Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.

Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.

Gastric lavage: a simple method to obtain IgA-rich intestinal secretions from the rabbit.

A lavage procedure was developed to obtain intestinal secretions from rabbits. The procedure facilitated the repeated monitoring of the intestinal IgA immune response of these animals to enteric infection with Campylobacter jejuni. This non-invasive technique was easily performed, reproducible and yielded consistent levels of IgA from rabbit intestinal secretions. It is anticipated that this procedure will aid in the study of the intestinal immune response of rabbits to other enteric pathogens.

Antibodies to Campylobacter flagellin recognize epitopes common to phase 1 and phase 2 flagella.

We used a simple method to obtain purified flagellin from Campylobacter, suitable for an immunization procedure in mice. Western blot analysis of cross-reacting antibodies showed that there were epitopes common to phase 1 and 2 flagellins. Analysis by ELISA suggested that certain common flagellar epitopes are conformational, and antibody immobilization tests confirmed that common surface-exposed epitopes exist in a region of flagella necessary for conferring motility to the bacteria.

Adherence, enterotoxigenicity, invasiveness and serogroups in Campylobacter jejuni and Campylobacter coli strains from adult humans with acute enterocolitis.

Two hundred Campylobacter jejuni and Campylobacter coli strains from the same number of adult Swedish patients with acute enterocolitis were tested regarding adherence to and invasiveness in HEp-2 cells and for enterotoxigenicity by the CHO-cell assay. The serogroup characteristics, heat-stable and heat-labile, for each strain were also investigated. Eighty-four percent of the strains were classified as C. jejuni and 16 percent as C. coli. All of the strains were adherent to HEp-2 cells, 39% were invasive and 31.5% enterotoxigenic. We found significantly more invasive strains in the non-enterotoxigenic group than in the enterotoxigenic one. There would seem to be no correlation between enterotoxigenicity or invasiveness and serogroup. The results of this study suggest the existence of multiple mechanisms for C. jejuni- and C. coli-induced diarrhoea and that the mechanisms may differ from one strain to another.

Serological analysis of the heat-stable antigens involved in serotyping Campylobacter jejuni and Campylobacter coli.

Analysis with serotyping antisera showed that carbohydrate determinants were the dominant heat-stable antigens of Campylobacter jenuni/coli involved, whereas proteins did not contribute to the serological reactions. Lipopolysaccharide (LPS) along with a polysaccharide extract from whole bacteria (PS(WB] conferred strain serospecificity. In general, analysis with monoclonal antibodies in passive haemagglutination and co-agglutination tests showed the existence of similar antigenic determinants in LPS and PS(WB) of the same strain. However, in some strains determinants were detectable in LPS but not in PS(WB) using monoclonal antibodies, in other strains the situation was reversed. All of these monoclonal antibodies reacted with LPS in the more sensitive immunoblotting technique. The presence of 2-keto-3-deoxyoctonic acid in PS(WB) preparations, in the absence of endotoxin, supported the conclusion that PS(WB) was derived from LPS during extraction. The lack of detection of a reaction by monoclonal antibodies with LPS in passive haemagglutination, in contrast to immunoblotting, was suggested due to the presence of low concentrations of the relevant epitopes because of the procedure used to prepare the LPS tested.

Plasmids and serogroups in Campylobacter jejuni.

For epidemiological purposes identification of Campylobacter strains is usually based on surface antigen characteristics. Two different systems, one for heat-stable (HS) and one for heat-labile (HL) antigen have dominated. In earlier studies we found a great variability in the two antigen systems. The aim of the present investigation was to analyse the frequency of plasmids in Campylobacter strains in the light of their possible use as an epidemiological tool as well as the relation between the presence of plasmids and surface antigens (HS and HL). Two hundred and forty-two strains from the same number of patients with diarrhea were analysed. In 70 (28.9%) plasmid(s) were found, in general one or two. Most of the plasmids were found in the molecular weight interval between 21-40 Md. There was no relation between the presence or size of plasmids and serogroup. We conclude that plasmid determination can be used as a complement to serotyping in epidemiological studies.

Campylobacter like organisms on the gastric mucosa: culture, histological, and serological studies.

Biopsy samples were taken from the gastric mucosa of 50 patients attending a gastroscopy clinic; blood was also taken for serological studies. A campylobacter like organism was grown from 31 patients (62%) and the organism was seen in sections from 27 biopsies. Antibody was found in 31 patients by complement fixation and in 27 by bacterial agglutination. There were strong positive correlations between the presence of the organism, detectable antibody, and histological gastritis. Antibody to the campylobacter like organism was comparatively uncommon in patients without gastritis and in samples from blood donors and antenatal patients.

Detection of campylobacter by immunofluorescence in stools and rectal biopsies of patients with diarrhoea.

Rabbit antiserum, elicited by the intravenous injection of a strain of Campylobacter jejuni heated to 100 degrees C, cross reacted strongly with all other thermophilic campylobacters tested as well as with "C pyloridis" and could be detected by indirect fluorescence with labelled anti-rabbit serum. Antisera to formalin killed cells did not do so. The correlation of positive stool culture with positive immunofluorescence of stools and rectal biopsies from patients with diarrhoea was 70-80%. Some inconsistent, weak reactions showing differently shaped organisms have been seen with some strains of Bacteroides fragilis. Wolinella spp reacted weakly, but one strain of Vibrio cholerae tested did not. Other intestinal organisms, commensals, and pathogens tested were negative.

The serotype distribution of Campylobacter in patients with diarrhoea in Kuwait.

Fifty one strains of Campylobacter jejuni/coli isolated from patients with diarrhoea, at the Amiri Hospital, Hawally, Kuwait were classified on the basis of the heatstable-HS-antigens and the heat-labile-HL-antigens, by using 20 and 23 hyperimmune antisera for the two methods, respectively. The ages of the patient ranged from 3 months to 60 years, and 72.6% of the strains were from children less than 4 years. With the number of antisera used 78.4% of the HS antigens and 96.1% of the HL antigens could be identified. About half of the strains had one of five HS antigens (4, 8, 13, 5 or 25) and 70.5% of the strains had one of five HL antigens (1, 36, 2, 6, or 21). The study shows that the most common HS and HL antigens among Campylobacter strains from Kuwait also are the most frequent antigens of strains from other parts of the world. A limited number of antisera are sufficient to identify the majority of the strains.

Serotyping of campylobacters by co-agglutination on the basis of heat-stable antigens.

A new and simple method of serotyping campylobacters has been developed which utilises co-agglutination to detect the presence of heat-stable antigens. Campylobacters are heated at 75 degrees C for 30 min to destroy antigenic protein and allowed to react on a glass slide with staphylococci coated with antibody. Of 74 isolates, 67 gave the same result by co-agglutination and the previously described passive haemagglutination method. The co-agglutination technique may be used as a rapid screening test before serotyping by passive haemagglutination.

Cross-protection of infant mice against intestinal colonisation by Campylobacter jejuni: importance of heat-labile serotyping (Lior) antigens.

An association of the heat-labile antigens detected by the Lior serotyping scheme with ability to protect infant mice against gastrointestinal colonisation with Campylobacter jejuni has been established. Overall, 39 (57%) of 68 infant mice challenged with a heterologous strain of the same Lior serotype as the vaccine strain were protected, compared with 40 (85%) of 47 infants protected against a homologous challenge. In contrast, none of the infant mice challenged with a strain carrying the same heat-stable antigens (i.e., of the same Penner serotype as the vaccine strain) were protected.

The mechanism of protection of infant mice from intestinal colonisation with Campylobacter jejuni.

BALB/c mice, vaccinated intraperitoneally with a heat-killed (62 degrees C) suspension of Campylobacter jejuni before mating, completely protect c. 90% of their own infants from intestinal colonisation. This protection has now been investigated further in fostering experiments. Fostering by vaccinated dams within the first 24 h of life prevented intestinal colonisation in 50% of infants from non-vaccinated dams, and reduced colonisation in a further 25%. Infants from vaccinated dams, even if allowed to receive their own mothers' colostrum and milk, became susceptible to challenge when subsequently fostered by non-vaccinated dams. Immunity in experimentally infected infant mice depended upon the consumption of immune milk at and after the time of challenge. High concentrations of IgG antibodies specific for C. jejuni were found in the serum and mammary secretion of vaccinated dams, but there was very little specific IgA antibody.

Role of murine macrophages and complement in experimental Campylobacter infection.

The roles of macrophages and the complement system as potential host defence mechanisms in mice against campylobacter infection were studied in vivo, by depleting the murine serum-complement or the phagocytic cells. Macrophage-depletion was performed by intraperitoneal (i.p.) injection of silica dust, Liquoid or dextran sulphate. During 5 days after infection, such mice showed a significant increase in mortality, compared with controls. In contrast, mice that were previously decomplemented by i.p. injection of Cobra Venom Factor showed no significant increase in mortality. The results with combined macrophage depletion and decomplementation did not differ from those with macrophage depletion alone. These experiments suggest that macrophages seem to be more important than complement in the defence of mice against experimental campylobacter infection.

Anti-enterobacteria antibodies in psoriatic arthritis.

The occurrence of certain antibacterial antibodies was studied in the sera of 22 healthy donors (HD) and 66 patients with different diseases. The cases investigated included 22 rheumatoid arthritis (RA), 22 non-arthritic-psoriasis (NAP), and 22 psoriatic arthritis (PA) patients. A complement fixation test was used with Yersinia enterocolitica 0:3 type (YEC), Yersinia pseudotuberculosis (YPT), Campylobacter jejuni (CJ), and Campylobacter fetus (CF) antigens; the detection of anti-Chlamydia trachomatis (CT) antibodies was carried out using an immunoperoxidase colorimetric slide test that allowed the detection of isotypes of specific antibodies. It was found that the synthesis of anti-CF, CJ, YEC, and YPT antibodies in NAP patients does not differ significantly from that of the HD group; on the contrary, the antibody levels were statistically higher in PA than in the other disease groups or in the healthy controls, although only anti-CF antibodies seemed to significantly differentiate (p = 0.000003) the PA group from the others. Anti-CT IgA antibody titers were found to be significantly higher in the PA as well as in the RA groups when compared with the controls, while the antibody levels in NAP patients showed no clear-cut difference with respect to those of either the arthritic patients or the healthy controls. By showing that anti-enterobacterial antibodies are increased in PA but not in NAP patients, our data furnish additional support to the thesis of a pathogenic role of bacterial infections in psoriatic arthritis.

The prevalence of antibodies reactive with Campylobacter jejuni in the serum of homosexual men.

Samples of serum from 187 homosexual and 169 heterosexual men were examined by means of an indirect immunofluorescent antibody test for the presence of antibody reactive with Campylobacter jejuni. Antibody of the IgG class was detected in the serum of 19 (10.2 per cent) and 15 (8.9 per cent) homo- and heterosexual men respectively. The prevalence of serum antibody in men attending a sexually transmitted diseases clinic was higher than in a previously reported control group.

An examination of Campylobacter fetus subsp. fetus by restriction endonuclease analysis and serology.

Restriction endonuclease analysis was used to examine 70 different strains of Campylobacter fetus subsp. fetus, which were isolated from aborted sheep foetuses. The strains could be divided into seven types based on the DNA fragment patterns obtained by electrophoresis after digestion with the enzyme BstEII. With one exception, digestion with the enzyme XhoI allowed the strains to be grouped identically to that for BstEII. Antisera were made against formalized whole cells representative of each of the seven different restriction types. Analysis of these sera by tube agglutination tests using whole cells revealed five different serogroups. Examination of the 70 strains with absorbed antisera demonstrated a complex relationship between the restriction type and the external antigens of C. fetus subsp. fetus.

Monoclonal antibodies specific for Campylobacter fetus lipopolysaccharides.

Four monoclonal antibodies (mAbs) (M1357, M1360, M1823 and M1825) which reacted with Campylobacter fetus lipopolysaccharide (LPS) core region epitopes were produced and characterized. Reactivity of these mAbs with C. fetus core LPS epitopes was determined by enzyme-linked immunosorbent assay (ELISA) with whole cell proteinase K digests and phenol-water extracted LPS, and by immunoblotting with proteinase K digests. The specificities of the four mAbs were evaluated using an indirect ELISA. One of the mAbs reacted with 42 and three of the mAbs reacted with 41 of the 42 C. fetus strains examined. No reaction was observed between the four mAbs and 32 non-C. fetus bacteria tested, with the exception of one mAb with one organism. The four mAbs reacted with serotype A and B strains indicating the presence of shared epitopes in C. fetus LPS core oligosaccharides. The specificities of three mAbs previously produced to C. fetus LPS O-antigens (M1177, M1183 and M1194) were also evaluated and no reaction was observed with these mAbs and the 32 non-C. fetus bacteria tested. Strong immunofluorescence reactions were observed with the anti-O chain mAbs and selected C. fetus strains of the homologous serotype. These anti-LPS core oligosaccharide and anti-LPS O chain mAbs are highly specific for C. fetus and are potentially useful as immunodiagnostic reagents for detection, identification and characterization of C. fetus.

Role of the S-layer proteins of Campylobacter fetus in serum-resistance and antigenic variation: a model of bacterial pathogenesis.

Campylobacter fetus are microaerophilic gram-negative bacteria that are pathogens of animals and humans. These organisms possess paracrystalline surface (S-) layers, composed of acidic high molecular weight proteins. C. fetus strains possessing S-layers are resistant to C3b binding, which explains both serum and phagocytosis-resistance. C. fetus strains also can vary the subunit protein size, crystalline structure, and antigenicity of the S-layer it expresses. Therefore, its S-layer permits C. fetus to resist complement and antibodies, two of the key defenses against extracellular pathogens. C. fetus possesses several full-length genes encoding S-layer proteins with both conserved and divergent sequences, which permits gene rearrangement and antigenic variation.

Immunization in heifers with dual vaccines containing Tritrichomonas foetus and Campylobacter fetus antigens using systemic and mucosal routes.

Vaccines against both bovine venereal campylobacteriosis and trichomonosis were tested. Heifers were assigned to three groups. Groups 1 (n = 21 heifers) and group 2 (n = 20) received a commercial or experimental vaccine, respectively, containing both Campylobacter fetus and Tritrichomonas foetus antigens. Group 3 (n = 21) received adjuvant alone. Preparations were injected SQ in groups 1 and 3 at days -60 and -30 (day 0 was considered the first day of a 90-day breeding period), and in group 2 SQ at days -30 and +11 and into the vaginal submucosa at day -9. Heifers were exposed to two pathogen-infected bulls for 90 days (from day 0 to day +90); furthermore, half of the heifers in each group were challenged at day +39 by an intravaginal instillation of C. fetus venerealis and T. foetus. Pregnancy diagnosis, vaginal culture, and determination of systemic IgG for both organisms were performed. Compared to controls, vaccinated heifers resisted or quickly cleared both pathogens, had a higher pregnancy rate and a higher systemic immune response during and after the breeding period. Overall, the experimental vaccine was superior to the commercial vaccine (groups 2 and 1, respectively). In conclusion, an experimental vaccine containing both C. fetus and T. foetus antigens, given both SQ and intravaginal immediately before breeding and early in the breeding season, yielded superior protection for heifers exposed to bulls harboring C. fetus and T. foetus.