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Differences in virulence attributes between cytolethal distending toxin positive and negative Campylobacter jejuni strains

¹ Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226014, India

² Department of Pathology, King George's Medical University, Lucknow 226001, India

Correspondence
Kashi Nath Prasad
knprasad{at}sgpgi.ac.in

Received 29 March 2007
Accepted 30 November 2007

Abbreviations: CDT, cytolethal distending toxin; CDT^–, CDT-negative; CDT⁺, CDT-positive; FCS, fetal calf serum.

The GenBank/EMBL/DDBJ accession number for the cdt* primers is DQ882648.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Campylobacter is a major cause of food-borne bacterial enteritis worldwide. It is estimated that the true Campylobacter infection rate in the USA and UK is as high as 1 % of the population per year (Linton et al., 1997). In the developing countries the infection rate varies from 5 to 20 % in children with diarrhoeal illness (Oberhelman & Taylor, 2000). The clinical spectrum ranges from a mild self-limiting, non-inflammatory diarrhoea to severe inflammatory bloody diarrhoea with abdominal pain and fever (Ketley, 1997; Ismaeel et al., 2005).

The pathogenesis of Campylobacter infection is multifactorial and complex. Various virulence factors such as adherence, invasive capabilities and toxin production have been implicated (Konkel et al., 2001). Adherence of bacteria to the epithelial cell surface is probably an important determinant for colonization and may increase the local concentration of secreted bacterial products; however, invasion is accompanied by pronounced cytopathic effects and is thought to be a primary mechanism of damage to the colonic mucosa, leading to inflammation (Russell et al., 1993; Wooldridge & Ketley, 1997). Campylobacter strains invade the gastrointestinal tract, and damaged epithelial cells are exfoliated into the lumen (Russell et al., 1993). Invaded cells become swollen and rounded, indicating changes in ion transport regulators, probably due to the production of cytotoxins (Friis et al., 2005). Toxins have been considered important factors for the pathogenesis of Campylobacter infection. The best-characterized of the toxins attributed to Campylobacter spp. is cytolethal distending toxin (CDT). The C. jejuni cdt operon consists of three adjacent genes, cdtA, cdtB and cdtC, that encode proteins with predicted molecular masses of 27, 29 and 20 kDa, respectively.

CdtB is proposed to be the enzymically active subunit of the holotoxin (Lara-Tejero & Galan, 2001; Smith & Bayles, 2006). Recent studies have shown that the cdtB gene is commonly present in Campylobacter jejuni rather than Campylobacter coli (Bang et al., 2001; Eyigor et al., 1999). CDT belongs to a family of bacterial protein toxins with the ability to block the cell cycle process, with resulting cell cycle arrest and cell death (De Rycke & Oswald, 2001). However, differences in the adherence and invasion capabilities of CDT-positive (CDT⁺) and negative (CDT^–) strains of C. jejuni have not been studied. The strains of Escherichia coli, Shigella spp. and Campylobacter spp. that show CDT activity are associated with diarrhoeal disease (Bouzari et al., 1992; Johnson & Lior, 1987, 1988). Several in vitro and in vivo models have been described to study the role of CDT in the pathogenesis of E. coli and Shigella infection (Johnson & Lior, 1987; Okuda et al., 1997).

Although CDT production is more common in C. jejuni strains isolated from patients with inflammatory diarrhoea, how CDT⁺ and CDT^– strains differ in their virulence properties is not yet known. The present study was planned to determine the difference between CDT⁺ and CDT^– C. jejuni strains in their adherence and invasive properties on the HeLa cell line and biological activities in the suckling mouse model.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains. A total of 49 Campylobacter strains (41 C. jejuni, 7 C. coli and 1 C. lari) isolated from the rural community of Lucknow district, India, were tested for the presence of the cdtB gene. Details of the species identification of these isolates have been described (Jain et al., 2005). Ten C. jejuni strains (five CDT^– and five randomly selected CDT⁺ C. jejuni strains) were subjected to adherence and invasion assay on HeLa cells. Culture supernatants from these C. jejuni strains were tested for CDT activity on HeLa cells.

Cell line. The HeLa (human cervical adenocarcinoma) cell line was used for the adherence, invasion and cytotoxin assay of C. jejuni strains. The cell line was maintained in Eagle's minimum essential medium (MEM) with 10 % fetal calf serum (FCS).

Detection of cdtB in Campylobacter species by PCR. Genomic DNA was extracted from Campylobacter strains by the alkaline lysis method (Sambrook et al., 1989). The DNA was subjected to PCR using specific primers for the cdtB gene. cdtB* gene primers, developed in our laboratory using Biosoftware (Primer Premier, PREMIER Biosoft International) were as follows: upstream 5'-GCGTTGATGTAGGAGCTAATCGTG-3' and downstream 5'-GGTTGATCGCGTTGAGTTCGTT-3'. The primer sequences have been submitted to GenBank (accession no. DQ882648). Another set of primers (upstream 5'-GTTAAAATCCCCTGCTATCAACCA-3' and the downstream 5'-GTTGGCACTTGGAATTTGCAAGGC-3') for the cdtB gene reported by Bang et al. (2001) was used to confirm the results of our laboratory-developed cdtB* gene primers.

All PCR amplifications were performed in a 50 µl volume containing 10x assay buffer, 200 µM each dATP, dCTP, dGTP, dTTP, 0.1 µM each primer, 1.5 units Taq DNA polymerase (Bangalore Genei, India) for 30 cycles with the following programs. cdtB* primers: 1 min at 94 °C, 1 min at 60 °C, 1 min at 74 °C followed by extension of 10 min at 74 °C; cdtB primers (Bang et al., 2001): 1 min at 94 °C, 2 min at 42 °C, 3 min at 72 °C and extension of 10 min at 72 °C.

Adherence and invasion assay. Tissue culture plates (Nunc, 24-well) were seeded with 5x10⁴ cells ml^–1. The plates were incubated at 37 °C in a humidified 5 % CO₂ incubator (Sanyo) until semi-confluent monolayers were obtained. Prior to the experiment, the cells were washed twice with phosphate-buffered saline (PBS) and incubated with MEM containing 2 % FCS.

Suspensions of CDT⁺ and CDT^– C. jejuni strains were prepared in PBS (pH 7.0) from cultures grown on charcoal cefoperazone deoxycholate agar (CCDA) under microaerophilic conditions at 37 °C for 48 h. The suspension was centrifuged at 10 000 g for 10 min at 4 °C. The bacterial pellet was resuspended in MEM with 2 % FCS, and the inoculum was adjusted to 10⁷–10⁸ bacteria ml^–1 by measuring the OD₆₀₀. The adherence and invasion assays were done by methods described earlier (Konkel et al., 1992; Prasad et al., 1996). The inoculum (0.5 ml) was added to the HeLa cell monolayer in duplicate. The plates were incubated at 37 °C in a 5 % CO₂ incubator for 3 h. The monolayers were washed three times with MEM containing 2 % FCS. In one of the duplicate wells, gentamicin (250 µg ml^–1) was added and plates were further incubated for 3 h. C. jejuni being sensitive to gentamicin, the antibiotic killed the bacteria that adhered to the surface of the tissue culture, while the bacteria that had already invaded and internalized remained unaffected. In the second well, medium without antibiotic was added to determine the number of bacteria that had adhered to and invaded the cell lines. The monolayers were lysed using 0.01 % Triton X-100. The lysed monolayer suspensions were diluted (10^–1 to 10^–4) in PBS, and 100 µl of each dilution was uniformly plated on CCDA. The number of viable bacteria was determined by counting the c.f.u. on the plates, multiplied by the dilution factor. Viable bacteria recovered from the first well (with gentamicin) and from the second well (without gentamicin) represented the intracellular (invasion) and the extracellular+intracellular (adherence+invasion) bacterial counts, respectively. The adherence was calculated by the formula [(c.f.u. ml^–1 from the second well at particular dilution) – (c.f.u. ml^–1 from the first well at the same dilution)xdilution factor].

Cytotoxicity assay on HeLa cells. Cell-free bacterial culture supernatants from all the strains were prepared according to the method described by Florin & Antillon (1992) with minor modifications. Briefly, each of five CDT⁺ and CDT^– strains was harvested from CCDA plates into MEM cell culture medium. The volume of medium used was adjusted so that the OD₆₀₀ of the bacterial suspension was 0.125 (2x10⁸ c.f.u. ml^–1). The bacterial count measured by optical density was subsequently confirmed by colony counts using different dilutions on solid media. Bacterial strains suspended in MEM tissue culture medium were lysed by sonication (4x30 s bursts with 30 s intervals between each burst). Cell debris and unlysed bacteria were then removed by centrifugation at 10 000 r.p.m. for 20 min at 5 °C and filter (0.22 µm) sterilized. The culture supernatant was then dialysed 10-fold using polyethylene glycol 600 (SRL, India) and finally the concentrated supernatant was collected and stored at –20 °C until use.

HeLa cells were seeded into 24-well tissue culture plates (Nunc) at a density of 2x10⁴ cells per well in 0.5 ml medium. Doubling dilutions of culture filtrates were prepared in MEM and 0.5 ml of each dilution was added to the HeLa cells and incubated for 3 days at 37 °C in an atmosphere of 5 % CO₂. The cells were examined under an inverted microscope every 24 h up to 72 h for demonstration of morphological changes. CDT activity titre was defined as the reciprocal of the highest dilution that produced distension in >50 % of the cells. Adherence, invasion and cytotoxicity experiments were done in triplicate and the mean results were considered for further analysis.

Suckling mice and sample inoculation. Suckling mice (2 days old) were obtained from the animal house, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow. Prior to the sample inoculation, mice were starved by separating them from their mother for 10 h at 23 °C. A total of 20 suckling mice were included in the study and divided into two groups of 10. Each group of mice was inoculated with culture supernatants of five CDT⁺ C. jejuni and five CDT^– C. jejuni strains, each strain in duplicate animals. Culture supernatant mixed with Evans blue dye (0.04 %, v/v) was administered into the stomach of each suckling mouse. The mice were observed for the development of diarrhoea and were sacrificed within 48 h.

Histopathology. Parts of the small and large intestines (stomach, jejunum, ileum and colon) were dissected from the suckling mice and fixed in 10 % formalin. Multiple representative areas from each anatomical region were processed for paraffin-embedded sections; 4 µm sections were stained with haematoxylin and eosin for morphological examination. Histopathological evaluation was done blinded to the inoculation subgroups. The extent of inflammation and mucosal damage was categorized as mild (+), moderate (++) or severe (+++).

Statistical analysis. All the statistical analyses were done with SPSS statistical software, version 12.0 (SPSS Inc.). Comparison of adherence and invasion assay among CDT⁺ and CDT^– C. jejuni strains was done using the Kruskal–Wallis test.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Detection of the cdtB gene in Campylobacter isolates by PCR

Among the 41 C. jejuni, 7 C. coli and 1 C. lari isolates, the cdtB gene was present in 36 C. jejuni and 1 C. coli; the lone C. lari strain was negative for cdtB (Table 1). Significantly higher proportions of C. jejuni strains contained cdtB as compared to C. coli (36/41 vs 1/7; P<0.001). The results of both sets of primers (cdtB* and cdtB) were identical.

Intragastric challenge of suckling mice with culture supernatant containing CDT activity

Watery diarrhoea within 24 h was observed in 6 of the 10 mice inoculated with supernatant containing CDT activity; the remaining 4 mice had no sign or symptoms of diarrhoea. Mice inoculated with supernatant lacking CDT activity did not show any sign of diarrhoea.

Histopathology

Histology was done on all 10 mice. A varying degree of inflammatory reaction was observed in different parts of the gastrointestinal tract. The changes were categorized as mild when a mild inflammatory infiltrate with no destructive changes was present. Moderate inflammation was defined as presence of moderate inflammatory exudates with partial necrosis of mucosa and luminal exudates. Tissues were categorized as having severe inflammation when the mucosa was almost completely denuded, with a prominent inflammatory infiltrate, necrosis and luminal exudates.

A remarkable change in pathology was observed in gastrointestinal tissues of the mice challenged with supernatant containing CDT⁺ activity. Moderate to severe inflammation was observed in all parts of the gastrointestinal tract in six mice, while in the remaining four, the stomach was spared. The sections from stomach showed partial denudation of mucosa, and fibrinous exudates in the lumen, with prominent mixed inflammatory infiltrate extending to the submucosa (Fig. 1b). The jejunum showed disruption of the mucosal lining with intense panmural inflammatory infiltration (Fig. 1d). In the ileum, the mucosa was mostly denuded, broken glands were evident in the lumen and the lining was replaced by macrophages mixed with neutrophils and lymphocytes. Panmural inflammation was present (Fig. 1f). Sections from colon also showed denuded mucosa and mucosal inflammation with macrophages, neutrophils and lymphocytes; luminal necrotic exudate was also evident (Fig. 1h). All 10 mice inoculated with supernatant lacking CDT activity showed no significant pathology in the stomach, jejunum or ileum, with mild inflammation in the colon in four animals (Fig. 1a, c, e, g). The destructive damage and intense inflammatory response noted in intestinal tissues of mice challenged with supernatant containing CDT⁺ activity was not evident in any member of the CDT^– group. The pathological changes in tissues of the large and small intestine of mice are summarized in Table 3.

View larger version (93K): [in this window] [in a new window]   Fig. 1. Histopathological evaluation by haematoxylin and eosin staining of the gastrointestinal tract of mice challenged with culture supernatants of CDT+ and CDT– C. jejuni strains. (a, b) Stomach: (a) CDT–, showing intact mucosa, minimal inflammation; (b) CDT+, showing partially denuded mucosa, fibrinous exudates in the lumen and mixed inflammatory infiltration. (c, d) Jejunum: (c) CDT–, showing normal jejunal morphology; (d) CDT+, showing partially denuded mucosa with intense inflammatory infiltrate. (e, f) Ileum: (e) CDT–, showing normal ileal morphology; (f) CDT+ showing denuded mucosa with broken glands in lumen and replaced by macrophages, mixed with neutrophils and lymphocytes. (g, h) Colon: (g) CDT–, showing intact mucosa with mild inflammation; (h) CDT+, showing denuded mucosa with a few remnant glands (arrow), inflammation with macrophages, neutrophils and lymphocytes and luminal necrotic exudates. Magnification: (g) x125; other panels x525).

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The above findings suggest that the cdtB gene is present in the majority of C. jejuni strains. To prove the role of cdtB as virulence marker we assayed the adherence and invasion properties of cdtB⁺ and cdtB^– C. jejuni strains on HeLa cells. Interestingly, all cdtB⁺ strains adhered to (range 8.0x10² to 7.2x10⁴ c.f.u. ml^–1, mean 2.7x10⁴±3.5x10⁴) and invaded (range 1.2x10² to 3.1x10³ c.f.u. ml^–1, mean 1.0x10³±1.3x10³) HeLa cells, while the level of adherence (range 1.3x10¹ to 5.4x10² c.f.u. ml^–1, mean 2.7x10²±1.9x10²) and invasion (range 100 to 0.7x10² c.f.u. ml^–1, mean 1.4x10¹±3.1x10¹) by cdtB^– C. jejuni strains was significantly lower (only one CDT^– strain showed a low level of invasion). Biswas et al. (2006) recently reported that there was no difference in adhesion to HeLa cells of cdt mutants compared to wild-type, but that they did have reduced levels of invasion. The ability of mutants to colonize birds either directly or by horizontal transfer was unchanged. The discrepancy in adherence properties between the studies might be related to the bacterial strains, as in the present study strains deficient in cdtB genes were used. All cdtB⁺ strains also showed cytotoxicity to HeLa cells, while none of the cdtB^– strains did so. The greater level of adherence, invasion and cytotoxicity of cdtB⁺ versus cdtB^– C. jejuni strains indicates the role of CDT as a virulence marker of this pathogen. Different cell lines such as CHO, J774, Int 407 and Vero have been used to determine the virulence properties of C. jejuni, with variable results. One of the important explanations for this variation was that the cytotoxic effects on HeLa cells were more prominent in freshly seeded cells than in semi-confluent monolayers (Lee et al., 2000). Freshly seeded cells may undergo active growth and protein synthesis and therefore may be more susceptible to the toxin. In the present study all the cdtB⁺ strains caused rounding in freshly seeded HeLa cells, suggesting that HeLa cells (freshly seeded) are good markers for detection of cytotoxic activity of C. jejuni strains.

CDT induces various types of responses (such as elongation, rounding and lethality) in HeLa cells and the mechanism(s) responsible for these responses is (are) still not known. Various in vivo models have been described to investigate the mechanism of pathogenesis associated with C. jejuni infection. In the present study, we chose the suckling mouse model because it is economical and various types of response can be monitored in the whole animal. We followed the protocol recommended by Okuda et al. (1997), who found that a 10 h starvation period of mice was found essential to obtain reproducible results. Watery secretion was observed in 6 of the 10 mice inoculated with supernatant having CDT activity while none of the animals administered supernatant lacking CDT activity had diarrhoea. These six mice died within 24 h, after commencement of diarrhoea, and were dissected immediately to collect tissues. All other mice were sacrificed at 48 h.

Although diarrhoea was not observed clinically in 4 of the 10 mice in the CDT⁺ group, remarkable changes in pathology were observed in tissues of all mice administered supernatant of CDT⁺ C. jejuni strains. Panmural inflammation with mucosal denudation and necrosis affecting the jejunum, ileum and colon was observed in all cases. The mice inoculated with supernatant lacking CDT activity only had mild inflammation in the descending colon, while the rest of the gastrointestinal tissues had normal histology. Similar studies have reported that CDT-producing E. coli and Campylobacter spp. induced inflammatory responses in the small intestines of rabbits (Bouzari et al., 1992) and rats (Johnson & Lior, 1988), respectively. However, Okuda et al. (1997) reported that the tissues of the small intestines remained apparently intact. The discrepancy in the results may be due to differences in the sensitivities of the animals. Literature suggests that heat-stable and heat-labile E. coli enterotoxin and cholera toxin stimulate the mucosal cells of the small intestine through enzymic actions without tissue damage (Kaper et al., 1995; Knoop & Owens, 1992; Spangler, 1992). This appears to be the first study demonstrating biological activity of culture supernatant of CDT⁺ and CDT^– C. jejuni in the suckling mouse model. We also for the first time observed histopathological evidence of damage in the stomach and jejunum due to CDT.

In conclusion, the cdtB gene was more frequently present in C. jejuni than in C. coli. The presence of cdtB in C. jejuni was associated with increased adherence to, invasion of and cytotoxicity towards HeLa cells. The major pathological changes in the colons of mice administered supernatant containing C. jejuni CDT suggest that CDT is an important virulence attribute and the colon is its major target site.

	ACKNOWLEDGEMENTS

 
Deepika Jain acknowledges financial assistance from the Council of Scientific and Industrial Research, New Delhi, India. This study was supported by a grant from the Indian Council of Medical Research, New Delhi, India (Project no. 5/3/3/2/2000-ECD-I).

	REFERENCES

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Al-Mahmeed, A., Senok, A. C., Ismaeel, A. Y., Bindayna, K. M., Tabbara, K. S. & Botta, G. A. (2006). Clinical relevance of virulence genes in Campylobacter jejuni isolated in Bahrain. J Med Microbiol 55, 839–843.[Abstract/Free Full Text]

Bang, D. D., Scheutz, F., Ahrens, P., Pedersen, K., Blom, J. & Madsen, M. (2001). Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers. J Med Microbiol 50, 1087–1094.[Abstract/Free Full Text]

Biswas, D., Fernando, U., Reiman, C., Willson, P., Potter, A. & Allan, B. (2006). Effect of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in cultured cells and in colonization of the chicken gut. Avian Dis 50, 586–593.[CrossRef][Medline]

Bouzari, S., Vatsala, B. R. & Varghese, A. (1992). In vitro adherence property of cytolethal distending toxin (CLDT) producing EPEC strains and effect of the toxin on rabbit intestine. Microb Pathog 12, 153–157.[CrossRef][Medline]

Dassanayake, R. P., Zhou, Y., Hinkley, S., Stryker, C. J., Plauche, G., Borda, J. T., Sestak, K. & Duhamel, G. E. (2005). Characterization of cytolethal distending toxin of Campylobacter species isolated from captive macaque monkeys. J Clin Microbiol 43, 641–649.[Abstract/Free Full Text]

De Rycke, J. & Oswald, E. (2001). Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation. FEMS Microbiol Lett 203, 141–148.[Medline]

Eyigor, A., Dawson, K. A., Langlois, B. E. & Pickett, C. L. (1999). Detection of cytolethal distending toxin activity and cdt genes in Campylobacter spp. isolated from chicken carcasses. Appl Environ Microbiol 65, 1501–1505.[Abstract/Free Full Text]

Florin, I. & Antillon, F. (1992). Production of enterotoxin and cytotoxin in Campylobacter jejuni strains isolated in Costa Rica. J Med Microbiol 37, 22–29.[Abstract/Free Full Text]

Friis, L. M., Pin, C., Pearson, B. M. & Wells, J. M. (2005). In vitro cell culture methods for investigating Campylobacter invasion mechanisms. J Microbiol Methods 61, 145–160.[CrossRef][Medline]

Ismaeel, A. Y., Senok, A. C., Bindayna, K. M., Bakhiet, M., Al Mahmeed, A., Yousif, A. Q. & Botta, G. A. (2005). Effect of antibiotic subinhibitory concentration on cytolethal distending toxin production by Campylobacter jejuni. J Infect 51, 144–149.[CrossRef][Medline]

Jain, D., Sinha, S., Prasad, K. N. & Pandey, C. M. (2005). Campylobacter species and drug resistance in a north Indian rural community. Trans R Soc Trop Med Hyg 99, 207–214.[CrossRef][Medline]

Johnson, W. M. & Lior, H. (1987). Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of Shigella spp. FEMS Microbiol Lett 48, 235–238.[CrossRef]

Johnson, W. M. & Lior, H. (1988). A new heat-labile cytolethal distending toxin (CLDT) produced by Campylobacter spp. Microb Pathog 4, 115–126.[CrossRef][Medline]

Kaper, J. B., Morris, J. G., Jr & Levine, M. M. (1995). Cholera. Clin Microbiol Rev 8, 48–86.[Abstract]

Ketley, J. M. (1997). Pathogenesis of enteric infection by Campylobacter. Microbiology 143, 5–21.[Free Full Text]

Knoop, F. C. & Owens, M. (1992). Pharmacologic action of Escherichia coli heat stable (STa) enterotoxin. J Pharmacol Toxicol Methods 28, 67–72.[Medline]

Konkel, M. E., Corwin, M. D., Joens, L. A. & Cieplak, W. (1992). Factors that influence the interaction of Campylobacter jejuni with cultured mammalian cells. J Med Microbiol 37, 30–37.[Abstract/Free Full Text]

Konkel, M. E., Monteville, M. R., Rivera-Amill, V. & Joens, L. A. (2001). The pathogenesis of Campylobacter jejuni-mediated enteritis. Curr Issues Intest Microbiol 2, 55–71.[Medline]

Lara-Tejero, M. & Galan, J. E. (2001). CdtA, CdtB, and CdtC form a tripartite complex that is required for cytolethal distending toxin activity. Infect Immun 69, 4358–4365.[Abstract/Free Full Text]

Lee, A., Smith, S. C. & Coloe, P. J. (2000). Detection of a novel Campylobacter cytotoxin. J Appl Microbiol 89, 719–725.[CrossRef][Medline]

Linton, D., Lawson, A. J., Owen, R. J. & Stanley, J. (1997). PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. J Clin Microbiol 35, 2568–2572.[Abstract]

Oberhelman, R. A. & Taylor, D. N. (2000). Campylobacter Infections in Developing Countries, 2nd edn, pp. 139–153. Edited by I. Nachamkin & M. J. Blaser. Washington, DC: ASM Press.

Okuda, J., Fukumoto, M., Takeda, Y. & Nishibuchi, M. (1997). Examination of diarrheagenicity of cytolethal distending toxin; suckling mouse response on the products of the cdtABC genes in Shigella dysenteriae. Infect Immun 65, 428–433.[Abstract]

Prasad, K. N., Dhole, T. N. & Ayyagari, A. (1996). Adherence, invasion and cytotoxin assay of Campylobacter jejuni in HeLa and HEp-2 cells. J Diarrhoeal Dis Res 14, 255–259.[Medline]

Russell, R. G., O'Donnoghue, M., Blake, D. C., Jr, Zulty, J. & DeTolla, L. J. (1993). Early colonic damage and invasion of Campylobacter jejuni in experimentally challenged infant Macaca mulatta. J Infect Dis 168, 210–215.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, vol. 3, 2nd edn, pp. 18.1–18.86. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Smith, J. L. & Bayles, D. O. (2006). The contribution of cytolethal distending toxin to bacterial pathogenesis. Crit Rev Microbiol 32, 227–248.[CrossRef][Medline]

Spangler, B. D. (1992). Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 56, 622–647.[Abstract/Free Full Text]

Wooldridge, K. G. & Ketley, J. M. (1997). Campylobacter–host cell interactions. Trends Microbiol 5, 96–102.[CrossRef][Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Jain, D. Articles by Husain, N. Search for Related Content PubMed PubMed Citation Articles by Jain, D. Articles by Husain, N. Agricola Articles by Jain, D. Articles by Husain, N.