Pathophysiology of Campylobacter jejuni infections of humans

Microbes and Infection, 1, 1999, 1023−1033 © 1999 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved

Review

Pathophysiology of Campylobacter jejuni infections of humans Trudy M. Wassenaara, Martin J. Blaserb* a

Johannes Gutenberg University, Institute of Medical Microbiology and Hygiene, Hochhaus am Augustusplatz, D-55101 Mainz, Germany b Division of Infectious Diseases, Vanderbilt University School of Medicine and VA Medical Center, Nashville, Tennessee 37232, USA

ABSTRACT – Campylobacter jejuni and closely related organisms are major causes of human bacterial enteritis. These infections can lead to extraintestinal disease and severe long-term complications. Of these, neurological damage, apparently due to the immune response of the host, is the most striking. This review examines current knowledge of the pathophysiology of the organism. Diversity of C. jejuni isolates in genotypic and phenotypic characteristics now is recognized and clinically relevant examples are presented. Expected future directions are outlined. © 1999 E´ditions scientifiques et médicales Elsevier SAS Campylobacter / microbial pathogenesis / bacterial toxins / enteric diseases / emerging infections

1. Introduction More than twenty years ago, microbiologists became aware of the importance of the Gram-negative bacteria Campylobacter jejuni (figure 1) and the closely related C. coli as causative agents of human enteritis. Since then, substantial insights have been gained about the ecology, environmental spread, epidemiology, and pathogenicity of the organisms. Nevertheless, this knowledge has not (yet) resulted in a reduction in the incidence of campylobacteriosis in most countries, which is one of the goals of future research. Moreover, it now is recognized that Campylobacter infections pose a risk of extraintestinal sequelae that can be life-threatening. Better insight into the pathogenesis of these diseases may result in improved prevention and therapy. This review considers the heterogeneity amongst C. jejuni strains, and Campylobacterinduced diseases, the bacterial virulence factors that have been recognized to be pertinent, the host responses that have been discovered, and the directions in which future research should head.

2. Diversity of C. jejuni C. jejuni isolates are strikingly diverse compared to many other enteropathogens. Both phenotypic and genotypic diversity have been described (table I). The two generally applied serotyping schemes detect a wide diversity in serotypes: the extended heat-labile typing scheme

* Correspondence and reprints Microbes and Infection 1999, 1023-1033

of Lior [1] now recognizes over 100 serotypes of C. jejuni, C. coli, and C. lari. The heat-stable typing scheme of Penner and Hennessy, based on lipopolysaccharide O-antigens, detects more than 60 serotypes [2]. Genotypic diversity has been demonstrated by several genetic methods, e.g., pulsed-field gelelectrophoresis [3–8], Restriction fragment length polymorphism analysis of the PCR-amplified flagellin locus [9–13], random arbitrarily primed DNA PCR [14–17], ribotyping [18, 19] and amplified fragment length polymorphism [20, 21]. The bacterial subtypes recognized by one phenotypic or genotypic technique mostly do not correlate with those determined by the other techniques [22–30], which demonstrates even further the complexity within the species. Diversity between C. jejuni strains has also been observed at the phenotypic level for almost every characteristic that has been implied in pathogenicity. However, by comparison of clinical and nonclinical isolates, this phenotypic variation does not always coincide with observed or predicted differences in virulence. Examples of phenotypic diversity observed with C. jejuni isolates are plentiful and a selection is given here. Adherence to intestinal cells, as determined in vitro with cultured HeLa cells, as well as invasion into HEp-2 cells, varies among isolates of C. jejuni [31, 32]. Toxin production was found extremely variable, either in the type of toxin produced or in the degree of toxicity detected [33–38]. Most C. jejuni isolates are serum sensitive; however, different degrees of serum resistance have been demonstrated for C. jejuni isolates [39]. The potential to colonize chickens, which are a major vehicle of transmission to humans, also varies between C. jejuni strains [40]. 1023

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Figure 1. C. jejuni as visualized by transmission microscopy. Bar represents 1 µm. Photograph courtesy of Benjamin Fry.

The occurrence of this diversity may be partially explained by the observation that C. jejuni cells are naturally competent to take up DNA [41]. In an experimental setting, chromosomal DNA fragments carrying an antibiotic resistance marker were taken up and incorporated into the genome of different strains of C. jejuni [42]. Given the ubiquitous presence of C. jejuni in the intestines of animals, and the high levels of (multiple-strain) colonization, the possibilities of exchange of genetic material are substantial. Although it remains to be proven that transformation indeed occurs in a natural setting, the limited data available from genotyping naturally occurring strains suggest that it does occur [43]. Genetic diversity, with phenotypic diversity as a possible consequence, can furthermore result from genome plasticity. This term describes the phenomenom that the order of genes on the chromosome is not conserved between isolates of the same species. Gene-order plasticity 1024

Table I. Diversity of C. jejuni strains. Characteristic Heat-stable serotypes Heat-labile serotypes Other phenotypic characteristics

Genotypes

Described diversity > 60 different serotypes > 100 different serotypes Diversity in adherence properties, invasive properties, toxin production, serum resistance, chicken colonization potential, aerotolerance, temperature tolerance Genotypic diversity has been detected by PFGE, RAPD, ribotyping, PCR-RFLP, AFLP

PFGE, pulsed-field gel electrophoresis; RAPD, random arbitrarily primed DNA; RFLP, restriction fragment length polymorphism analysis; AFLP, amplified fragment length polymorphism. Microbes and Infection 1999, 1023-1033

Pathophysiology of Campylobacter jejuni infections of humans

has been described for Salmonella typhi [44] and Helicobacter pylori [45], and evidence is accumulating that genome plasticity occurs in C. jejuni as well [46]. This would explain the differences observed in the two genomic maps produced for C. jejuni strains [47, 48] and the complete genomic map deduced from the genomic sequence which is now available [49]. Intramolecular recombinations of large genomic fragments are the most likely mechanism for genome plasticity; however, the requirements and frequency of such recombinations are as yet unclear. From these observations it is clear that C. jejuni does not represent a limited number of clones, but that a wide variety of strains exists with many different characteristics. This point has often been ignored in the past, with confusing consequences for the interpretation of experimental data. The degree to which differences in virulence are related to the observed genetic diversity of C. jejuni remains to be established.

3. Campylobacter colonization of animals and humans C. jejuni and C. coli, for simplicity called C. jejuni here, can be regarded as normal biota in most mammals and birds. Although diarrhea may be incidentally observed in young animals (cats, dogs, calves), colonization in these hosts is generally harmless. In contrast, the accidental colonization of humans often results in disease. The symptoms observed in humans vary from slightly loose stools to a severe mucoid and bloody diarrhea. In most nonimmune hosts, an inflammatory diarrhea develops, which typically lasts for three to five days. An important question is what determines the difference in virulence of these organisms in humans compared with their usual animal hosts. To address this issue, pathogenic characteristics need to be identified and differentiated from colonization mechanisms. In general, animal models are critical for such studies. Unfortunately, no suitable animal models to mimic human campylobacteriosis are available for general use. The applied models are either a poor reflection of the human disease, e.g., the infant-mouse model, or the animals of the described model are not generally available, as in the case of ferrets or primates. In contrast, several colonization models have been developed in birds (chickens) and mammals. Experimental infection of rodents, such as rats and mice, has been useful for immunological studies. However, none of these models teach much about the events in humans, regardless of the high colonization levels obtained. Therefore, it is instructive to use clinical and epidemiological observations in humans to outline pathogenic events in C. jejuni infections.

4. Clue to C. jejuni pathogenesis from clinical epidemiologic observations in humans Information gained from natural (and experimental) C. jejuni infection of humans can aid in understanding Microbes and Infection 1999, 1023-1033

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pathogenesis. Although in volunteer challenges, inoculations with large doses (105 to 108 colony forming units) were required for high attack rates [50, 51], waterborne outbreaks indicate that low doses can induce illness, often on a wide scale [52]. From point-source outbreaks, we have learned that the incubation period may vary from one to seven days, although 24 to 48 h is usual [53, 54]. Taken together, these data indicate that most infections are due to exposure to relatively low numbers of organisms that must multiply in the host to achieve a clinically apparent outcome. From surveillance studies, it is clear that not all infections are symptomatic [55, 56]. Incidence of C. jejuni infection is 40 to 100 times higher in persons with AIDS than in the background population [57]. These data indicate that immunocompromised persons are more susceptible to C. jejuni, and suggest that in developed countries, the population is commonly exposed to low inocula of organisms that will not sicken most normal hosts but can regularly induce disease in compromised hosts. That a prodrome of fever and other constitutional symptoms may precede the onset of diarrheal disease by up to 48 h, suggests that the inflammatory effects of the infection precede the loss of intestinal epithelial cell function that causes the diarrhea. Examination of colonic biopsies shows an acute inflammatory response with infiltration of the epithelium and lamina propria with neutrophils and mononuclear cells. Among affected persons in developed countries, both leukocytes and erythrocytes are nearly always present in stools, indicating the universality of the inflammatory process, even when stools are watery and not grossly bloody. Thus, Campylobacter colitis and enteritis must be considered an inflammatory illness. Even without antibiotic treatment, the duration of illness usually is brief (less than one week), as is convalescent excretion of the organism (mean 16 days). In up to 20% of the cases a relapse, usually mild, may occur within a few days of spontaneous remission. Such cases may indicate an incomplete immune response. Immunocompromised hosts, especially those with immunoglobulin deficiency, often have severe, extraintestinal, prolonged, and relapsing illnesses, sometimes with permanent inability to clear the organism [58–60]. Such observations indicate the central role of acquired immunity in terminating the infection. That the intestinal illness may be accompanied by bacteremia and systemic infection, especially in persons at the extremes of age or who are immunocompromised, indicates the importance of the immune response for confining the infection to the intestine. An interesting, and presently unexplained feature, is the tendency for pregnant women to become bacteremic, usually with benign outcome. In developing countries, C. jejuni infection is extremely common in early childhood, in that 5 to 10 separate episodes may occur in the first two years of life [55, 56, 61]. Early life infections are most likely to be symptomatic; however, the infection-to-illness ratio and duration of colonization fall with age. By late childhood, few symptomatic infections occur. These trends are consistent with the progressive development of immunity, which correlates with specific serum anti-C. jejuni IgA [62]. Further1025

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more, these data indicate that C. jejuni strains must possess cross-reactive antigens that are immunogenic, suggesting that vaccine development is feasible. An important sequelum of C. jejuni infections is the development of the Guillain Barré Syndrome (GBS), an acute neurological disease marked by ascending paralysis (see [63] for a recent review). GBS results from demyelination of peripheral nerves, and has long been considered to be immunologically mediated. In the United States, an estimated 1 in 2 000 cases of C. jejuni infections is followed about 7 to 21 days later by GBS [63]. This timing suggests that the immune response to C. jejuni, rather than the acute toxicity of the infection, is responsible for the disease. For infections with serotype O:19 strains [64], the risk may be as high as 1 in 150 [63]. Such observations suggest that both strain and host differences are determinants of development of GBS following C. jejuni infection.

5. Bacterial virulence factors Since many human Campylobacter infections arise from ingestion of contaminated animal products (raw milk, poultry), we know that the organisms that harmlessly colonize animals can make humans ill. However, this phenomenon does not exclude the possibility that variation in virulence exists between bacterial populations or strains. It is possible that the pathotypes of C. jejuni are as diverse as those of Escherichia coli, but the genetic tools and model systems to differentiate strains according to pathogenic potential are not yet available. Diversity could in part explain the differences in disease observed in patients. However, from outbreak investigations and studies using human volunteers, we know that a single strain can produce no, or only mild, symptoms in one individual and severe illness in another [50, 51, 53, 54], observations indicating that infection outcome results from the interaction between host and bacterium. Of the potential virulence factors shared by all C. jejuni strains, flagella are the best studied. At the other extreme, toxin production varies substantially between strains as do invasive properties. The structure of lipopolysaccaride (LPS) varies between heat-stable serotypes, and the difference between serotypes in eliciting sequalae has already been mentioned. Finally, the extraintestinal spread of the bacteria depends on their ability to overcome defense mechanisms, which may be determined by bacterial factors as well as by the host. 5.1. Flagella

C. jejuni contains one or two polar flagella that cause the typical darting motility observed by microscopy, and the moist appearance of colonies on agar plates. As in other bacteria, the flagellar filament consists of multimers of the protein flagellin and is attached by the hook protein to a basal structure, which is embedded in the membrane and serves as a motor for rotation. The expression of flagella is an important virulence determinant, and subject to phase variation. Campylobacters must be flagellated to colonize and cause disease [50, 65–67]. The motility resulting from functional flagella presumably helps the bac1026

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teria overcome the clearing movement of peristalsis, and enables them to enter and cross the mucous layer covering the epithelium. Flagella also play an active role in the process of invasion into epithelial cells; thus motility, needed to reach the cells and establish close contact, is not the only consequence of flagellation [68, 69]. Flagella are a target of human immunity as demonstrated by the immunodominance of flagellin, and seroconversion occurring after infection [70, 71]. The resulting antibodies are protective against the homologous strain, but no, or only partial, cross-protection against other strains occurs [72]. The gene encoding flagellin is present on the genome in two copies, and the tandem arrangement of these genes, each with its own promoter, seems to be highly conserved between strains. 5.2. Toxin production

One of the mechanisms for virulence proposed in the early days of Campylobacter research was the production of toxins, which are important in diarrheal diseases due to Vibrio cholerae and Clostridium difficile, for example. Research concentrated on exotoxins (as opposed to the toxic effects of LPS) which were either called enterotoxins or cytotoxins. Although the terminology has not always been used consistently, in most cases, an enterotoxin represents a cytotonic toxin with homology to the enterotoxins of V. cholerae and E. coli, whereas cytotoxins refer to all other (proteinaceous) factors, secreted in the medium or surface-bound, that display toxicity to cells. There has been much effort to assess enterotoxin production by C. jejuni. Although cytotonic activity could be demonstrated for some strains at low titer, enterotoxin genes were not identified. It therefore is likely that the observed lowlevel cytotonic effects were caused by other toxin(s), possibly with modes of action similar to enterotoxin [73]. Many publications described toxic effects of Campylobacter products on cells, ascribing these to the action of one or several cytotoxins. Most investigations did not include controls based on the other investigations in the field; this lack of cross-referencing experiments has obfuscated the issue of toxin production. In a recent review [73] at least six different toxins, or classes of toxins, have been proposed to exist: a 70-kDa toxin active on HeLa cells but not on Vero or animal cell lines [74]; a cytotoxin active on HeLa and Vero cells [37]; a cytolethal distending toxin, Cdt [36]; a shiga-like toxin [75]; cytotoxin(s) that produce hemolytic effects; and a hepatotoxin [76]. Of these, the Cdt is the first toxin for which the genetic locus has been cloned and its sequence analyzed [77]. A strong homolog is present in E. coli, and the E. coli toxin has been shown to block HeLa cell division in the G2/M phase, ultimately leading to cell death [78]. A similar function has been proposed for C. jejuni Cdt [79]; however a direct role of Cdt in disease remains to be demonstrated. A seemingly conflicting observation is that all studied C. jejuni isolates contain the cdt gene locus, although differences in expression of Cdt exist [36, 77]. This observation suggests that the presence of cdt is not sufficient for expression; however, the genetic basis for the observed variation in expression has not yet been cleared. The strong conservation of the cdt locus in strains that are scored Cdt-negative in Microbes and Infection 1999, 1023-1033

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toxicity tests leaves the possibility open that toxicity is not the sole function of this locus. 5.3. Cell association

C. jejuni can either survive as free-living bacteria in the mucous layer, or invade the epithelium. Prior to invasion, the bacteria must attach to the epithelial cells. Fimbriaeassociated adhesins have not been found on campylobacters (but see below). Instead, adhesive properties have been ascribed to other structures on the surface of the bacteria. Flagella possess adherent properties in vitro [69, 80], corroborating earlier in vivo observations [67]. LPS also was found to play a role in adherence to INT-407 cells [81]. Four proteins isolated from C. jejuni outer membranes directly adhered to HEp-2 cells [82]. It is likely that one of these resembles an immunogenic protein, PEB1 [82, 83], which is identical to CBF1, found to be a major adhesin to epithelial cells [84]. Ablation of PEB1 affects adherence to and invasion of epithelial cells, as well as colonization of mice [85]. Binding to fibronectin, a component of the extracellular matrix, has been proposed as an early event in colonization, followed by the de novo protein synthesis required for invasion [86]. Recently, a surfaced-exposed C. jejuni protein (CadF) was shown to bind to fibronectin; the coding gene cadF shows homology to outer membrane proteins of other bacteria [86]. Although classical fimbriae are absent in C. jejuni, hairlike fimbrial structures have been observed after exposure of the bacteria to bile salts [87]. Whereas the gene encoding the subunit of these fimbrial filaments has not been identified, inactivation of pspA, a gene proposed to be involved in fimbriae biosynthesis, resulted in afimbrial mutants. This mutation had no effect on bacterial attachment in vitro, or on ferret colonization; however, the mutants were attenuated in causing disease in ferrets [87]. Thus, while several bacterial components have been shown to have adhesive properties (LPS, flagella, fimbrial filaments, surface-exposed proteins), the relative importance of these structures for adhesion in vivo as a requirement for colonization and invasion remains to be determined. 5.4. Invasion

Bacterial invasion of epithelial cells in vivo ultimately results in cellular injury and consequent loss of cellular function and diarrhea. Therefore, invasion has been proposed as an important pathogenic mechanism for C. jejuni, and experimental studies in monkeys [88] support this hypothesis. Numerous investigators have used the model of bacterial invasion of tissue culture cell lines to demonstrate strain differences in invasive properties, to determine effects of mutations on invasion, and to characterize the mechanism of invasion with the use of specific cellular inhibitors (for a recent review, see [89]). INT-407, HEp-2, and Caco-2 are the cell lines preferentially used. Several studies indicate that bacterial protein synthesis is required for invasion. The use of radiolabeling techniques permitted identification of at least 14 de novo synthesized proteins when bacteria were grown in the presence of cells [90]; nine of these were immunogenic, and antiseMicrobes and Infection 1999, 1023-1033

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rum directed against these proteins inhibited internalization [91]. However, there is no consensus about the mechanism of invasion. For example, in several (e.g., [92, 93]) but not in all studies [94, 95], cytochalasin inhibited invasion, due to inhibition of actin polymerization. Such differences could be bacterial strain-dependent, since the latter two studies used an identical strain, not previously studied. At least two mechanisms of invasion apparently are relevant to C. jejuni. Either microfilament-dependent pseudopods are formed, suggesting an entry mechanism similar to that of Salmonella typhimurium or enteropathogenic E. coli; however, the presence of condensed actin is not always inhibited by cytochalasin [92]. Alternatively, microfilament reorganisation does not take place: instead microtubules are involved in the uptake mechanism [94]. Both clathrin-coated pits [94] and clathrin-independent caveolae [96] have been implicated in endocytosis. Contrasting findings in such studies using different bacterial strains should be examined directly by exchange of strains involved in the studies. Two genetic approaches have been followed to identify bacterial genes involved in invasion. After the introduction of mutations (either site-directed or at random) a decrease in invasion potential can be assessed. Transposon mutagenesis has not yet been successful for C. jejuni, but an alternative high-frequency insertional mutagenesis strategy yielded five noninvasive, fully motile mutants (mutations affecting motility also affect invasion) [69]. The characterization of four of these mutants resulted in the identification of cheY, which inhibits adherence and invasion when the gene is duplicated, whereas adherence and invasion properties are increased when CheY is inactivated in C. jejuni strain 81–176 [97]. The role of CheY in pathogenesis is not completely understood. The second approach is that of complementation; noninvasive organisms have been transformed with genomic C. jejuni fragments and resulting invasiveness assessed. Since it is most likely that several gene products are required to permit invasion, the introduced genomic fragments must be large. One such an approach, using a cosmid library of C. jejuni DNA to transform a noninvasive E. coli host, has identified several invasive clones, but their characterization is not yet complete [98]. 5.5. Lipopolysaccharide

As with other Gram-negative bacteria, the lipid A component of C. jejuni LPS has endotoxic activity [99]. Systemic infection can lead to sepsis and shock, presumably due to LPS release. C. jejuni LPS possesses one of many possible O-antigens, based on the saccharide structure. These O-antigens either may represent polysaccharide or oligosaccharide side chains. Thus, as expected, C. jejuni strains may produce LPS, or LOS, or both, but the regulation of LPS/LOS chain length is not understood at present. Unlike for E. coli, the C. jejuni core oligosaccharides are quite diverse. The core oligosaccharides may contain N-acetylneuraminic (sialic) acid) [63, 100], which is uncommonly found in bacterial LPS, but is a common constituent of vertebrate glycoproteins and gangliosides. Interestingly, flagellar proteins of C. jejuni also may be sialylated [101]. As mentioned before, flagella and other 1027

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surface structures display adherence properties [67, 80]. The contribution of sialylation of C. jejuni flagella to these adherence properties has not yet been established. Interest in the role of LPS in C. jejuni pathogenesis has been stimulated by the recognition that the resemblance of core or side-chain saccharide structures to gangliosides [100] may be a key feature in the development of GBS and related neuropathies [64]. It now is believed that LOS structures on the surface of particular serotypes of C. jejuni (e.g., O:19 and O:41) can elicit an autoimmunemediated attack against host peripheral neural tissue via molecular mimicry. GBS is believed to be antibodymediated, which is consistent with the beneficial effect of plasmapheresis and the lack of benefit from corticosteroid treatment. In one model, binding of antibodies directed against ganglioside self-antigens on the surface of Schwann cells is an initial step in GBS pathogenesis. As a consequence of antibody binding, complement may be activated, forming transmembrane pores, leading to tissue damage and loss of neural activity [64]. Not all patients infected with the uncommon serotype O:19 develop GBS. Microheterogeneity of the LOS structure within serogroups could partly explain this phenomenon. For example, the core structure of two O:19 strains isolated from GBS patients differed from each other and from the O:19-type strain [100]. Since O:19 strains represent a highly clonal population [102, 103] with enhanced serum resistance [104], but only a small percent of persons infected with these strains develop GBS, host factors must be strong determinants for illness.

6. Factors determining deep infection The intracellular fate of C. jejuni once it crosses into intestinal cells has not been thoroughly examined. Most in vitro invasion studies have concentrated on the mode of entry, but the fate of bacteria and of the epithelial cells over the course of the interaction have not been described. Since C. jejuni is taken up by M cells [105], the in vitro observation of invasion in nonspecialized epithelial cells may not be particularly germane to their entry into deeper tissue components. The issue of bacteria expressing toxins, even at low titers, while surviving intracellularly has not been addressed experimentally, but could be relevant to pathogenesis. Once bacteria have invaded the epithelium by whichever means, they can reach deeper tissues as well [106]. Translocation of Campylobacter species across polarized Caco-2 cell monolayers may occur quickly [107]. Thus, Campylobacter cells could reach the bloodstream at an early phase of infection. However, since in general the thermophilic campylobacters are sensitive to complement-mediated lysis, both by the classical and the alternative pathways, they are rapidly killed even in the absence of specific antibodies [108]. Occasional C. jejuni strains that are highly resistant to serum have been detected, although the mechanisms for resistance have not been clarified [39]. Most circulating bacteria eventually will be phagocytosed by leukocytes or macrophages in the reticuloendot1028

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helial system. Survival of C. jejuni within monocytes has been observed in vitro for up to seven days [109], and this intracellular survival has been used to explain the complication of long-term bacteremia, since such a niche could protect the bacteria from complement-mediated killing. In that study, differences in survival rates between strains were not observed. However, sustained bacteremia is an uncommon consequence of C. jejuni infection except in immunocompromised hosts. More recent studies on the behavior of C. jejuni after phagocytosis in vitro used monocytes stimulated with cytokines and allowed to differentiate to an activated state before they were exposed to bacteria. Under such conditions, the macrophages were able to kill all tested strains with high efficiency [110], findings in agreement with the self-limiting nature of most C. jejuni infections.

7. Host responses The outcome of C. jejuni infection most often is spontaneous clearing. Association of C. jejuni with colonic epithelial cells induces the secretion of IL-8 [111], a proinflammatory cytokine that recruits phagocytic cells. Such innate host responses may help limit the extent of the infection but may be partially responsible for the symptoms. Asymptomatic colonization occurs in adults and children who have been highly exposed previously, indicating that the human host also can provide an adaptive barrier to limit disease development. This barrier is most likely dependent on the development of specific immunity, as described above, and is supported by the observation that immunocompromised patients generally suffer from more prolonged, severe, relapsing, and extraintestinal disease. Similar to other acute infectious diseases, serum titers of IgG, IgM, and IgA rise after infection and rapidly decline over the next two months. Studies indicate that a humoral response occurs in the intestine as well, but characterization has been limited [50, 112]. In addition to the humoral response, cellular immunity also is important to limit and clear the bacterial infection, as demonstrated by the higher incidence, severity and relapse of C. jejuni infections in HIV-infected patients [57]. One highly immunogenic protein of C. jejuni is flagellin; that this protein is encoded by two genes led to speculations of the role of diversity in immune avoidance. The two genes cannot only be alternatively expressed, but also can recombine to yield new combinations of epitopes; in addition, the protein is posttranslationally modified [113–115]. Each of these processes can lead to a change in antigenicity, but there is no direct evidence that this occurs during natural infection. Rather, the selflimiting nature of infection suggests that the organisms are not well-adapted to avoid the immune response of humans. Generally, only the breakdown of immune defenses or a specific local host deficiency (e.g., atheromatous aorta) can lead to prolonged, as opposed to transient, systemic infection. For instance, nearly all cases of bacteremia included in a long-term study were in patients with underlying conditions, of which HIV infection and other immunosuppression were most common [116]. Malnutrition and young age are other risk factors. Microbes and Infection 1999, 1023-1033

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In developing countries, children are repeatedly infected and ill in their first years (with a mortality not to be neglected), but at later ages become asymptomatic transient carriers after exposure [55, 56, 61]. A similar resistance may develop in adults who are highly exposed, such as farmers, regular consumers of raw milk, or slaughterhouse workers [53]. The number of natural infections required to induce full cross-protective immunity is not known.

8. Conclusions and perspectives We still understand little of the pathogenesis of C. jejuni infection. The consensus is that campylobacteriosis is an inflammatory disease in which the invasive and possible toxigenic properties of the organism are mainly responsible for enterocyte malfunction. However, current evidence of the role of toxins in pathogenesis is not convincing. At least for Cdt, the production of knockout mutants and their testing in an appropriate animal model seems to be a matter of time. Comparison of cdt expression amongst clinical isolates may further indicate the importance of this toxin in causing disease in humans. It is becoming increasingly clear that the interplay of C. jejuni with the host immune response is an important determinant of whether infection leads to disease, and the location, duration, and severity of consequences. Since epidemiologic observations in developing countries suggest that high-level exposure leads to immunity with protection from disease, the development of a vaccine is feasible [117]. However, in view of possible immunological sequelae (such as GBS), a vaccine should be carefully designed and a subunit vaccine may be preferable to a whole cell vaccine [118]. The phenotypic and genotypic diversity observed amongst C. jejuni isolates deserves renewed attention now that genotypic methods are becoming useful. It will be interesting to understand how the phenotypic diversity observed is reflected by genotypic differences. Detailed genotypic analysis of isolates from different reservoirs should indicate whether animals and humans share the same bacterial population, or whether sub-populations of C. jejuni exist in animals which are not, or only opportunistically, virulent to humans. Moreover, the long-term use of genotypic methods will indicate whether, and how frequently, genetic instability occurs in naturally occurring bacterial populations. A deeper understanding of the pathogenesis of human infection is expected now that the genomic sequence of C. jejuni is available. A UK consortium has taken the initiative to determine the complete chromosomal sequence of C. jejuni strain NTCT 11168 [49]. For example, if genetic homology is sufficient, toxin genes present on the chromosome could be identified (strain 11168 was described to produce cytotoxic activity to HeLa and Vero cells, but no enterotoxin could be detected [119]). Since the sequenced strain also is invasive, gene loci involved in invasion with homology to known invasion genes could be recognized. Similarly, the structural gene for the fimbrial appendices might be identified. In the next phase, Microbes and Infection 1999, 1023-1033

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putative functions of identified genes should be proven by mutagenesis, followed by specific phenotypic characterization, and complementation analysis. Such a combination of genetic methods with available in vitro and in vivo model systems is expected to be the major future approach to understanding the pathogenesis of C. jejuni.

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