Animal models of Campylobacter jejuni colonization and disease and the lessons to be learned from similar Helicobacter pylori models

Summary, 57S 2. Introduction, 57S 3. Criteria for animal models, 58S 4. Naturally acquired human campylobacteriosis and volunteer studies, 58S 5. Naturally acquired campylobacteriosis in animals, 59S 6. Experimental challenge of laboratory animals, 59S 6.1 Non-human primate models, 59S 6.2 Ferret models, 59S 6.3 Pig models, 60S 6.4 Rodent models, 60S 6.5 Rabbit models, 61S 6.6 Chicken model of colonization, 61S 7. Can we learn anything from animal models of Helicobacter pylori? 62S 7.1 Manipulation of the bacterium, 62S 7.2 Use of related bacterial species and their natural hosts, 63S 7.3 Manipulation of the host, 63S 8. Conclusions, 64S 9. Acknowledgements, 64S 10. References, 64S The mechanisms by which Campylobacter jejuni induces disease in human beings remain unknown. Identification of campylobacter virulence factors requires appropriate animal models. Several in vivo models of disease have been described. Models in non-human primates are closest to the disease in humans but are excluded on ethical grounds. An oral ferret model, inducing mild diarrhoea, has proved promising in the investigation of virulence factors but may be difficult to use widely. The same is true for piglet models. Rabbit models reported generally involve surgical intervention and abnormal routes of administration, and are consequently of limited accessibility and usefulness. Several mouse models have recently been described; one model with orally challenged SCID mice induced diarrhoea. However, the frequency of disease was low. Moreover, immune-compromized mice would have restricted usefulness for immunological studies. The use of alternative challenge routes in mice, such as via the nasal mucosa, may be of value if the effects are reproducible. In summary, models of campylobacteriosis remain unavailable. Acceptable models of colonization have been developed; the most frequently used is the orally challenged one-day-old chick. Identification of bacterial factors important in the colonization of chickens may lead to the development of targeted intervention strategies to reduce contamination of the food chain by this pathogen. However, the value of models of avian colonization to investigate human infection has yet to be established. Comparison of animal models of the related pathogens C. jejuni and Helicobacter pylori has been informative. Although faced with similar problems, acceptable animal models of several disease manifestations of H. pylori infection are now available. This experience suggests that appropriate models of campylobacteriosis can be developed. Approaches involving the manipulation of both the pathogen and host are suggested which may enable the virulence of C. jejuni to become detectable in animal models in the near future. Campylobacter jejuni and Helicobacter pylori have many common morphological, biochemical and physiological features. The relationships between these bacteria and their hosts are, in many respects, comparable. They colonize the gastrointestinal tracts of human beings, both inducing clinical disease in some individuals, and acting like commensals in others. On the other hand, the diseases, ecological niches and pathophysiology of these infections are quite different. The importance of C. jejuni and H. pylori infections in human beings has been recognized for about the same length of time: since 1977 (66) and 1984 (49), respectively. Nevertheless, considerably more progress has been made on understanding the molecular basis of the latter than the former. One reason for this has been the development and extensive use of a spectrum of animal models of H. pylori colonization and disease. These models have recently been comprehensively reviewed (30; 20; 44). In contrast, there has been a paucity of similar models for C. jejuni (29). In this review the current status of animal models of C. jejuni infection, and what lessons can be learned to develop further in vivo models using H. pylori as a paradigm, will be discussed. Acceptable animal models of bacterial pathogens, such as C. jejuni, are essential to: a) confirm the identification and enable the characterization of putative bacterial virulence factors b) validate in vitro models of pathogenic mechanisms c) investigate the role of host mechanisms in the induction of clinical symptoms d) determine host immune responses and investigate surrogates of protective immunity e) measure and test the efficacy of therapeutic or prophylactic treatments such as vaccines or antibiotics Optimally, in vivo models, in order to be reproducible and verifiable, should utilize small laboratory animals, which are specific-pathogen free, genetically homogenous, of known immune status and widely available. Obviously the model should mimic the human disease as closely as possible. The route of challenge, infective dose, clinical symptoms, colonization characteristics and pathophysiology (79) are all important. Such characteristics should mimic those in human beings, if possible, without the need to compromize host defences. Because the model is likely to be required to investigate host immunity, either as a surrogate of vaccine development or as an indicator of immunopathological events, a well-characterized immune system is preferable. Similarly, if the model is to be used to investigate therapeutic agents, then the pharmacokinetics of these agents should be similar to that in human beings. Moreover, in the postgenomic era, now relevant for both C. jejuni and H. pylori, animal models will be an essential component of strategies to understand functional genomics. In particular such studies will be used to: a) screen defined bacterial mutants for reduced colonization and virulence properties b) positively select bacterial genes expressed upon infection (in vivo expression technology; IVET) c) generate biomass for transcriptosomes and proteomes to detect mRNA and all polypeptides expressed during infection d) produce immunological reagents to identify bacterial components that are antigenic during infection Thus, for current research purposes, models which can detect quantitative differences in the virulence and colonization potential of wild-type strains and their mutants would be optimal. With many bacterial pathogens the choice of animal model is predictable from the observation of naturally occurring disease. The clinical features of human campylobacteriosis are complicated. In developing countries, where it is assumed that exposure is frequent, infection rarely causes disease symptoms except in children under 2 years of age. However, in industrialized countries, asymptomatic infection is rare and C. jejuni infection is generally characterized by 1–3 d of prodromal fever, followed by 5–7 d of acute diarrhoea with watery or bloody stools (72). This epidemiological picture indicates that prior exposure and the subsequent host immunological response can significantly affect the clinical outcome of infection. Such naturally acquired immune responses appear to protect from disease but not necessarily from colonization. Campylobacter-associated enteritis is frequently accompanied by fever and severe abdominal pain. Normally the infection is self-limiting and excretion is terminated within 10–14 d. Because of this, little information is available about the histopathology of such infections in man. Susceptible individuals, especially those who are immunocompromized, may develop chronic infections. Occasionally, severe complications, including bacteraemia, extra-intestinal infections and abortion, can arise apparently from host failure to limit the infection to the gut. Immunopathological sequelae are also recognized including arthropathies and neuropathies. Campylobacteriosis is now recognized as a common antecedent to the polyneuropathy, Guillain–Barré Syndrome (2). Other postinfectious symptoms, potentially requiring investigation in animal models, have been reported, including irritable bowel syndrome (50). Without doubt, human volunteer studies provide the best model of the human disease. Oral challenge of human volunteers with as few as 800 cfu organisms can induce diarrhoea (9). Such studies have confirmed the virulence of at least one of the laboratory strains, 81176, in regular use for in vivo models. These studies indicate that there is no clear relationship between dose and symptoms. Volunteers reported a spectrum of disease symptoms, from asymptomatic or mild to dysenteric illness. The severity of illness appears to be strain related. In ill individuals histopathology, using sigmoidoscopy biopsies of the rectal mucosa, indicated inflammatory cell infiltrate, with neutrophils in the crypts and lymphoid cells in the muscularis mucosa. The induction of protective immunity from disease on rechallenge, at least by homologous bacteria, was confirmed in these volunteer studies (9). These results also suggest that the immune response may not protect from colonization, confirming the epidemiological observations from developing countries. With the recent attempts to develop whole cell vaccines against C. jejuni (65), this human volunteer model has become crucial (59). Such a model is essential for testing vaccine toxicity and efficacy, and detecting immunological responses. Campylobacters can be isolated from the faeces of a wide range of wild, domestic and laboratory species. However, such colonized animals rarely demonstrate clinical signs of disease. The reason for this paradox is unknown. This lack of disease may reflect infection with strains that lack appropriate virulence factors and are therefore non-pathogenic, or the development of protective immunity following frequent exposure, as in humans in the developing world, or a lack of susceptibility, for example, as a consequence of the absence of appropriate host receptors for toxins. Nevertheless, outbreaks of campylobacter-associated disease can occur in some animals (52; 29). Campylobacter-associated enteritis was reported in captive breeding groups of non-human primates (62). Diarrhoea in companion animals, especially puppies and kittens, is not infrequent and is a well-recognized source of human infection (72). Enteritis associated with campylobacter infection in young domestic animals, such as piglets, calves and lambs, has also been reported but the organism is also found in healthy animals (67). Descriptions of naturally occurring disease in small mammals of laboratory status are rare but endemic campylobacteriosis has been reported in hamsters and ferrets (52; 29) and recently in rats (54). The rarity of these events suggests as yet unrecognized physiological components of the disease. Although birds, especially poultry, are naturally colonized by huge numbers of organisms, disease in avians has rarely been convincingly described, excepting occasional outbreaks of hepatitis which appear to be associated with C. jejuni infection. One further exception to this is campylobacter-associated death and enteritis in young ostriches (78). A wide range of laboratory animals have been challenged with C. jejuni in attempts to identify reproducible models of disease and/or colonization. Many of these models have been reviewed previously (52; 29). These early studies clearly indicated that, following oral challenge, colonization was relatively easy to establish in most laboratory mammals, especially in young animals such as infant mice, puppies and piglets. This experimental colonization was usually chronic and resulted in long-term intermittent excretion. Moreover, colonization, regardless of extent, was almost always asymptomatic. Over the last 10 years or so efforts have concentrated on the development of models which demonstrate a disease outcome from infection. Several such models have now been described with non-human primates, piglets, ferrets, rabbits and mice. The closest model to the human infection is oral infection of non-human primates, especially Macaca nemestrima (63). Challenged infant M. nemestrima, but not M. fascicilaris, developed vomiting and diarrhoea, with blood in their stools. Most animals had a bacteraemia. The onset and duration of disease was similar to that in human beings. The gross pathology and histology of these infections was also similar to that seen in human beings with acute campylobacter-associated colitis. Experimental infection induced acquired, and apparently protective, immunity. This model has been used to test the safety and immunogenicity of whole-cell, killed campylobacter vaccines (5), providing evidence for human clinical trials. Although this is apparently a good model of the naturally occurring human disease, there are sufficient problems to preclude the use of non-human primate models. In particular, the availability of such animals for experimental purposes, the facilities and skills required to house these animals, the variability in their immune status and ethical considerations, all prevent routine use. One promising model has been developed in 3–6-week-old ferret kits challenged orally with C. jejuni (31; 7, 8). In a modified version of this model, at about 24 h postchallenge, animals excreted greenish mucoid stools, frequently with occult blood, occasionally accompanied by anorexia, dehydration and bacteraemia (19). These symptoms were self-limiting and the infection induced acquired immune responses which appeared to be partly protective. The model was used to assess the virulence potential of mutants of pspA, a gene associated with the expression of a C. jejuni pilus-like appendage (19), and cheY (85). Recently the oral ferret model was used to compare the virulence of C. jejuni strains 81–176 and NCTC 11168, the genome sequence strain (4). The results of this study indicated that NCTC 11168 was poorly virulent, which may be due to the absence of certain gene(s). Thus this model appears to be an extremely useful in vivo tool in the study of virulence factors. However, the relationship to human disease remains debatable. The use of tincture of opium to suppress peristalsis, the high doses (1010–1011 cfu), and the short-lived symptoms of soft, but not diarrhoeic, stools raises a number issues. More importantly, for most laboratories availability for routine testing, for example mutants, is restricted due to price, seasonal breeding and a lack of campylobacter-free ferret breeding colonies. International importation into the United Kingdom is subject to rabies control legislation. This, plus the general paucity of ferret immune reagents and the generally subjective analysis of the diarrhoea, means that the model requires considerable skill to establish and interpret. Because of the similarity between the human and porcine gastrointestinal tracts, pigs have frequently been used as animal models of human enteric infections. Although C. jejuni and the closely related organism, C. coli, colonize piglets, the outcome of infection is largely dependant on the status of the animal. Experimental infection of was generally asymptomatic animals developed diarrhoea which was occasionally bloody and mucoid and for to d and an abnormal histopathology are to the In piglets, clinical and effects were also These effects and of the and with watery diarrhoea from about 2 to These results suggest that piglets, in the absence of provide a useful model of The symptoms and histopathology appear to be with human disease. However, few studies have been to assess the of the Moreover, a of wild-type strains or mutants with this model has not yet been Many attempts have been made to induce disease in Although oral of C. jejuni induced colonization of the gastrointestinal tracts of both and infant mice, disease was rarely However, reported diarrhoea in about of mice orally challenged with clinical of C. This was accompanied by However, were to detect in challenged SCID mice, This lack of may be a of the of the but to indicate that the disease symptoms in the are not a consequence of the lack of host immune Thus such a model may be difficult to This oral mouse diarrhoea model requires in other laboratories it can be used to test bacterial virulence with such a of disease, it is due to gene would be Although the natural route of for C. jejuni is oral challenge, alternative routes to virulence potential have been of mice death within d The of with the dose, mouse strain and campylobacter doses of there was of mice with C. jejuni strain from mice challenged with this strain were from a of and extra-intestinal a infection, and colonization the Thus, this appears to be a model of acute infection involving extra-intestinal colonization and is also a model of immune and was used to assess vaccine efficacy The of to with such mice are the relationship of this model to the human disease remains The abnormal high dose and high are a of human disease symptoms. Moreover, the of this model in other laboratories has yet to be established. challenge of mice with to cfu bacteria generally resulted in infection with from and but no symptoms of disease in strains of mice of the challenge dose with virulence Such induced a in of diarrhoea, and These results were reproducible in laboratory and differences in virulence were between bacterial However, the of this model is especially as challenge with organisms induced significantly symptoms were several early of disease induced by experimental challenge of hamsters with C. jejuni The outcome of infection was diarrhoea and the disease in However, this outcome was either by surgical into the or by oral followed by with and The oral of C. jejuni to rabbits resulted in colonization but no evidence of disease in animals However, rabbits are an appropriate for animal models in which the bacteria can be into an The diarrhoea model, developed to investigate the of and the of by a C. jejuni in this for about h induced a occasionally diarrhoea within d inflammatory and death were also frequently The model differences between strains, and mutants Because the is the animals can be to investigate effects and may be to induce detectable immune responses. Such studies have indicated the development of against An alternative to the model is the test In this model of were into The to were with bacterial The were h The pathology an acute inflammatory The model which appears to be associated with host factors such as of and Thus this model may mimic some of the responses to infection. of these models considerable surgical skills and and are consequently not widely available. The relationship between the observations in these models to human disease is the route of is the effects of surgical are and the severe effects occasionally poorly reflect human disease. However, in particular, to be models of host and acute inflammatory responses as as the via Because numbers of organisms can be in a the model has also been useful for biomass to investigate in vivo expression of it is generally that C. jejuni has to colonize the avian gut. this colonization is both extensive and asymptomatic the organism appears to as a in this the has become an important model for the investigation of bacterial colonization factors. orally challenged with as few as cfu of of C. jejuni, colonization within d at of to cfu birds, to of were to experimental colonization. This colonization was chronic and to or Although the of colonization was the organisms were from the gastrointestinal as as the and that some infection The and of colonization in some models on the of the the challenge strain and the of laboratory of the strain and responses and of colonization by these had a in to rechallenge, that this immune response was partly protective models have also been developed for testing vaccine The results indicated that with killed whole cell or vaccines may induce this efficacy was not reproducible One with killed or vaccine was the or of to effects at avian in not appear to provide detectable from experimental the oral model has proved useful for the of the colonization potential of defined mutants and One C. jejuni a of colonization providing a quantitative This model has been useful for detecting the colonization of or so in this model have and and a reduced the colonization the effects were either the whole range of doses for a restricted The a model of campylobacter colonization in the avian gut. The relationship between this and colonization is unknown. Experimental challenge of chickens of all appears to induce an outcome to that during natural infection, C. jejuni like an avian In studies of strains from a of and at of cfu colonization is asymptomatic regardless of strain The reason such colonization not usually induce disease in is unknown. expression of bacterial virulence factors or lack of receptors for such factors. Nevertheless, there are some of disease associated with experimental infection, including diarrhoea, hepatitis and occasional death Moreover, there are occasional of between natural campylobacter infections and avian disease. a syndrome to death in naturally young ostriches and symptoms of in chickens have been described. Such symptoms may be related to immune and of the these are not yet reproducible. The Helicobacter a of bacterial species the gastrointestinal tracts of a number of However, the of is H. pylori which the human mucosa. This colonization is associated with chronic of the In about of colonized individuals this may progress to more disease, including lymphoid H. pylori appears to have a for human and non-human However, this organism has been usually by in vivo to colonize the of other orally challenged animals such as mice, and In animal models other Helicobacter either in their hosts or modified for hosts, have been developed. of such models H. in ferrets and H. in mice. these models have recently been reviewed (30; 20; model has With the exception of non-human primates, excluded as previously all these models poorly reflect the spectrum of human Nevertheless, most of the models have been used for the identification and of virulence investigation of host immune testing therapeutic agents and developing vaccines what can we learn from the development and use of these models which would be useful in similar campylobacter bacterial strains of H. pylori colonized piglets, which were or and This colonization was by in vivo Similarly, in vivo also the of an H. pylori to colonize the of mice appeared to have been from a of from the of several Because in vivo were and the was it likely that the organisms to host by the of several during the of the chronic infection. and therefore appears to be a of H. pylori and may be in the of this bacterial species. C. jejuni is also and may have similar mechanisms for host Although infection with this organism in human beings is usually in avians the colonization is more chronic for Thus in vivo may the of C. jejuni strains to colonize in vivo of strains of C. jejuni colonization by to This was in vitro that it was a consequence of However, colonization in this model not appear to be with virulence sufficient to disease, at least in In mammals the may be different. example in vivo of C. jejuni apparently virulence in challenged infant mice, to acute disease and death the such as and high usually to during their routine and from clinical it is not that are and that bacterial of certain physiological is C. jejuni expressed during colonization that in vivo may induce the of the expression of appropriate colonization factors which may have become during laboratory in vitro Such bacterial components may for the evidence that are more than laboratory strains in the SCID mouse model in vitro may need to be developed to the expression of appropriate bacterial factors. In summary, to both colonization and virulence should be Such the in vivo of C. jejuni strains appropriate animal models, the use of strains isolated by such as with laboratory and challenge with and/or studies many domestic and animals for evidence of of H. Although these studies the host of H. pylori for the of humans and non-human primates, a of other were These into H. and H. on the basis of and These alternative bacterial and their hosts, have a of information related to general of colonization and One of the most models used the H. organism, H. in the ferret the of colonization from that of H. pylori in human beings, this model has been used for vaccine studies. H. has the experimental of a host range than the H. Although natural colonization with this or similar organisms restricted use in animal models such as and H. has been in laboratory mice. In this model the disease was more severe than that induced by H. pylori proved for testing vaccine and and to investigate and are also a of Campylobacter species other than C. Many of these colonize the and can be associated with enteric disease. C. can enteritis in human beings but is frequently associated with infections in In such infections may lead to In this organism can also the inducing infection to in A similar outcome of infection is occasionally with C. jejuni in C. has an experimental model which has been used for investigation of the role of C. may therefore be used to model some of C. jejuni diseases, especially those associated with and infection. C. jejuni is frequently isolated in severe infections in developing of all C. jejuni were from blood in with of C. jejuni jejuni, 1. This suggested that C. jejuni is more than most C. jejuni C. is also a common of bacteraemia this virulence of these alternative is in animal models has yet to be and provide into the pathogenic mechanisms in has become from animal model studies of H. pylori, that associated with this infection are to both bacterial and host factors. The host factors appear to at the species and strain host species challenged with the same strain of Helicobacter developed significantly This research to be more with potential animal models. Recently has been with which challenged with H. pylori developed and Although used C. jejuni to investigate many of the then available laboratory animals, it may now be to this in host response are between strains or of strains, challenged with H. developed severe strains such as developed mild or strains such as few mouse strains have been with C. jejuni to indicate this is an appropriate for research but it is clear that some mice strains were more to this infection than likely that host immunological response to disease during Helicobacter infections Thus, the use of animals is an understanding of the disease the such studies are in mice where the and immunological are available. from some studies in SCID mice few attempts have been made to investigate the effects of immune on C. jejuni colonization and Such effects may be by of some of the bacterial strain manipulation suggested Although mice are now frequently used for Helicobacter colonization studies their usefulness for virulence studies is by the of the human disease. One between the human and animal infections is the lack of of H. pylori to mouse as a consequence of the absence of appropriate bacterial receptors on this mice were developed which expressed putative These mice apparently developed more severe disease This the of host to those defined features of the infection not naturally C. enteritis be to those animals for example for the may provide a further for animal models. A further for the mouse model is the use of human or non-human primate gastrointestinal into mice. This was used to investigate Helicobacter colonization studies that of human be for the investigation of bacterial Although or non-human has a restricted many mice can be from the can be for some this may provide a model for the of putative virulence factors of C. years of no reproducible animal model of human campylobacteriosis has been developed. This has attempts to identify and potential virulence factors and to on the by the recently genome Nevertheless, it is clear from a with similar studies to develop models for that we have not yet all to both the organism and the host should be Models of C. jejuni colonization are available. The most useful at is the oral model which is to investigate putative colonization factors and will become a tool for studies of functional genomics. Although the of avian colonization to that in the human has yet to be such studies may provide the information to understand colonization in the and enable appropriate intervention strategies to be developed to reduce campylobacter contamination in the food the of and for and and for