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Campylobacter fetus is a Gram negative bacterium recognized for its virulence in animals and humans. This bacterium possesses a paracrystalline array of high molecular weight proteins known as surface-layer proteins covering its cell surface. A mathematical model has been made of the outer membrane of this bacterium, both with its surface-layer proteins (S+) and without (S-). Monte Carlo computer simulation was used to understand the stability of the surface-layer protein structure as a function of ionic concentration. The interactions of an electrically-charged antimicrobial agent, the cationic antimicrobial peptide protamine, with surface-layer proteins and with the lipopolysaccharides of the outer membrane were modeled and analyzed. We found that (1) divalent ions stabilize the surface-layer protein array by reducing the fluctuations perpendicular and parallel to the membrane plane thereby promoting adhesion to the LPS region. This was achieved via (2) divalent ions bridging the negatively-charged LPS Core. The effect of this bridging is to bring individual Core regions closer together so that the O-antigens can (3) increase their attractive van der Waals interactions and "collapse" to form a surface with reduced perpendicular fluctuations. These findings provide support for the proposal of Yang et al. [1]. (4) No evidence for a significant increase in Ca(2+) concentration in the region of the surface-layer protein subunits was observed in S+ simulations compared to S- simulations. (5) We predicted the trends of protamine MIC tests performed on C. fetus and these were in good agreement with our experimental results., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We report here the first demonstration of intra- and interspecies conjugative plasmid DNA transfer for Campylobacter fetus. Gene regions carried by a Campylobacter coli plasmid were identified that are sufficient for conjugative mobilization to Escherichia coli and C. fetus recipients. A broader functional range is predicted. Efficient DNA transfer involves the virB9 and virD4 genes of the type IV bacterial secretion system encoded by a pathogenicity island of C. fetus subsp. venerealis. Complementation of these phenotypes from expression constructions based on the promoter of the C. fetus surface antigen protein (sap) locus was temperature dependent, and a temperature regulation of the sap promoter was subsequently confirmed under laboratory conditions. Gene transfer was sensitive to surface or entry exclusion functions in potential recipient cells carrying IncPα plasmid RP4 implying functional relatedness to C. fetus proteins. The virB/virD4 locus is also known to be involved in bacterial invasion and killing of cultured human cells in vitro. Whether specifically secreted effector proteins contribute to host colonization and infection activities is currently unknown. Two putative effector proteins carrying an FIC domain conserved in a few bacterial type III and type IV secreted proteins of pathogens were analyzed for secretion by the C. fetus or heterologous conjugative systems. No evidence for interbacterial translocation of the Fic proteins was found.

The objective of the present study was to characterize the phenotypic and molecular aspects of Campylobacter fetus strains isolated from bovine herds with reproductive problems. Thirty-one Brazilian field isolates, together with one reference strain of each subspecies of C. fetus, were analyzed. The strains were submitted to phenotypic identification followed by subspecies characterization using the polymerase chain reaction (PCR) and numeric evaluation of restriction fragment length polymorphism (RFLP) separated by pulsed-field gel electrophoresis (PFGE). Phenotypically, 4 isolates (12.1%) were classified as C. fetus subsp. fetus, and 29 isolates (87.9%) were classified as C. fetus subsp. venerealis. However, according to molecular analysis, only 1 isolate (3.0%) was classified as C. fetus subsp. fetus (the reference strain), whereas 32 isolates (97.0%) were considered C. fetus subsp. venerealis. SalI digestion of C. fetus genomic DNA, obtained from the 33 strains, yielded 7-10 DNA fragments ranging in size from 40 to 373kb, with 12 distinct patterns. Furthermore, the numeric analysis by neighbor-joining of the DNA from the 33 strains resulted in a dendrogram in which 2 distinct groups were identified. It was concluded that phenotypic characterization of C. fetus subspecies might lead to erroneous classification of field isolates. Although RFLP-PFGE is a powerful and reliable technique to characterize C. fetus, it has the inconvenience of being time consuming and laborious. Whereas PCR, besides providing rapid results, was found to be reliable and convenient for the characterization of field isolates of C. fetus.

The Campylobacter fetus surface layer proteins (SLPs), encoded by five to nine sapA homologues, are major virulence factors. To characterize the sapA homologues further, a 65.9 kb C. fetus genomic region encompassing the sap locus from wild-type strain 23D was completely sequenced and analysed; 44 predicted open reading frames (ORFs) were recognized. The 53.8 kb sap locus contained eight complete and one partial sapA homologues, varying from 2769 to 3879 bp, sharing conserved 553-2622 bp 5' regions, with partial sharing of 5' and 3' non-coding regions. All eight sapA homologues were expressed in Escherichia coli as antigenic proteins and reattached to the surface of SLP- strain 23B, indicating their conserved function. Analysis of the sap homologues indicated three phylogenetic groups. Promoter-specific polymerase chain reactions (PCRs) and sapA homologue-specific reverse transcription (RT)-PCRs showed that the unique sapA promoter can potentially express all eight sapA homologues. Reciprocal DNA recombination based on the 5' conserved regions can involve each of the eight sapA homologues, with frequencies from 10(-1) to 10(-3). Intragenic recombination between sapA7 and sapAp8, mediated by their conserved regions with a 10(-1)-10(-2) frequency, allows the formation of new sap homologues. As divergent SLP C-termini possess multiple antigenic sites, their reciprocal recombination behind the unique sap promoter leads to continuing antigenic variation.

A collection of Campylobacter fetus strains, including both C. fetus subsp. fetus and C. fetus subsp. venerealis, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.

Phase variation of Campylobacter fetus surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique sap promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the C. fetus sap inversion system is RecA dependent. When we challenged a pregnant ewe with a recA mutant of wild-type C. fetus (strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the aphA insertion into recA was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the sap locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these recA mutants the sap inversion frequency is reduced by 2 to 3 log(10) units compared to that in the wild type. Thus, although the major sap inversion pathway in C. fetus is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.

Precipitate of Campylobacter jejuni cytotonic enterotoxin(CE) performed in an 80% saturated solution of ammonium sulfateit indicated that there were some little molecular proteins except the 68 kD main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), whereas the eluate from GM1 ganglioside affinity column chromatography exhibited only one 68 kD band on SDS-PAGE. The results suggest that CE mainly be consisted of 68 kD protein. The toxin is heat-labile, pH dependent and resistant to trypsin, It could be completely inactivated by heating at either 56 degrees C and 60 degrees C for 30 min or 100 degrees C for 15 min. The activity was maximum at pH 6.0 and was completely inactivate at pH 3.0 and pH 9.0, and rapidly reduced after storage over 3 d at 4 degrees C. The anti-LT serum could completely inhibited the activity of CE.

The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by sapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapA homologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by Escherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system.

Campylobacter (C.) jejuni from persons suffering from diarrhoea, from organs of poultry, C. jejuni and C. fetus ssp. fetus from the gastrointestinal tract of calves and adult cattle as well as a number of reference strains were examined for cytotoxin formation in a CHO-K1 cell culture test. During evaluation, three morphologically different pictures were observed. The first cytotoxin caused a formation of strikingly large, rounded or polymorphic and elongated cells which was associated with reduced growth. The progressive morphological changes corresponded to those described for the Cytolethal Distending Toxin (CLDT) and were assigned to it. The second cytotoxin produced a rounding of cells without a change in their size while at the same time, growth was reduced. In analogy to CLDT, this toxin was termed Cytolethal Rounding Toxin (CLRT). A third morphological picture consisted of cell changes characterized by enlarged polymorphic as well as by small rounded cells. These cell changes were considered as being distinct from the above mentioned ones and referred to as CLTD/CLRT effect. In none of the 39 Campylobacter strains isolated from humans and calves with diarrhoea, a noteworthy cytotonic activity could be detected that would indicate the presence of an enterotoxin.

Campylobacter jejuni strains are able to produce at least two different cytotoxins called "cytolethal distending toxin" (CLDT) and "cytolethal rounding toxin" (CLRT). In this study, we investigated the corresponding changes in CHO-K1 cells using the cell counter and analyzer system CASY 1. Determination of the cell volume after toxin treatment of the cells is a useful criterion for differentiation between the cytotoxic activities produced by Campylobacter strains. Incubation of the cells with crude CLDT resulted in a decrease in the cell count combined with a dramatic increase of the mean cell volume in comparison to the control culture. A decrease in the cell count was also seen as a response to CLRT preparations, while this toxin had no effect on the mean cell volume determined. It was shown that only CLDT caused histone-associated DNA fragments in the cytoplasm of CHO-K1 cells indicating an apoptotic pathway of cell death. In addition, the polymerase chain reaction (PCR) was employed to screen Campylobacter strains for the presence of the cdtB gene sequence, which was detectable in all strains investigated.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

The flagellins of Campylobacter spp. differ antigenically. In variants of C. coli strain VC167, two antigenic flagellin types determined by sero-specific antibodies have been described (termed T1 and T2). Post-translational modification has been suggested to be responsible for T1 and T2 epitopes, and, using mild periodate treatment and biotin hydrazide labelling, flagellin from both VC167-T1 and T2 were shown to be glycosylated. Glycosylation was also shown to be present on other Campylobacter flagellins. The ability to label all Campylobacter flagellins examined with the lectin LFA demonstrated the presence of a terminal sialic acid moiety. Furthermore, mild periodate treatment of the flagellins of VC167 eliminated reactivity with T1 and T2 specific antibodies LAH1 and LAH2, respectively, and LFA could also compete with LAH1 and LAH2 antibodies for binding to their respective flagellins. These data implicate terminal sialic acid as part of the LAH strain-specific epitopes. However, using mutants in genes affecting LAH serorecognition of flagellin it was demonstrated that sialic acid alone is not the LAH epitope. Rather, the epitope(s) is complex, probably involving multiple glycosyl and/or amino acid residues.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.

Cytolethal distending toxin (CLDT) production by Campylobacter fetus subsp. fetus isolated from calves was examined using CHO cells. Twenty-five of the 26 strains tested were positive for CLDT with titer of ranging 1:8 to 1:2,048. CLDT positive strains were divided into low and high cytotoxin titer groups. Isolates from diarrhea cases tended to produce a significant amount of CLDT compared with isolates from liver. CLDT produced by C. fetus showed no effect on Y-1 cells and was heat-labile and trypsin-sensitive.

Wild-type Campylobacter fetus strains possess high-molecular-weight S-layer proteins (S+) and are highly resistant to serum-mediated killing and phagocytosis. Spontaneous mutant strains lacking these proteins (S-) are serum and phagocytosis sensitive and have reduced virulence in a mouse model. Intact S+ cells were treated with pronase, which made them S- although genotypically S+ and had essentially no effect on other cellular proteins or on viability. Treatment with pronase, but not buffer alone, rendered these cells serum and phagocytosis sensitive and reduced mouse virulence to the level observed for the S- mutant cells. In related studies, purified S-layer proteins diminished neutrophil chemoluminescent responses to a heterologous particulate antigen. Finally, passive administration of antiserum to the 97-kDa S-layer protein partially protected mice against lethal challenge with the S+ strain. These studies define the contribution of the S-layer proteins to C. fetus virulence.

Campylobacter fetus strains may be of serotype A or B, a property associated with lipopolysaccharide (LPS) structure. Wild-type C. fetus strains contain surface array proteins (S-layer proteins) that may be extracted in water and that are critical for virulence. To explore the relationship of S-layer proteins to other surface components, we reattached S-layer proteins onto S- template cells generated by spontaneous mutation or by serial extractions of S+ cells with water. Reattachment occurred in the presence of divalent (Ba2+, Ca2+, Co2+, and Mg2+) but not monovalent (H+, NH4+, Na+, K+) or trivalent (Fe3+) cations. The 98-, 125-, 127-, and 149-kDa S-layer proteins isolated from strains containing type A LPS (type A S-layer protein) all reattached to S- template cells containing type A LPS (type A cells) but not to type B cells. The 98-kDa type B S-layer protein reattached to SAP- type B cells but not to type A cells. Recombinant 98-kDa type A S-layer protein and its truncated amino-terminal 65- and 50-kDa segments expressed in Escherichia coli retained the full and specific determinants for attachment. S-layer protein and purified homologous but not heterologous LPS in the presence of calcium produced insoluble complexes. By quantitative enzyme-linked immunosorbent assay, the S-layer protein copy number per C. fetus cell was determined to be approximately 10(5). In conclusion, C. fetus cells are encapsulated by a large number of S-layer protein molecules which may be specifically attached through the N-terminal half of the molecule to LPS in the presence of divalent cations.

Campylobacter fetus strains with type A lipopolysaccharide (LPS) and a surface array protein layer (S+) have been found to be pathogenic in humans and animals. Spontaneous laboratory mutants that lack surface array proteins (S-) are sensitive to the bactericidal activity of normal human serum. The ability of lectins to determine the presence of the S-layer and differentiate LPS type was assessed. We screened 14 lectins and found 3 (wheat germ agglutinin, Bandeiraea simplicifolia II, and Helix pomatia agglutinin) that agglutinated S- C. fetus strains with type A LPS but not S- strains with type B or type C LPS or S+ strains. However, the S+ type A strains were agglutinated after sequential water extraction, heat, or pronase treatment, all of which remove the S-layer, whereas there was no effect on the control strains. Specific carbohydrates for each lectin and purified LPS from a type A C. fetus strain specifically inhibited agglutination of an S- type A strain. In a direct enzyme-linked lectin assay, binding to the S- type A LPS strain was significantly greater than binding to the S+ strain (P = 0.01) or to a Campylobacter jejuni strain (P = 0.008). Consequently, these results indicate that the three lectins bind to the O side chains of C. fetus type A LPS but that the presence of the S-layer on intact cells blocks binding.

A 68,000-molecular-weight protein was isolated by polyacrylamide gel electrophoresis from the organism-free filtrate of a fully virulent clinical strain of Campylobacter jejuni. The eluted protein was heat labile, was inactivated at either pH 3.0 or 9.0, was sensitive to trypsin, and was lethal for fertile chicken eggs. It also had toxic effects on chicken embryo fibroblast, Chinese hamster ovary (CHO), and intestinal 407 (Int407) cells. A monoclonal antibody (CETPMAb4) raised to this eluted toxic protein (ETP) from C. jejuni abolished these toxic activities. Homology between C. jejuni ETP and Vibrio cholerae toxin was not observed in that specific antisera to each did not block their respective toxic activities. In enzyme-linked immunosorbent assays, ETP, unlike chlorea enterotoxin, did not bind to GM1 ganglioside. Furthermore, the C. jejuni toxin had cytotoxinlike properties and induced rounding of CHO cells. Binding of ETP to Int407 and primary chicken embryo fibroblast cells was maximal after 2 h as assessed by enzyme-linked immunosorbent assay, and this toxin adherence to host cell membranes was significantly reduced by prior treatment of the cells with proteolytic enzymes, neuraminidase, or glutaraldehyde but not by treatment with beta-galactosidase, lipase, Nonidet P-40, or sodium metaperiodate. In competitive binding assays, sugars, lectins, or GM1 ganglioside did not adversely influence uptake of ETP by these cells. These results suggest that the ETP produced by C. jejuni is a cytotoxin which binds to Int407 cells via a protein- or glycoproteinlike receptor on cell membranes and possesses properties dissimilar to those of V. cholerae toxin.

To understand the role of Campylobacter jejuni surface proteins in the interaction of C. jejuni with cultured mammalian cell lines in vitro, we developed a ligand-binding assay. This procedure allowed us to antigenically identify C. jejuni outer membrane proteins (OMPs) that attach to intact host cell membranes. OMPs isolated from an invasive strain and a less invasive strain were antigenically indistinguishable. However, we found that proteins with molecular masses of 28 and 32 kilodaltons (kDa) from just the invasive strain bound to HEp-2 cell monolayers. Binding of the 32-kDa OMP was cell line specific and correlated directly with the ability of the invasive C. jejuni strain to penetrate. Such a correlation was probably also true for the 28-kDa OMP. We also investigated the binding of glycine acid extracts with cell line HEp-2. We identified four proteins with apparent molecular masses of 28, 32, 36, and 42 kDa in the invasive strain extracts that bound to HEp-2 cells. In contrast, only the 36-kDa protein from the less invasive strain bound to HEp-2 cells. Our data suggest that binding of these surface exposed proteins may play a key role in C. jejuni-host cell interactions and ultimate invasion.

A synthetic medium for production of Campylobacter jejuni enterotoxin was developed for the purposes of its purification by modifying syncase medium, replacing sucrose with glucose, and supplementing with 0.025% sodium pyruvate, 0.25% sodium metabisulphite, 0.001% ferric chloride and 0.1% L-cysteine, adjusted to pH 6.7. Culture filtrates of a human diarrhoeal and a chicken isolate, grown in this medium caused fluid accumulation ranging between 0.50-0.70 ml/cm of rat ileal loop. The kinetics of toxin production indicated a peak at 36 h and decline by 72 h, coinciding with the period of release of protease by the organism. At least 0.4 rat ileal loop units of enterotoxic activity was recovered per ml of culture filtrates and one unit of this toxin contained only 14 micrograms of protein. The toxin is heat-labile, pH dependent, nonhaemolytic, resistant to trypsin, sensitive to papain and pronase and may show subunit molecular weight analogy with CT subunits.

A set of chemically defined media has been developed for the cultivation of Campylobacter jejuni strains of human origin. A minimal medium, a complete medium and 5 different nutrient-deficient media (NDM1-NDM5) are described. Some of the strains investigated required L-methionine(lacking in NDM1), L-cystine and L-cysteine (NDM2), K2HPO4 (NDM 3), KH2PO4 (NDM4) and NAD, thiamine and calcium pantothenate (NDM5). 57.7% of the strains investigated required L-methionine. The strains grew at pH 6.6-7.7. The media described are not suitable for C. intestinalis.

Maximum growth of Campylobacter fetus subsp. jejuni, strain C-61, occurred when the cultures were incubated with shaking in atmospheres containing approximately 30% hydrogen, 5% oxygen, and 10% CO2. Suspensions of cells grown under these conditions consumed oxygen with formate as the substrate in the presence of 0.33 mM cyanide, which completely inhibited respiration with ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine and with lactate. Spectroscopic evidence with intact cells suggested that a form of cytochrome c, reducible with formate but not with lactate or ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine, can be reoxidized by a cyanide-insensitive system. Analysis of membranes from the cells showed high- and low-potential forms of cytochrome c, cytochrome b, and various enzymes, including hydrogenase, formate dehydrogenase, and fumarate reductase. The predominant carbon monoxide-binding pigment appeared to be a form of cytochrome c, but the spectra also showed evidence of cytochrome o. The membrane cytochromes were reduced by hydrogen in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide at concentrations which prevented the reduction of cytochrome c with succinate as the electron donor. Reoxidation of the substrate-reduced cytochromes by oxygen was apparently mediated by cyanide-sensitive and cyanide-insensitive systems. The membranes also had hydrogen-fumarate oxidoreductase activity mediated by cytochrome b. We conclude that C. fetus jejuni has high- and low-potential forms of cytochrome which are associated with a complex terminal oxidase system.

The effects of sub-inhibitory concentrations (MIC 50 and MIC 25) of clindamycin, erythromycin and chloramphenicol on the adherence of Campylobacter jejuni to INT 407 cells were studied. The results indicated that the adhesion was inhibited at various extents, mostly by MIC 50, and that the inhibition increased with time. The interpretation of our data is that the antibiotics can interfere with the adhesin(s) synthesis.

Campylobacter jejuni/coli strains were isolated from the faeces of 240 patients suffering from acute enteritis. The following characteristics were investigated: (i) growth at different temperatures, and on different substrates under either microaerophilic conditions or anaerobically, with fumarate or nitrate as terminal electron acceptors; (ii) production of H2S in cysteine-containing broth; (iii) hydrolysis of hippuric acid; (iv) DNase; (v) alkaline phosphatase; (vi) beta-lactamase; (vii) presence of menaquinone; and (viii) reduction of selenite. Based on characteristics (ii)-(v), the strains could be divided in 9 phenotypical groups. Most of the strains represented group 2 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%), and groups 8 (DNase-, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%). The other groups were of minor importance. On the other hand, most of the isolates from the United States (Weaver, 1981) fitted well into group 1 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase+) which might demonstrate geographical variations among C. jejuni/coli.

Studies were performed on a cytotoxin (CT) from human strains of Campylobacter jejuni isolated in Malaysia. CT was detected by cytopathic effect (CPE) on HeLa cells at titres from 8 to 32, in culture filtrates from 14 (48%) of 29 human isolates. The CPE correlated well with a quantitative 51Cr-release assay where a specific release of 54-68% was noted. CT production was lost after 5-7 subcultures. CT activity was also detected in 5 (26%) of 19 faecal filtrates from which CT-producing isolates were subsequently obtained. The mol. wt of CT was estimated by Sephadex G-50 chromatography to be greater than 30,000. In a suckling-mouse assay, CT consistently failed to demonstrate fluid accumulation after intragastric inoculation of culture filtrate. The Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) assay was also used. Rabbits given CT-producing strains of C. jejuni developed bacteraemia and severe watery mucus-containing diarrhoea for the duration of the experiment with death of some animals. Rabbits given CT non-producing strains had less severe disease and none died. Rabbits given partially-purified CT had diarrhoea for 3 days but none died.

One hundred and twelve Campylobacter strains comprising 15 species and subspecies were examined for their ability to hydrolyze indoxyl acetate. All strains of C. coli, C. cryaerophila, C. fennelliae, and C. jejuni hydrolyzed the compound, whereas three strains of C. cinaedi were negative and a fourth was weakly positive. Representatives of all other species were negative. Organisms that hydrolyzed indoxyl acetate did so regardless of the medium used.

A total of 101 Campylobacter isolates from Nigerian children with or without gastroenteritis were biotyped and serogrouped by using the Lior typing schemes (H. Lior, J. Clin. Microbiol. 20:636-640, 1984; H. Lior, D. L. Woodward, J. A. Edgar, L. J. Laroche, and P. Gill, J. Clin. Microbiol. 15:761-768, 1982). Fifty-three (52.5%) of the isolates were Campylobacter jejuni biotype I, 29 (28.7%) were C. jejuni biotype II, 10 (9.9%) were Campylobacter coli biotype I, and 9 (8.9%) were C. coli biotype II. Serogroup 36 was the most common (20.7%) in this study, in contrast with serogroup 1 (18.5%) earlier reported from Canada (Lior et al., J. Clin. Microbiol. 15:761-768, 1982).

The oxygen and carbon dioxide requirements of different biotypes of thermophilic campylobacters were investigated by means of (a) quantitative studies, and (b) total growth studies. Oxygen tolerance of the five test organisms differed markedly and varied with the carbon dioxide concentration. At most carbon dioxide concentrations tested, Campylobacter jejuni strains NCTC 11168 and NCTC 11392 tolerated 21% oxygen (growth reduced), C coli NCTC 11353 tolerated 15% oxygen (growth reduced), and C jejuni ATCC 3036 and (nalidixic acid resistant thermophilic campylobacter) NCTC 11352 tolerated 10% oxygen (growth not reduced). Total growth studies indicated that 10% oxygen was the optimal concentration for growth of the five test organisms. All exhibited a requirement for carbon dioxide, and only C jejuni strains NCTC 11168 and NCTC 11392 tolerated its absence (growth reduced), when the oxygen concentration was low. The studies indicated that atmospheres containing 5% to 10% oxygen and 1.0% to 10% carbon dioxide are suitable for growth of the various biotypes of thermophilic campylobacters. The oxygen and carbon dioxide concentrations produced in anaerobic jars by variations of the evacuation-replacement technique were determined and suitable practices identified.

To evaluate the capacity of enzyme-linked immunosorbent assays (ELISAs) to identify pathogenic strains among clinical fecal isolates of Campylobacter jejuni, 40 consecutively obtained strains from 39 sick patients and 1 asymptomatic person were tested by respective ELISAs for enterotoxin production in culture filtrates and for the invasive virulence antigen of bacterial cells. Of the 40 strains, 14 produced the enterotoxin; 15 strains, two of which were also enterotoxigenic, were invasive; and 11 strains had no detectable virulence property. The presence or absence of these virulence properties was confirmed by the demonstration that viable cells of all 12 randomly selected enterotoxigenic or invasive strains tested, but none of 9 nonpathogenic strains tested, caused fluid secretion in rat ligated ileal loops. All 12 patients examined who were infected with an invasive strain had grossly or microscopically evident blood cells in their stools or both, whereas none of those infected with an enterotoxigenic strain had overtly bloody diarrhea, and only 1 of 8 patients examined had microscopically evident blood cells in the stool. Twelve of the invasive, five of the enterotoxigenic, and three of the nonpathogenic strains also produced small amounts of cytotoxin, but there was no correlation between cytotoxin production and an abnormal response in rat ligated ileal loops. These observations show that enterotoxin production or invasiveness or both can be detected by ELISAs in three-fourths of C. jejuni fecal isolates and that there is usually a relationship between the specific pathogenic property of the infecting strain and the clinical mainfestations.

The binding of C-reactive protein to a variety of species of bacteria with potential clinical significance was studied to assess the potential function of C-reactive protein in nonimmune defense against infection. Purified, radioiodinated human C-reactive protein bound to all Streptococcus pneumoniae tested and to some viridans streptococci, but not to group A or group B streptococci or to any of eight different gram-negative rods and cocci.

During a seven-month survey, Campylobacter jejuni was isolated from 9.6% of the 116 acute diarrhoeal cases admitted to the Infectious Diseases Hospital, Calcutta. In six of the 11 cases, C. jejuni occurred together with V. cholerae biotype El Tor (Ogawa) while in one case it was found in association with Shigella sonnei. No age or sex specific incidence was observed. A distinct clinical profile in cases suffering from Campylobacter enteritis was not discernible. Biochemically all strains isolated in this study conformed to the typical reported characteristics of C. jejuni. Survival of the organism in positive stool samples held at 4 degrees C without any transport medium was limited. This preliminary study indicates that C. jejuni is an important aetiological agent of acute diarrhoea and must be routinely monitored in enteric laboratories in India.

A method using an ammonia electrode is being developed for investigating the deamination of amino acids and amides by bacteria. Application of this method to Campylobacter jejuni and C. coli has led to the demonstration of D-asparaginase activity in some strains. This has allowed the subdivision of both species into D-asparaginase-positive and -negative biotypes. Even though the method is in the developmental stage, it was found to be generally reproducible and easy to perform. Areas for further improving the procedure have been identified. The ammonia electrode offers the theoretical possibility of investigating the breakdown of any amino acid by bacteria. It thus opens up a new and practical approach for separating species and strains, particularly in those bacterial groups that are difficult to subdivide by conventional means.

Surface proteins of nine Campylobacter jejuni strains belonging to three different serovia were extracted with lysozyme/ethylenediamine-tetraacetic acid. The preparations bound to isolated murine small intestinal cells and to a membrane fraction (MF) of isolated brush borders obtained by detergent treatment with Triton X-100 and Nonidet P40. Binding was demonstrated by an enzyme-linked immunosorbent assay procedure. Using lipopolysaccharide (LPS)- and flagella-specific antisera the contribution of flagella and LPS, present in the protein preparations, to the total binding to MF was investigated. Only up to approximately 10% of the total binding of each strain was found to be mediated by LPS, 10%-33% of binding was flagella dependent. The preparations of four strains (serovar HS2) bound in a trypsin-sensitive manner (45%-85% reduction), while the others (serovaria HS1 and HS13) were hardly influenced by trypsin treatment. Binding to MF was not impaired by preincubation of the bacterial surface protein preparations with several sugars and lectins.

Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide-insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha-ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger.

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.

Wolinella succinogenes cells and extracts reduced nitric oxide, and cells, but not extracts, reduced nitrous oxide. Formate-reduced W. succinogenes extracts generated the 573-nm peak in difference spectra seen previously in response to nitric oxide in denitrifiers. The type strains of several Campylobacter species did not reduce either gaseous oxide. Cells, but not extracts, of C. fetus subspecies (fetus and venerealis) reduced nitrous oxide; acetylene inhibited reduction. Neither cells nor extracts reduced nitric oxide.

Dimetridazole, a nitroimidazole drug reported to act only on obligately anaerobic micro-organisms, is widely used for the prevention and treatment of swine dysentery. Forty-four strains of the microaerophilic bacterium Campylobacter coli isolated from either healthy or diseased pigs, and a strain of Campylobacter fetus, were all sensitive to dimetridazole. The sensitivities (minimal inhibitory concentration less than 10 microng per ml) were similar to those of anaerobic bacteria. Dimetridazole inhibited growth of campylobacters in a shaken culture in air, but did not inhibit uptake of oxygen. Inhibition of growth appeared to result from an inhibition of nucleic-acid synthesis and does not seem to depend upon interference with electron transport in the catabolism of pyruvate.

Examination of catalase-positive Campylobacter-like organisms from cattle and pig abortions, indicated the existence of two distinct biochemical groups. The group 1 strains, predominantly of intestinal origin, were identified as C fetus subspecies intestinalis whereas the group 2 strains appeared similar but not identical to C fetus subspecies venerealis. This second group of organisms was recovered from fetal and placental tissue of animals which aborted, however, the majority of strains were isolated only at 30 degrees C in media not generally employed for Campylobacter isolation. All of the strains from group 2 became aerotolerant on subculture.

The addition of bovine superoxide dismutase to Brucella broth or Brucellar agar greatly echanced the oxygen tolerance of Campylobacter fetus subsp. jejuni strain H840 (ATCC 29428). Catalase also enhanced oxygen tolerance, but to a lesser extent. These enzymes must act externally to the bacteria. All of the diverse compounds which enhance oxygen tolerance of C. fetus, including nor-epinephrine and a combination of ferrous sulfate, sodium metabisulfite, and sodium pyruvate, share the ability to quench either superoxide anions or hydrogen peroxide. On the basis of these and other data, we propose that C. fetus is more sensitive to exogenous superoxide anions and hydrogen peroxide than are aerotolerant bacteria, despite the occurrence of superoxide dismutase and catalse activities in C. fetus. Compounds that enhance oxygen tolerance in C. fetus appear to act by quenching superoxide anions and hydrogen peroxide which occur spontaneously in the culture medium.

A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrhea or from poultry in King County, Washington. Of these strains, 98 (52 hippurate-positive strains and all 46 of the hippurate-negative strains) were selected for further phenotypic characterization and genetic classification. Hippurate hydrolysis, the test typically used to differentiate Campylobacter jejuni and C. coli, did not always correlate with the genetic classification. All hippurate-positive strains were classified as C. jejuni. Of the hippurate-negative strains, 20% were C. jejuni, 78% were C. coli, and 2% were C. laridis. Assuming that the remaining hippurate-positive strains were all C. jejuni, then hippurate-negative C. jejuni represented a small percentage (9 of 556 or 1.6%) of C. jejuni strains but a significant percentage (9 of 46 or 20%) of hippurate-negative strains. This finding suggests that hippurate hydrolysis should not be used as the sole criterion for differentiating thermophilic Campylobacter species, particularly when describing the disease states associated with these organisms.

Strains of Campylobacter jejuni and Campylobacter coli were characterized and grouped by their distinct reaction patterns with lectins. Heating of the Campylobacter cultures to 100 degrees C and holding for 30 to 60 min greatly enhanced their reactivity with lectins and permitted the grouping of all but 3 of 155 cultures tested in this study without interference of autoagglutination and other nonspecific activities. The lectin reaction patterns of the heated cultures were stable and reproducible. They were strain specific and independent of the heat-stable antigenic types. The lectin-reactive sites of C. jejuni and C. coli may be useful as additional markers for strain characterization. Based on these observations, a simple slide agglutination procedure is described for differentiating strains of C. jejuni and C. coli by their interaction with a selected group of commercially available lectins.

Agglutination tests with lectins indicated differences in the surface composition of strains of the thermophilic (optimum temperature 42 degrees C) Campylobacter species C. coli, C. faecalis, C. hyointestinalis, C. jejuni and C. laridis. All strains examined were agglutinated by the protein-reactive agglutinins of Mangifera indica (mango) and Persea americana (avocado) and a large proportion was also agglutinated by the carbohydrate-reactive lectins of Canavalia ensiformis (Jack bean) and Triticum vulgaris (wheat germ). Reactions with other lectins varied widely between strains, even of the same species and serotype. Lectin agglutination may be useful as a supplementary procedure for characterizing individual Campylobacter isolates for epidemiological purposes.

The 45-kilobase tetracycline resistance plasmids pMAK175 and pUA466 from Campylobacter jejuni were examined using curing and mating experiments. However, these plasmids encoded neither cytotoxin production, as determined in Vero cells, nor enterotoxin activity, as determined in Chinese hamster ovary cells.

One hundred strains of Campylobacter jejuni/coli isolated from faeces of children with diarrhoea were characterised. Frequency of isolation of these microorganisms from faeces of children with enterocolitis symptoms was evaluated. In this group Campylobacter jejuni/coli constituted 11.4% of all isolates, being the dominant etiologic agent of these infections. Biotype pattern of 100 Campylobacter jejuni/coli strains was determined. Biotype I C.jejuni prevailed and C. coli constituted as much as 35% of all isolated strains. All isolated strains were characterised serologically according to typing scheme of Lior. Seventy four strains were typed and 22 were untypable, out of which four were rough. Two new serotypes were isolated: LIO 71 and LIO 72, LIO 4 and LIO 72 serotypes were the most frequently isolated. Frequency of isolation of Campylobacter jejuni/coli strains were also determined in the period from january 1985 to august 1987.