Human serum antibody response inCampylobacter jejuni enteritis as measured by enzyme-linked immunosorbent assay

An ELISA for detection of IgG, IgA, and IgM antibody using an acid-glycine extract from Campylobacter jejuni as antigen was developed. To determine the value of this assay for the diagnosis of acute Campylobacter jejuni infections, the IgG, IgA, and IgM immune response against Campylobacter jejuni was investigated at various timepoints after infection in patients with culture-proven infection. A total of 112 sera from 46 patients and 78 sera from a control group were tested. All but one of the 46 patients with culture-proven Campylobacter jejuni enteritis developed IgG antibodies against Campylobacter jejuni. IgA and IgM ELISA both showed 97% specificity, and sensitivity of 63% and 30% respectively. IgG antibody titers generally remained at a constant level for more than 50 days, whereas IgA and IgM antibody titers declined more rapidly to normal values within 30 to 50 days after onset of clinical symptoms. Detection of Campylobacter jejuni specific IgA antibodies in a single serum sample provided the most useful assay for serological diagnosis of Campylobacter jejuni enteritis. The presence of Campylobacter jejuni specific IgM antibodies was the sole diagnostic criterion in three cases. Serological diagnosis of Campylobacter jejuni enteritis should therefore include both IgA and IgM antibody determination.     

Antibody responses to Campylobacter infections determined by an enzyme-linked immunosorbent assay: 2-year follow-up study of 210 patients

An enzyme-linked immunosorbent assay (ELISA) was adapted to measure immunoglobulin G (IgG), IgM, and IgA classes of human serum antibody to Campylobacter jejuni and Campylobacter coli. Heat-stable antigen, a combination of C. jejuni serotype O:1,44 and O:53 in the ratio 1:1, was used as a coating antigen in the ELISA test. A total of 631 sera from 210 patients with verified Campylobacter enteritis were examined at various intervals after infection, and a control group of 164 sera were tested to determine the cut-off for negative results. With a 90th percentile of specificity, IgG, IgM, and IgA showed a sensitivity of 71, 60, and 80%, respectively. By combining all three antibody classes, the sensitivity was 92% within 35 days after infection, whereas within 90 days after infection, a combined sensitivity of 90% was found (IgG 68%, IgM 52%, and IgA 76%). At follow-up of the patients, IgG antibodies were elevated 4.5 months after infection but exhibited a large degree of variation in antibody decay profiles. IgA and IgM antibodies were elevated during the acute phase of infection (up to 2 months from onset of infection). The antibody response did not depend on Campylobacter species or C. jejuni serotype, with the important exception of response to C. jejuni O:19, the serotype most frequently associated with Guillain-Barré syndrome. All of the patients infected with this serotype had higher levels of both IgM (P = 0.006) and IgA (P = 0.06) compared with other C. jejuni and C. coli serotypes.     

Production of a Shiga-like cytotoxin by Campylobacter

Cell lysates and culture supernatants of 36 Campylobacter isolates from patients with enteritis were tested for cytotoxic activity on HeLa cells. Cytotoxic activity was considered Shiga-like if neutralized by monoclonal antibody to the B subunit of Shiga-like toxin I of Escherichia coli and rabbit anti-Shiga toxin. Fifteen of the Campylobacter isolates produced no detectable cytotoxin, 10 produced a non-neutralizable cytotoxin, and 11 produced low levels of a cell-associated SLT. However, under low stringency conditions no hybridization was observed between a DNA fragment containing cloned SLT-I genes and restriction enzyme-digested total DNA from a Campylobacter strain that produced low levels of a Shiga-like toxin I. The Shiga-like toxin neutralizing titers in sera from 15 patients with C. jejuni infections, 5 patients infected with S. sonnei, and 20 healthy persons were then determined. No rise in neutralizing titer between acute and convalescent sera of patients with C. jejuni infection or S. sonnei infection was observed, although 27% of C. jejuni-infected patients, 40% of S. sonnei-infected patients, and 30% of the healthy controls had neutralizing activity in their sera. These data indicate that low levels of Shiga-like toxin are produced by some Campylobacter isolates but that SLT is genetically distinct from the SLT-I toxin produced at high levels by certain E. coli. The findings also suggest that exposure to SLTs is common in the adult population but not as a consequence of infection with C. jejuni or S. sonnei.     

Immune response to Campylobacter jejuni and Campylobacter coli in a cohort of children from birth to 2 years of age.

A cohort of 111 children from Bangui, Central African Republic, was surveyed for enteric Campylobacter infections from birth to the age of 2 years; stools were examined biweekly in these children until 6 months of age and at least four times per year thereafter until 2 years of age and after each diarrheal episode. Blood samples were obtained at birth and at 3, 6, 9, 12, 18, and 24 months of age. Antibodies against glycine-extracted membrane antigens, purified flagella, and cholera toxin (CT) were assayed by an enzyme-linked immunosorbent assay. The results showed that titers of antibody against the three tested antigens increased in children between 6 and 12 months of age and that nearly all children were immunized by the age of 2 years. A significant fall in anti-flagellum (P less than 0.001) and anti-glycine extract antibodies (P less than 0.001) occurred between birth and age 3 months, and children who had Campylobacter infections during the first 6 months of life had significantly (P less than 0.02) less anti-flagellum antibodies at birth than did those who did not have Campylobacter infections during that time. Three-month-interval stratification showed that CT antibody titers at birth were significantly lower in children who developed Campylobacter infection than in controls (P = 0.05). Comparison of the immune response to a single Campylobacter episode showed that 46.6% of children with asymptomatic carriage did not respond to CT while only 5% of children with diarrhea-producing infection did not respond to CT (P less than 0.01), compared with 30% (P = 0.065) and 56% (P less than 0.01), respectively, of the age-matched controls. Antibodies to flagella seem to protect against enteric colonization by Campylobacter jejuni and Campylobacter coli.     

Identification of Campylobacter jejuni and Campylobacter coli antigens with mucosal and systemic antibodies.

The development of a rapid and specific diagnostic assay for Campylobacter infections is important in determining the etiology of acute diarrhea in humans. Studies have shown that sonicated whole bacteria or partially purified antigens cross-reacted with antibodies against other closely related bacteria. To solve the problems of specificity, we identified specific antigens of Campylobacter jejuni and Campylobacter coli for use in diagnostic assays. We investigated the responses of serum, urine, and intestinal lavage antibodies in infected (fed live bacteria) and parenterally immunized (intraperitoneal injection of sonicated whole bacteria with adjuvant) mice directed against C. jejuni or C. coli by Western blot (immunoblot) analysis. Antibody responses were examined weekly for up to 28 days. Fewer antigens were detected by urinary and intestinal lavage fluid immunoglobulin A (IgA) than serum IgG and IgM for both parenterally immunized and infected mice. Serum from parenterally immunized mice detected more antigens than that from infected mice. Two high-molecular-weight antigens (62,000 and 43,000) were predominantly detected by serum, urine, and intestinal lavage fluids of both parenterally immunized and infected mice. Serum antibodies from 28-day parenterally immunized mice detected one antigen specific to C. coli with a molecular weight of 38,000 and one antigen specific to C. jejuni with a molecular weight of 27,000. An immunodominant protein with a molecular weight of 31,000 common to both C. jejuni and C. coli was also recognized by serum antibodies from parenterally immunized mice.     

Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers.

The pathogenesis of campylobacter infection in man is largely unknown, although cytolethal distending toxin (CDT) has been incriminated as a virulence factor. However, little is known about the cdt genes in Campylobacter spp. isolated from broiler chickens. A total of 350 cloacal swabs was collected and tested by conventional culture and PCR. Of the 114 Campylobacter isolates obtained, 101 (88.6%) were identified as C. jejuni and 13 (11.4%) as C. coli by conventional methods. cdt genes were detected by PCR in all the isolates except one C. jejuni isolate. Cytotoxic effects were produced in a Vero cell line, by 100 of the C. jejuni isolates. In contrast, 10 C. coli isolates produced much lower levels of toxin and 3 produced no detectable toxin. These results confirm the common occurrence of campylobacter infection in chickens and indicate that cdt genes are commonly present in both C. jejuni and C. coli isolates from broilers, but that there are distinct differences in CDT production in these two closely related species.     

Characterization of cytolethal distending toxin of campylobacter species isolated from captive macaque monkeys

An association between certain Campylobacter species and enterocolitis in humans and nonhuman primates is well established, but the association between cytolethal distending toxin and disease is incompletely understood. The purpose of the present study was to examine Campylobacter species isolated from captive conventionally raised macaque monkeys for the presence of the cdtB gene and for cytolethal distending toxin activity. The identity of each isolate was confirmed on the basis of phenotypic and genotypic analyses. The presence of cytolethal distending toxin was confirmed on the basis of characteristic morphological changes in HeLa cells incubated with filter-sterilized whole-cell lysates of reference and monkey Campylobacter isolates and examinations by light microscopy, confocal microscopy, and flow cytometry. Although cdtB gene sequences were found in both Campylobacter jejuni and Campylobacter coli, the production of cytolethal distending toxin correlated positively (P < 0.0001) only with C. jejuni. We concluded that cytolethal distending toxin activity is a characteristic of C. jejuni. Our C. jejuni cdtB gene-specific PCR assay might be of assistance for differentiating toxigenic C. jejuni from C. coli in clinical laboratories.     

Experimental Campylobacter jejuni infection in Macaca nemestrina.

Experimental infection of four specific-pathogen-free Macaca nemestrina monkeys (aged 3.5 and 4.5 months) with Campylobacter jejuni 81-176 caused acute diarrheal illness, characterized by fluid diarrhea, bloody stools, and fecal leukocytes, which lasted for approximately 7 to 11 days. Histologic examination of intestinal biopsies showed acute colitis characterized by infiltration of the mucosa with neutrophils and lymphocytes, and cryptitis. There were no histologic changes in the small intestine. Excretion of C. jejuni was demonstrated for 2 to 4 weeks postchallenge. Plasma antibodies to C. jejuni group antigen were elevated after challenge. Only mild diarrhea occurred after rechallenge with the same strain or with a heterologous C. jejuni strain (79-168) followed by further elevation in specific immunoglobulins A, M, and G. Four 1-year-old juvenile M. nemestrina monkeys which had experienced multiple infections with Campylobacter spp. did not exhibit illness when challenged with C. jejuni 81-176. All had elevated immunoglobulin A, M, and G plasma antibodies prior to challenge, and these humoral antibody levels were indicative of the immunity to challenge. The results demonstrate that C. jejuni infection in M. nemestrina caused colitis with clinical and pathologic results similar to those found in humans and indicate that prior infection protects against subsequent challenge.     

Guillain-Barré syndrome- and Miller Fisher syndrome-associated Campylobacter jejuni lipopolysaccharides induce anti-GM1 and anti-GQ1b Antibodies in rabbits

Campylobacter jejuni infections are thought to induce antiganglioside antibodies in patients with Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) by molecular mimicry between C. jejuni lipopolysaccharides (LPS) and gangliosides. We used purified LPS fractions from five Campylobacter strains to induce antiganglioside responses in rabbits. The animals that received injections with LPS from GBS-associated strains developed anti-GM1 and anti-GA1 antibodies. Animals injected with LPS from one MFS-related C. jejuni strain produced anti-GQ1b antibodies. Rabbits that were injected with Penner O:3 LPS had a strong anti-LPS response, but no antiganglioside reactivity was observed. The antiganglioside specificity in the rabbits reflected the specificity in the patients from whom the strains were isolated. In conclusion, our results indicate that an immune response against GBS- and MFS-associated C. jejuni LPS results in antiganglioside antibodies. These results provide strong support for molecular mimicry as a mechanism in the induction of antiganglioside antibodies following infections.     

Kinetics of anti-Campylobacter jejuni monomeric and polymeric immunoglobulin A1 and A2 responses in serum during acute enteritis

The intensity and kinetics of the serum polymeric and monomeric immunoglobulin A1 (IgA1) and IgA2 antibody responses to Campylobacter jejuni were analyzed. A rapid and marked serum IgA antibody response involving both the monomeric and polymeric components of IgA was observed after C. jejuni infections. IgA antibodies reached a peak of activity in serum during week 2 after the first symptoms of enteritis, about 10 days before the peak of IgG activity. Polymeric IgA accounted for most of the anti-C. jejuni activity at the peak of the IgA response (median, 90%; range, 44 to 98%) but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time. Anti-C. jejuni IgA detected in the serum of healthy blood donors was mainly monomeric (median, 83%; range, 17 to 94%). In both the patients and the positive controls, IgA1 was the predominant (greater than 85%) subclass involved, even when the IgA antibody response was mainly polymeric. Our results suggest that polymeric IgA antibody responses are linked to a strong or persisting antigenic stimulation or both. Polymeric IgA antibodies appear to be a potential marker of acute C. jejuni infections, and their determination could provide a useful tool for the serological diagnosis of recent C. jejuni infections.     

Major outer membrane proteins from many Campylobacter species cross-react with cholera toxin

We have previously shown that Campylobacter jejuni strains do not produce a functional cholera toxin-like toxin (CTLT) detectable in a Chinese hamster ovary cell assay. Instead, the 53-kDa major outer membrane protein (OMP) of C. jejuni, PorA, reacts with cholera toxin (CT) antibody on immunoblots. Here, we have extended this observation to other species of Campylobacter, including C. coli, C. lari, C. fetus, C. hyointestinalis, and C. upsaliensis, the common 53-kDa OMP of which reacted with CT antibody in immunoblotting assays. There were additional reactive bands for C. fetus. As with C. jejuni, this finding may lead to the erroneous conclusion that these additional species produce a functional CTLT. However, this common cross-reactive OMP can be explored as a vaccine candidate to prevent campylobacteriosis.     

Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.

Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections.     

Bacteraemia caused by Campylobacter spp.

The genus Campylobacter has become increasingly recognised as the cause of various infections. Campylobacter jejuni and C coli cause acute gastroenteritis in man all over the world. C jejuni enteritis can lead to bacteraemia, but its actual incidence remains unknown. Seven cases of bacteraemia caused by C jejuni or C coli are reported, from the blood of seven patients: five immune deficient adults; a newborn baby; and a patient who had had abdominal surgery. Patients who develop diarrhoea as a result of Campylobacter infection are at risk of bacteraemia thereafter.     

Cytotoxic and cytotonic factors produced by Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis.

Complete toxigenicity studies were performed on 341 strains of Campylobacter spp., including 23 nonhuman isolates. Toxin profiles based on both cytotonic and cytotoxic factors were determined after analyzing responses in Vero, HeLa, CHO and Y-1 cells. Suckling mouse assays were consistently negative for all culture filtrates tested. Toxin-producing strains were frequently encountered among both the human and nonhuman strains of Campylobacter jejuni, C. coli, and C. laridis investigated. Strains isolated from outbreaks demonstrated parallels in serotype, biotype, and toxigenicity profile, although no clear association could be demonstrated. Biphasic culture conditions conducive to the production of both toxic factors were delineated for the propagation of test Campylobacter strains. Cytotonic effects of Campylobacter culture filtrates were determined in Vero and CHO cells, and cyclic AMP accumulation in cells exposed to these culture filtrates was compared with that in cells exposed to reference toxigenic strains of Vibrio cholerae and Escherichia coli. Partial neutralization of C. jejuni enterotoxin was demonstrated by using antitoxins to cholera toxin and E. coli heat-labile enterotoxin. No neutralization of C. jejuni cytotoxin could be achieved by using antitoxins to either Clostridium difficile cytotoxin or E. coli Verotoxin (0157:H7).     

Cytolethal distending toxin genes in Campylobacter jejuni and Campylobacter coli isolates: detection and analysis by PCR

Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity of Campylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni and Campylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between the cdt genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate C. jejuni and C. coli isolates on the basis of restriction patterns within their cdt genes.     

DNA relatedness among strains of Campylobacter jejuni and Campylobacter coli with divergent serogroup and hippurate reactions.

Eleven strains of Campylobacter from earlier fluorescent-antibody studies were examined by DNA hybridization to determine their species. Three of the strains hydrolyzed sodium hippurate, and eight did not. Four of the hippurate-negative strains were in Campylobacter jejuni serogroups, and the remaining strains were in both C. jejuni and Campylobacter coli serogroups. DNA relatedness to type strains of C. jejuni and C. coli indicated that all three of the hippurate-positive strains and two of the hippurate-negative strains were C. jejuni. The six remaining hippurate-negative strains were C. coli. Two of the hippurate-negative strains in C. jejuni serogroups were C. jejuni, and two were C. coli. Three of the strains in serogroups of both species were C. jejuni, and four were C. coli. These studies confirm that a few strains of C. jejuni are hippurate negative and show that identical or highly related antigens are found in C. coli and C. jejuni.     

Evidence of reinfection with multiple strains of Campylobacter jejuni and Campylobacter coli in Macaca nemestrina housed under hyperendemic conditions.

A prospective bacteriologic study of 18 infant pig-tailed macaques (Macaca nemestrina) housed in a nursery facility in which Campylobacter spp. are endemic was undertaken to determine the epidemiology of infection and reinfection. The isolates of Campylobacter jejuni and C. coli cultured from 8 of the 18 infants were characterized by serotyping, DNA hybridization, and polyacrylamide gel electrophoresis protein profiles. The chronology of infection was indicative of multiple reinfections with different strains of C. jejuni and C. coli during the 12-month study of each infant. The duration of infection with a particular strain was 3 to 4 weeks. Infants were also infected with nalidixic acid-resistant campylobacters. These observations indicated that long-term infections under endemic conditions are caused by continual reinfection. C. jejuni or C. coli infection correlated with diarrhea in 5 of the 18 infants at 1 to 4 months of age.     

Campylobacter, from obscurity to celebrity

After its successful isolation from stools in the 1970s, Campylobacter jejuni has rapidly become the most commonly recognised cause of bacterial gastroenteritis in man. Reported cases of human campylobacteriosis represent only a small fraction of the actual number. In industrialised countries, the incidence of C. jejuni/Campylobacter coli infections peaks during infancy, and again in young adults aged 15-44 years. Acute self-limited gastrointestinal illness, characterised by diarrhoea, fever and abdominal cramps, is the most common presentation of C. jejuni/C. coli infection. The introduction of selective media has made the diagnosis of Campylobacter enteritis a simple procedure. In general, Campylobacter enteritis is a self-limiting disease which seldom requires antimicrobial therapy, although one in 1000 infections may lead to the Guillain-Barré syndrome. In industrialised countries, most infections are acquired through the handling and consumption of poultry meat. In developing countries, where the disease is confined to young children, inadequately treated water and contact with farm animals are the most important risk factors. Many infections are acquired during travel. Fluoroquinolone resistance has been reported in C. jejuni since the late 1980s in Europe and Asia, and since 1995 in the USA. The use of fluoroquinolones to treat animals used for food has accelerated this trend of resistance. In Australia, where fluoroquinolones have not been licensed for use in food production animals, C. jejuni remains susceptible to fluoroquinolones. The public health burden of Campylobacter spp. other than C. jejuni/C. coli remains unmeasured. Better diagnostic methods may reveal the true health burden of these organisms.     

Immunological relationship of the B subunits of Campylobacter jejuni and Escherichia coli heat-labile enterotoxins.

The application of dissociation techniques, involving gel filtration in the presence of guanidine, to a semipurified preparation of Campylobacter jejuni heat-labile enterotoxin yielded a material whose functional and immunological properties resemble those of the B subunits of cholera toxin and Escherichia coli heat-labile toxin (LT). The C. jejuni toxin B subunit reacted with GM1 ganglioside in an enzyme-linked immunosorbent assay, but lacked the holotoxin's cytotonic activity in the Chinese hamster ovary tissue culture assay and its ability to cause fluid secretion in rat ileal ligated loops. The C. jejuni toxin B subunit showed lines of partial identity with the B subunits of both cholera toxin and LT in gel immunodiffusion; it appeared to be more closely related immunologically to the LT B subunit than to the cholera toxin B subunit in enzyme-linked immunosorbent assays that used antisera either to LT or to its B subunit. Rats immunized with LT B subunit were significantly protected against challenge with either the semipurified C. jejuni toxin or a viable enterotoxigenic strain of C. jejuni, although twice the immunization dosage was required to achieve protection comparable to that against the homologous toxin or viable bacteria. These observations indicate that the C. jejuni enterotoxin contains a B subunit that bears an immunological relationship with the B subunits of cholera toxin and LT.     

Cross-reactive antigens shared by Pseudomonas aeruginosa, Helicobacter pylori, Campylobacter jejuni, and Haemophilus influenzae may cause false-positive titers of antibody to H. pylori.

Cystic fibrosis (CF) patients suffer from many of the gastrointestinal conditions which occur in non-CF individuals, e.g., dyspepsia and peptic ulceration. These symptoms may be caused by Helicobacter pylori but could also be due to either pancreatic insufficiency or the intensive antibiotic treatment used in CF patients. Since CF patients chronically infected with Pseudomonas aeruginosa produce antibodies against a wide range of antigens, including antigens common to many other bacteria, e.g., GroEL and lipopolysaccharide, we studied, by the Western blot (immunoblot) technique, the specificity of immunoglobulin G antibodies to H. pylori in Danish CF patients chronically infected with P. aeruginosa, CF patients without P. aeruginosa infection but with Haemophilus influenzae infection, patients with dyspeptic ulcers associated with H. pylori, and patients recovering from acute Campylobacter jejuni or Campylobacter coli infection. Sera from CF patients with chronic P. aeruginosa or H. influenzae infection and patients recovering from acute C. jejuni infection cross-reacted with H. pylori antigens. A strong cross-reacting protein antigen at approximately 14 kDa and minor cross-reactive antigens at approximately 27, 30, and 60 kDa (the heat shock protein GroEL is equivalent to the common antigen of P. aeruginosa) could be demonstrated. The results of this study show that high immunoglobulin G antibody titers against H. pylori in CF patients cannot be regarded as indicating present or past H. pylori infection unless their specificity is proven by absorption studies.