number of already described that animal models of experimental infection with several did not quite fit the genus in which they had been classi- Helicobacter species. fied. For example, , which had been named successively pyloridis, then Natural Infections with Helicobacter Campylobacter pylori,was different from the other mem- Species bers of the Campylobacter genus, and it was the first to be This section will be divided in two parts: firstly, placed in this genus, while being renamed accordingly. Helicobacter species other than H. pylori, and secondly, H. During the past decade, H. pylori has become recognized pylori itself. as one of the most common human , colonizing Helicobacter species other than H. pylori, H. the gastric mucosa of almost all individuals exposed to poor hepaticus, H. bilis, etc.: Recently, a number of bacteria hygienic conditions from childhood. It also is often found, were observed in the liver of the mouse, and possibly also albeit with a lower frequency, in groups of higher socioeco- in other species. These include H. hepaticus, H. bilis, H. nomic status. H. pylori causes chronic-active and pullorum, and H. canis. Due to the lack of time, this pre- is a major factor in the pathogenesis of duodenal ulcers sentation will focus on H. hepaticus, saying only that H. and, to a lesser extent, of gastric ulcers. In addition, the bilis has a slightly different appearance than that of H. presence of this bacterium is now recognized as a risk fac- hepaticus, that it has periplasmic fibers, and that it has tor for gastric adenocarcinoma and lymphoma. Neverthe- multiple flagellae at both ends. The slides that I am going less, most infections appear to be without clinical conse- to show you have been loaned to me by Dr. Jerrold Ward, quences. Other organisms now classified in the Helicobacter who is at the National Cancer Institute in Frederick, Md. I genus have been described both in animals and in humans want to thank him for this loan. Also, I wanted to give you since the end of the 19th century. the address of his home page. It is www.ncifcrf.gov/vetpath/ The interest in this class of organisms started when Robin emerg.html, and it is an address where those of you who Warren, an Australian pathologist, observed that there was are Internet-fluent can seek additional information, and an almost 100% association between the presence of spiral also see and download some of the slides I am going to show organisms and in his patients. How- you. You will not regret your visit! In addition, Dr. Ward ever, the work really progressed when a clinician, Barry kindly agreed to have me illustrate these transcripts with Marshall, who was then a young clinical assistant, joined some of his figures. the laboratory of Warren. Together they confirmed that a H. hepaticus was isolated a few years ago at the Frederick large number of spiral organisms were present in virtually NCI Center and at the Massachusetts Institute of Technol- all patients with peptic ulcer disease. Marshall and War- ogy (MIT), Cambridge, Mass. Dr. Jerrold Ward and his ren then demonstrated that culture of a majority of the group, in collaboration with Dr. Jim Fox and his group at biopsies harvested from the stomachs of patients with pep- MIT, discovered that a fraction of the mice that were bred tic ulcer disease produced gram-negative in that colony developed acute hepatitis, which was accom- that were , catalase, and oxidase positive. panied by focal necrosis and focal non-suppurative inflam- My presentation today is focusing on animal models. mation, in 1- to 6-month-old mice. Older animals developed What I would like to show you is that in this case, in con- chronic hepatitis, characterized by hepatocytomegaly, cho- trast to what was presented earlier with Microsporidia, the langitis, bile ductular hyperplasia and oval cell hyperpla- sia, increased cell proliferation, and cirrhosis. Finally, liver tumors appeared in animals older than 12 months with Uniformed Services University of the Health Sciences, F. Edward Hérbert initially preneoplastic hepatocellular foci, hepatocellular School of Medicine, Bethesda, Maryland The opinions and assertions contained herein are the private ones of the adenoma, and eventually carcinoma. authors and are not to be construed as official or reflecting the views of the In animals infected with H. hepaticus, subacute hepati- Department of Defense or the Uniformed Services University of the Health Sciences. tis is characterized by foci of inflammation. Cholangitis is

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The structure of H. hepaticus can be analyzed by scanning elec- tron microscopy and by trans- mission electron microscopy (Fig- ure 3). These methods have shown that, while the majority of the or- ganisms have a single at each end and have a spiral shape, they can also appear as rods or even round coccoid- shaped organisms. Therefore, morphology is not an absolute criterion for recognizing this or- ganism. It is interesting to note that H. hepaticus can be stained using monoclonal antibodies against H. pylori, indicating that even monoclonal antibodies used to stain H. pylori are nonspecific, which can lead to some confusion when diagnosing these organ- isms in different species. H. hepaticus can be detected by polymerase chain reaction analy- sis. This method is very sensitive in demonstrating the presence of the organism in the cecum and in the feces of colonized ani- mals, but not in uninfected ani- mals. Therefore, shedding of this organism is a likely cause of fecal-oral transmission within the mice colonies. H. hepaticus colonization is accom- panied by secretion of specific antibodies in naturally infected Figure 1. Steiner stain showing H. hepaticus in bile canaliculi of mouse liver (downloaded from www.ncifcrf.gov/vetpath/emerg.html, with the permission of Dr. Jerrold Ward, NCI, Frederick, Md. mice that have been detected To view this photo in color, please visit http://www.aalas.org/pubs/laonline.htm on the web.). after a month as well as at 142 to 200 days. also often present. Silver stains are useful to demonstrate Finally, it is important to realize that mice from many the presence of H. hepaticus, as is the case for all commercial breeding colonies have been found to be colo- Helicobacter species. It is important to stress that H. nized by H. hepaticus, although a number of commer- hepaticus is an intraluminal organism, as it is found in cial sources now have eliminated the bacterium from biliary canaliculi and liver parenchyma, but never within their colonies. hepatocytes (Figure 1). H. muridarium: H. muridarium has been found in the This is also typical of all Helicobacter species, which can ileum of rats and the colon of mice. The organism is very be found exclusively in the lumen of the gut, and they seem characteristic in that it has ridges along the entirety of its unable to invade the gut wall. They cause intense inflam- body and flagellae at only one end. mation within the walls of the gut, but the immune de- H. mustelae: In this country, 99% of the ferrets are colo- fense that is sparked by this organism is unable to clear it nized by H. mustelae. Thus, the frequency of colonization is from most colonized hosts (Figure 2). higher than that of H. pylori, which, as stated earlier, colo- H. hepaticus can also be seen in homogenized hepatic nizes about 50% of the U.S. human population. biopsies, where it appears among cellular debris. Interest- Dr. Jim Fox from MIT has demonstrated the presence of ingly, the presence of H. hepaticus in the cecum and in the H. mustelae in the lumen of the gastric glands of a major- intestine of colonized mice also was demonstrated by scan- ity of the ferrets he examined. Similar to what is observed ning electron microscopy. When present in the intestines, in humans colonized by H. pylori, there is intense inflam- it causes inflammation that may represent a good model mation. A number of animals also present with gastric ul- for inflammatory bowel disease. cers. So for veterinarians, H. mustelae has an interest in

597 Vol 48, No 6 Laboratory Animal Science December 1998 that presence of ulcers can be caused by an infection in pet ani- mals such as ferrets, and that it can be cured or prevented by treating the animals against H. mustelae. H. felis: This organism was initially isolated from cats. Its morphology is very specific. There are single or double ridges spiraling along the length of the organism. By transmission elec- tron microscopy, these ridges ap- pear as black dots on the top of each turn of the spiral. H. heilmannii: In carnivores such as cats and dogs, a number of Helicobacter species have been observed. Several genetically dis- tinct organisms were initially classified as “Gastrospirillum hominis.” More recently, the name H. heilmannii has been proposed in the memory of the deceased German pathologist who was the first to describe it extensively. The change of name may be premature since several different subspecies probably do exist. Nonetheless, the name is more and more used, and I will use it for simplicity. The difficulty Figure 2. Severe cholangitis and bile ductular hyperplasia in liver of mice with H. hepaticus with this organism is that most infection (downloaded from www.ncifcrf.gov/vetpath/emerg.html, with the permission of Dr. Jerrold Ward, NCI, Frederick, Md. To view this photo in color, please visit http://www.aalas.org/ groups have been unsuccessful in pubs/laonline.htm on the web.). attempting to grow it in vitro. Only one group in Finland has been able to isolate the organism, and further progress will depend on that they are within the canaliculi of parietal cells and in- the confirmation of these results. side parietal cells. We don’t know whether the organism is The morphology of H. heilmannii is different than that of digesting the parietal cell or if the parietal cell is digesting most other Helicobacter species, and is closer to that of H. it. Although it is appears to colonize the of these felis. It is tightly spiraled, and its length is 10 to 15 ␮m. animals for life, there is much less inflammation, if any, than Morphologically similar H. heilmannii have been observed when the stomach is colonized by H. pylori (see below). in dogs, in pigs, in rhesus monkeys, and in humans. These H. acynonix: Cheetahs are carnivores in which several organism are observed floating freely in the mucus of the Helicobacter species have been described. The cheetahs stomach and near the surface of normal gastric mucus cells. studied by Dr. Kate Eaton at the zoo in Columbus, Ohio, Using electron microscopy, we can demonstrate that, al- were initially investigated for gastrointestinal bleeding and though they are in close association with the surface epi- were found to have gastric ulcers. Histology and culture of thelial cells, there is no direct attachment of the organism gastric biopsies demonstrated the presence of three bacte- to the cells, and that there is no specialized structure in ria: H. felis, H. Heilmannii, and a newly described the cell membrane opposite to the bacteria. In addition, Helicobacter species, H. acinonyx. This last organism is transmission electron microscopy demonstrates that the quite different from the other two, and the sequence of its microvilli are preserved, as opposed to what is happening 16S 16rRNA was found to be closer to that of H. pylori, with H. pylori (see below). H. heilmannii is also observed compared with other Helicobacter species. in the lumen of gastric glands in the acid-producing pari- H. nemestrinae: Pigtailed macaques (Macaca nemestrina) etal cell area of the stomach. It also can be seen overlying were found to harbor not only H. pylori, but also a different the parietal cells. By light microscopy, one cannot deter- organism, H. nemestrinae, which is very similar to H. pylori, mine whether H. heilmannii are within parietal cells, but the 16S rRNA sequence is sufficiently different to jus- whereas by transmission electron microscopy one can see tify its classification as a different species.

598 Animal Models of Helicobacter Infection

Figure 3: Ultrastructure (with 3D embossing) of mouse liver showing the presence of H. hepaticus in bile canaliculi (downloaded from www.ncifcrf.gov/vetpath/emerg.html, with the permission of Dr. Jerrold Ward, NCI, Frederick, Md.).

Helicobacter pylori: Only two animal species have been The source of the contamination in these Massachusetts colo- found to be naturally infected with H. pylori. nies is at present unknown. Felines: Recently, the presence of H. pylori colonizing the The immune system of the cat is very different from that of stomach of cats was described in animals bred in two Massa- nonhuman primates and of humans, but it provides a unique chusetts colonies. However, other investigators have been opportunity to study different types of immune reactions to unable to confirm this observation in other breeding colonies. an organism that is found in primates.

599 Vol 48, No 6 Laboratory Animal Science December 1998

Histology of the gastric mucosa of colonized cats shows the Gastroenterology, 1994, 106:1405–1417). presence of lymphoid aggregates deep in the lamina propria A seroepizootiology study of seropositivity for H. pylori of the mucosa. In contrast, the diffuse inflammation that char- was performed in the breeding colony from which rhesus acterizes H. pylori infection in primates is not present. monkeys were coming. Analysis was performed by gender Rhesus monkeys (Macaca mulatta) and cynomolgus mon- and over the age span of these animals. We demonstrated keys (Macaca fascicularis): We and others have described that that 60 to 70% of the animals were already infected by age rhesus monkeys can be naturally colonized by H. pylori. More one. So this infection occurs very early during the life of recently, in collaboration with Dr. Jim Reindel in Michigan, the animals, and the frequency increases with age. How- we have found that cynomolgus monkeys also can be natu- ever, 5 to 10% of the animals were H. pylori negative, an rally colonized by H. pylori. Another group in Japan also has observation similar to that in humans. Thus, some hosts described the presence of H. pylori in Japanese monkeys can develop defenses that can clear the infection, whereas (Macaca fuscata). the majority of them cannot. If some of the animals—and In the interest of time, I will discuss only our findings in this happens in humans, too; Barry Marshall was one of rhesus monkeys. As you will see, all observations are very them—can cure the infection spontaneously, then this is similar to those in humans, making this colonization a rel- an opportunity to use what nature can do on its own, and evant pre-clinical model. develop novel (immune or other) therapies to clear this in- Macroscopic gastritis can be observed during fection (for figures, see Dubois et al., Journal of Clinical videogastroduodenoscopies, allowing examination of the ap- Microbiology, 1995, 33:1492–1495). pearance of the stomach in rhesus monkeys. This demon- By sequencing a fragment of the 16S rRNA gene of H. strated that some animals can develop gastritis at a certain pylori, we have been able to demonstrate that a H. py- time, whereas at other times gastritis disappeared spontane- lori strain isolated from a monkey was 100% homologous ously. So there is a spontaneous fluctuation of macroscopic to two isolates from humans, and 99% homologous to an- gastritis, although microscopic gastritis appears to remain other isolate from humans (Drazek et al., Journal of constant (for figures, see Dubois et al., Gastroenterology, Clinical Microbiology, 1994, 32:1799–1804). The same 1991, 100:884–891). technique demonstrated that H. acinonyx was about 97% Microscopic gastritis is characterized by destruction or homologous to this isolate from monkey and also from damage of the superficial epithelial cells. Apical mucus of these two human isolates. H. felis, although very close to superficial epithelial cells is reduced or entirely lost, as H. pylori, is less related to it, and H. mustelae and H. demonstrated in H&E, gram, and PAS-stained slides in muridarium even less so. persistently colonized animals, although mucus in the crypts appears normal. By comparison, PAS-positive mu- Experimental Infection with cus is very dense on the superficial epithelial cells in H. Helicobacter Species pylori-negative animals. In the second part of this presentation, I would like to In addition, a few rhesus monkeys have been shown to discuss how can we use what we learned from these natu- develop gastroduodenal ulcers. However, these observations ral infections to develop reliable experimental models for were made before the discovery and description of H. py- H. pylori colonization. lori, and the biopsy blocks are no longer available. There- Models using Helicobacter species other than H. py- fore, we cannot prove that the animals were H. pylori posi- lori. Ferrets: Animals not colonized by H. mustelae are dif- tive, although the infiltration observed in the burgeoning ficult to find in this country, and only a few colonies are portion of the ulcer suggests colonization by this bacterium. breeding H. mustelae-free ferrets. Colonized animals can Finally, we recently observed a rhesus monkey with a be used to determine the effect of therapeutic immuniza- gastric ulcer accompanied by intestinal metaplasia in a tion, of novel treatments, or of superinfection with differ- gastric gland. Intestinal metaplasia was characterized by ent strains. For studies of the effect of colonization on the the presence of goblet-like cells, staining blue with the mucosa and/or on the immune response, one first has to Alcyan blue of the Genta stain. Genta stain combines H&E, cure the natural infection with . One also can a silver stain, and Alcyan blue to demonstrate concurrently use the model for studies of the prophylactic vaccination the structure of the gastric mucosa and the presence of H. on experimental colonization. pylori and of intestinal metaplasia. Importantly, intestinal Mice: Mice can be experimentally colonized by H. felis and metaplasia is considered a preneoplastic lesion in humans H. heilmannii, but it is much more difficult to successfully and therefore could represent a model for preneoplastic colonize them with H. pylori. The model of H. felis-infected lesions in rhesus monkeys. mice has been extensively used in the last 5 years, although Transmission electron microscopy shows that, although it tends to be less used now. H. felis is in close association the majority of H. pylori are floating in the mucus, a frac- with the surface, but it doesn’t attach to surface epithelial tion of the organisms are attached to the epithelial cells cells, which makes it different from H. pylori. However, the through the formation of pedestals similar those de- intense inflammation and the persistence of the coloniza- scribed in humans. At the same time, there is destruc- tion make this model a very useful one. The advantage of H. tion of the microvilli that are normally present on su- felis is that it can colonize 100% of inoculated mice, although perficial epithelial cells (for figures, see Dubois et al., the immune response and the organism are different.

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Models using H. pylori: Rats: Successful colonization Gnotobiotic piglets: Gnotobiotic piglets have been used of the stomach of the rat has been achieved, but most at- extensively by Dr. Steve Krakowka and Dr. Kate Eaton in tempts have been unsuccessful. Columbus, Ohio. They inoculated animals with H. pylori Mice: The mouse model of H. pylori infection received a and observed colonization in almost 100% of them. The lot of attention since the 1995 paper by the Iris-Chiron model was used to study the initial immune response and group from Siena, which described the fact that mice could also as a pre-clinical model to evaluate novel therapies for be colonized by H. pylori. This paper was important be- the treatment of H. pylori. cause most investigators had been unsuccessful in their Several factors limit the use of this model. First, pigs attempts to colonize mice with H. pylori. Drs. Martha grow very fast, and they rapidly overgrow the isolation sys- Marchetti, Paolo Ghiara, and Rino Rappuoli inoculated a tem; second, since they are gnotobiotic, their immune sys- mixture of multiple virulent strains to about 100 mice. tem is by definition quite different than that of normal pigs. About 5% of the mice were colonized. They then isolated Interestingly, pigs bred in a normal environment cannot the strains that had been successful in colonizing these mice be inoculated with H. pylori, possibly because the stomach and used these isolates to colonize other mice. After a few contains a number of other bacteria, and competition may passages, they selected a few strains that were able to con- prevent colonization by H. pylori. Thus, although this model sistently colonize a majority of mice. has a lot of interesting features, it also has, like any model, In England, the Glaxo group of Andrew McColm inocu- some limitations. lated multiple strains of mice with several H. pylori strains Nonhuman primates: Although some differences obvi- and were successful in colonizing a large proportion of ani- ously exist, nonhuman primates are the closest to humans mals. More recently, Adrian Lee in Australia initiated simi- genetically and immunologically. Chimpanzees and sev- lar experiments but immediately was successful in colo- eral macaques species have been used to study the effect nizing animals with a strain isolated from a patient with of experimental colonization by H. pylori. My laboratory duodenal ulcer that colonized most mice. He named the has focused on the rhesus monkey. In H. pylori-negative strain the “Sydney strain,” and he makes it available to animals, we observed no gastritis, just a few mononuclear any investigator upon request. By now the Sydney strain cells, and good preservation of the surface epithelial cells. has been widely used and published. In slides kindly pro- Within a few months of inoculation, persistent coloniza- vided by Adrian Lee, one can observe the effect of coloniza- tion by H. pylori is associated with marked gastritis with tion by the Sydney strain on the mouse gastric epithelium. mononuclear and polynuclear infiltration of the entire H. pylori is present in the lumen of gastric glands, where thickness of the lamina propria, some atrophy of the its presence is associated with mononuclear infiltration; glands, mucus depletion, and a few superficial erosions. however, no polymorphonuclear infiltrate is present, and These animals have been followed at regular intervals for inflammation is less than that observed in colonized pri- over a year, and gastritis similar to that seen in naturally mates and humans. The close association of these bacteria colonized animals was observed (for figures, see The Jour- with the surface of epithelial cells is best illustrated by nal of Infection and Immunity, 1996, 64:2885–2891). silver stains. Some of the H. pylori are observed very close In the initial colonization experiments, we used animals to microvilli, but, in contrast to the H. pylori observed in that had remained H. pylori negative despite having been humans and in monkeys, there is generally no destruction bred in a colony in which the majority of the adult animals of the microvilli, and no pedestal formation. In one case, were naturally colonized by H. pylori. We found that a num- however, there was specialized association. So it is possible ber of these animals could be colonized only transiently for that H. pylori can attach to the epithelial cells of mice. a few months before clearing the infection spontaneously. Based on these observations, the group of Jeff Gordon at We concluded that some animals may be resistant to colo- Washington University in St. Louis, Mo., has been creating nization, at least by some of the H. pylori strains. This is transgenic mice in which adhesion molecules are present on similar to the few cases of transient colonization that have superficial epithelial cells, thus allowing H. pylori to attach. been reported in humans following experimental or acci- A paper is currently in press, and this approach may be- dental ingestion of pure cultures of H. pylori. In an attempt come very fruitful in that it allows the study of specific at- to examine the question further, we used animals that had tachment mechanisms. When the genetic basis for attach- been naturally colonized by H. pylori and treated them with ment is determined, it may be possible to develop novel ap- a regimen of three antibiotics plus a proton pump inhibi- proaches to cure H. pylori colonization by preventing bacte- tor that is 95% effective in humans. All animals treated rial attachment. Indeed, attachment is believed to play a were cured. We observed that gastritis disappeared pro- pivotal role in persistence of H. pylori colonization. gressively over a 5-month period. After inoculating these Felines: After he observed that cats could be naturally animals with a mixture of seven human isolates, persis- colonized, Dr. Jim Fox and his group at MIT inoculated cats tent colonization was observed in all animals, antral gas- with H. pylori, and they observed persistent colonization tritis increased rapidly to a level similar to that observed in many animals. Thus, felines can be used as an animal before cure, and it remained at that level for the duration model to study H. pylori colonization. of the experiments. Corpus gastritis increased too, but more Furthermore, it may be useful for veterinarians to con- slowly. Plasma IgG and IgA also decreased progressively sider H. pylori colonization in the differential diagnosis of after cure of the colonization over the 5-month observation gastric ulcers. period. The drop of IgA was more precipitous than the one

601 Vol 48, No 6 Laboratory Animal Science December 1998 of IgG. Following inoculation, IgG jumped back within a tions, and that may explain why, once you go to the real month to a level similar to the pre-cure level, whereas IgA world and you don’t do a clinical study anymore, only 60% did not significantly rise over time. of the patients are cleared of the infection. As a result, Another interesting aspect of this model was that it al- strains resistant to many antibiotics have emerged. This is lowed us to inoculate the animals with a mixture of seven the case even with clarithromycin, which is the most effec- strains isolated from humans with various diseases: non- tive currently available, and it is estimated that ulcer dyspepsia, duodenal ulcer, or gastric ulcer. We used 14% of the strains colonizing humans in the U.S. are now random amplified PCR DNA fingerprinting to differenti- resistant to clarithromycin. The situation is even worse ate these seven strains and were able to determine which when considering resistance to metronidazole. strains were successful in colonizing the animals. In one So the use of animal models can help develop novel thera- animal, we found that at day 7, four of the seven strains pies. For instance, we have used the rhesus monkey model could be recovered. Over time, although strain C seemed to to study the effect of anti-adhesive therapy using oligosac- have some advantage, eventually strain B was the one that charides. This treatment could prevent the attachment of overwhelmed all the other ones, and it was the only one we H. pylori to the mucosa, or possibly detach H. pylori from could recover by 10 months. In two other animals, it seemed superficial epithelial cells, and allow the flushing of all the that strain C was winning over, but then by 3 months strain organisms by gastric peristalsis. B again became dominant. In the fourth animal, only strain Participant: There was a recent report of the presence A was found at day 7 post-inoculation, and in all subsequent of Helicobacter in dolphins. Does it seem possible? tests over the next 7 months. At 10 months’ post-inocula- Dr. Dubois: Yes, indeed, I have read this publication. tion, however, three-fourths of isolates were of the B type, This is intriguing. That would show that marine whereas the other fourth were of the A type (the type that can certainly be infected, and some also have been had predominated at all earlier times). This remarkable found to be infected with Helicobacter species different from outcome with this last monkey illustrated that a given strain H. pylori. The dolphin is an animal that is very close to can be maintained in an animal colonized by several strains, humans in many respects, and this observation warrants some of these being at a very low level (conservatively esti- further investigations. mated here as less than 5% of total population) for months, Participant: (Comments off mike.) and then suddenly emerge. (For figures, see Dubois et al. Dr. Dubois: Do you still have the blocks? Gastroenterology, 1999, 116:1-12) Participant: I will look for it. Dr. Dubois: It would be worthwhile, because you could Conclusions easily get sections, do a Genta stain, and try to detect The studies summarized above indicate that H. pylori Helicobacter in the slide. Thus, animal diseases of unex- organisms that appear to be identical to those observed in plained origin, especially any gastric disease or liver dis- humans can be isolated from animals. Thus, veterinarians ease, should be investigated to evaluate the possibility that can use the information collected in humans to further in- they are caused by the presence of such bacteria. This is a vestigate related pathology in animals. good example of cross-fertilization between clinical inves- Thank you. tigations and animal research, and each is then likely to Dr. Frenkel: Thank you, Dr. Dubois. Are there any ques- help the other, certainly leading to progress in veterinary tions? Please. medicine. Dr. Nakeeb: What have we learned from these animal Dr. Tabor: Do you know to what extent the original ob- models about the issue of resistance to therapy with anti- servations on H. hepaticus has been followed up in the de- biotics? velopment of animal models? Dr. Dubois: This is a very important question indeed. Dr. Dubois: Currently there are discussions on the sub- In humans, it is known that even the best therapies achieve ject at NCI-Frederick, because the people who breed mice permanent cure of the infection in only 90 to 95% of the want to eliminate H. hepaticus from the colony, whereas patients. This is similar to what we have observed in rhesus those who do research on H. hepaticus want to maintain monkeys. Although other models such as gnotobiotic pig- infected animals. There is currently one study trying to lets and mice can be useful for screening new treatments, infect monkeys with H. hepaticus. So far, intragastric, they may not always reflect results in the clinic. For in- intraduodenal, and intraperitoneal inoculations have been stance, mice can be cured permanently with one single unsuccessful. Dr. Jim Fox, on the other hand, is investigat- antibiotic, a regimen that is generally ineffective in hu- ing the possibility of using H. hepaticus-induced colitis as mans. These observations can be useful, however, because a model for inflammatory bowel disease. a better understanding of why it is so effective in animals Also, I have tried to trigger some interest in human pa- and not in humans could lead to progress in the treatment thologist liver specialists to do silver stains in patients with of this infection both in humans and in animals. diseases that appear different from A, B, or C hepatitis, Also, one has to realize that the quadruple therapy used and some investigations are ongoing. If positive, such an to clear H. pylori infection is not a pleasant treatment. It observation would represent an interesting development, consists of four medications; some of these medications can because bacteria have been excluded since viruses are rec- cause side effects such as nausea, and they can alter taste. ognized as the main cause of hepatitis. These organisms So a number of the patients are not taking all the medica- are not easy to find on histology section, and they are diffi-

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cult to grow in vitro; it would not be surprising if some of Helicobacter species by PCR using samples of liver and gall these had been missed. I tried to convince my hepatologist bladder of patients with hepatobiliary disease in Chile. colleagues to do silver stains in all the cases where they Dr. Barthold: To reinforce that, silver stains may also did biopsies, because it is such a simple thing to do, and pick up other Helicobacter species. they may discover something. Finally, Dr. Jim Fox just re- Dr. Dubois: Yes. ported that he was able to demonstrate the presence of

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