OBJECTIVE: Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168. METHODS: Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study. RESULTS: Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA. CONCLUSION FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.
Campylobacter jejuni/genetics* ; Bacterial Proteins/metabolism* ; Mutation ; Biological Variation, Population

In order to develop a rapid method which can check Campylobacter jejuni in animal and poultry foods nicely, an immunomagnetic capture-fluorescent PCR (IMC-FPCR) method was established in this paper. The reported method involves isolation of the target pathogen by immunocapture prior to the fluorescent PCR step, therefore the immunomagnetic-beads for Campylobacter were developed, and two groups of primer/probe, which targeted for the species special sequence of flaA gene and hipO gene for Campylobacter jejuni were designed. The immunomagnetic capture-fluorescent PCR assay amplification of the hipO gene and flaA gene for detection of Campylobacter jejuni was firstly reported in this paper. Result indicated that IMC-FPCR method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 24 h. The assay results are positive for all of the isolates of Campylobacter jejuni (3 isolates, including type strain ATCC 33560 and ATCC8341) with a detection limit of approximately 10 cfu/mL, are negative for Campylobacter coli and several other bacteria. IMC-FPCR assay provide not only a rapid, sensitive method for quantitative detection of Campylobacter jejuni, but also an important method for detecting of Campylobacter jejuni of viable but non-culturerable (VNC) state.
Campylobacter jejuni ; genetics ; isolation & purification ; Fluorescence ; Immunomagnetic Separation ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVEThis study was to simultaneously identify Campylobacter jejuni and Campylobacter coli isolates in China by Multi-PCR assay and to study the prevalence of six virulence and toxin genes on them.

METHODSA multi-PCR method with three sets of primers specifically designed for application of a 16S rRNA as a universal control, mapA, ceuE based on the specific sequence of C. jejuni and C. coli, was applied to detect 65 Campylobacter isolates from China. Another two separately PCR Primers were directed towards the hippuricase gene (hipO) characteristic of C.jejuni and glyA gene characteristic of C. coli were performed for further confirmation. The presence of the cadF, virB11, flaA, cdtA, cdtB, cdtC genes among these 65 strains were investigated by PCR.

RESULTSFrom multi-PCR detection, 42 isolates belonged to C. jejuni, other 23 isolates belong to C. coli. Data showing the identification were 100% in concordance with the separated PCR for hipO and glyA amplification. The efficiency (100%) of identification by these three primers multi-PCR method was higher than the biochemical test (83.1%). The cadF and flaA genes were detected from 100% (65/65) of the isolates and the PCR product of each gene were identical with each isolate. Only 10.8% (7/65) of the isolates were positive for virB11. The cdtA gene was found in 92% (60/65) of the isolates. 97.6% (41/42) of C. jejuni had cdtB gene, whereas no PCR product with this primers for all the C. coli isolates. cdtC was presented in all the isolates but the lengths of PCR products were different. For C. jejuni, it was 555 bp, for C. coli, it was about 465 bp.

CONCLUSIONThis three primers simultaneous multi-PCR method seemed to be useful for the identification of C. jejuni and C. coli isolates from China since cadF and flaA genes were widely spread in Campylobacter isolates in this country. The present report on virB11 was similar to previous reports from other countries, but the distribution of cdt gene cluster in Campylobacter species isolated from China might be different.

Campylobacter coli ; genetics ; isolation & purification ; pathogenicity ; Campylobacter jejuni ; genetics ; isolation & purification ; pathogenicity ; China ; DNA Primers ; Genes, Bacterial ; Polymerase Chain Reaction ; Virulence ; genetics

OBJECTIVETo construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization.

METHODSThe plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.

RESULTSThe fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05).

CONCLUSIONThe function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Animals ; Bacterial Proteins ; genetics ; Campylobacter jejuni ; genetics ; pathogenicity ; Chemotaxis ; genetics ; Gene Knockout Techniques ; Jejunum ; microbiology ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C

OBJECTIVETo investigate the protein expression profiles of the major food-borne pathogen Campylobacter jejuni NCTC11168.

METHODSMembrane and soluble cellular proteins were extracted from the genome-sequenced C. jejuni strain NCTC11168. Protein expression profiles were determined using two-dimensional gel electrophoresis (2-DE). All the detected spots on the 2-DE map were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis.

RESULTSA total of 537 and 333 spots were detected from the whole cell and membrane-associated proteins of C. jejuni NCTC11168 cultured on Columbia agar medium at 42 °C by 2-DE and Coomassie Brilliant Blue staining, respectively. Analyses of whole cell and membrane-associated proteins included 399 and 133 spots, respectively, which included 182 and 53 functional proteins identified by MALDI-TOF/TOF analysis.

CONCLUSIONThe comprehensive expression protein profiles of C. jeuni NCTC11168 obtained in this study will be useful for elucidating the roles of these proteins in further pathogenesis investigation.

Bacterial Proteins ; genetics ; metabolism ; Campylobacter jejuni ; classification ; genetics ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; methods ; Gene Expression Regulation, Bacterial ; physiology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods ; Transcriptome

OBJECTIVEA comparative study on the role of Campylobacter jejuni (CJ) HB9313 and galE mutant in inducing experimental sciatic nerve damage was conducted in guinea pigs in order to explore whether CJ lipo-oligosaccharide (LOS) is critical component associated with peripheral nerve lesions and find experimental evidence for the presumption of molecular mimicry on the pathogenesis of Guillain-Barre syndromes (GBS) with CJ antecedent infection.

METHODSA total of 32 guinea pigs were randomly divided into four groups: parental strain group (n = 10), galE mutant group (n = 10), control group (n = 6) and PBS group (n = 6), and immunized with the whole cell antigens of CJ HB9313 with Freund's adjuvant (FA), the whole cell antigens of galE mutant (without ganglioside-like structure) with FA, PBS with FA, and PBS alone, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to detect anti-LOS and anti-ganglioside GM1 antibodies in sera of these animals, and comparative morphologic studies of pathologic changes were carried out on the sciatic nerves, including examination of teasing fibers, examination of semithin sections made from epon-embedded tissue blocks under light microscope and transmission electron microscope.

RESULTSELISA results indicated that after immunization, the levels of anti-LOS IgG antibody were significantly elevated in animals from parental strain group and galE mutant group as compared with those before immunization (P < 0.01). No statistically significant difference was found between the two groups. However, the mean optical densities (ODs) of IgG antibody against GM1 at 14 and 28 day after immunization, in parental strain group, were 0.661 +/- 0.290 and 0.984 +/- 0.025, respectively, significantly higher than those of galE mutant group, which were 0.193 +/- 0.078 and 0.180 +/- 0.063 (P < 0.01). The results of morphologic examination on sciatic nerves showed that for teased-fiber study, incidence of pathologic abnormalities of teased fibers from animals of galE mutant group was 4.9% (98/2000), significantly lower than that from parental strain group, which was 16% (320/2000), characterized by predominantly axonal degeneration. The difference between them was highly significant statistically (P < 0.01). Examination of semithin sections of sciatic nerves also revealed that obvious pathological changes occurred in the animals from parental strain group, while only minimal abnormalities could be seen from galE mutant group, there was a significant differences between them (P < 0.01). In parental strains group, the predominant pathologicanl change was axonal degeneration with considerable variation in severity. These morphologic changes were confirmed by electron microscopy.

CONCLUSIONCompared with parental strain, galE mutant without ganglioside-like structure no longer could induce anti-GM1 antibodies, nor induce obvious immune damage of peripheral nerves in experimental guinea pigs. The results of this study provide a strong support to the hypothesis of molecular mimicry as a pathogenesis in patients with GBS following CJ antecedent infection.

Animals ; Antibodies, Bacterial ; blood ; Campylobacter jejuni ; genetics ; immunology ; pathogenicity ; G(M1) Ganglioside ; immunology ; Guillain-Barre Syndrome ; etiology ; Guinea Pigs ; Immunization ; Lipopolysaccharides ; immunology ; UDPglucose 4-Epimerase ; physiology

OBJECTIVETo explore the important role of the terminal residues containing sialic acid (SA) in Campylobacter jejuni (CJ) lipopolysaccharide (LPS) as the critical antigen to induce nerve damage, and also to identify immunopathological evidence for the hypothesis of molecular mimicry and cross-immunity between CJ LPS and gangliosides.

METHODSA mutant of Pen O:19 CJ with neuB1 gene inactivated and LPS outer core terminal residues losing SA was to be constructed. PCR and RT-PCR were used to confirm the mutant. Capability of CJ LPS binding to cholera toxin B subunit (CTB) was tested. Guinea pigs were systematically immunized with LPS of the wild and the mutant strains, respectively. Titers of anti-LPS and anti-ganglioside GM(1) IgG antibodies in sera of immunized guinea pigs were detected by ELISA. Pathological study for sciatic nerves of both Guinea pigs either immunized systematically or perineural injection with their immunized serum was finished.

RESULTS(1) The mutant of CJ O:19 strain with inactivated neuB1 gene was successfully constructed and lost transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. SA was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (2) Compared with the titers before immunization, the titers of anti-GM(1) IgG antibody increased in sera of guinea pigs immunized with LPS of the wild strain. However there were no detectable anti-GM(1) IgG antibody in sera of the animals immunized with mutant LPS and PBS. (3) The incidence of pathological fibers of sciatic nerves in wild CJ LPS group (17.3%) was significantly higher than the mutant CJ LPS group (chi(2) = 125, P < 0.01); the difference between the mutant CJ LPS group and control group was not statistically significant (chi(2) = 1.633, P > 0.05). (4) After perineural injection with immunized serum, the incidence of pathological fibers of sciatic nerves in wild strain group (67.8%) was also significantly higher than the incidence of mutant group (P < 0.01).

CONCLUSIONA mutant of CJ O:19 strain neuB1 gene inactivated and SA component of terminal structure of LPS lost was successfully constructed. And it no longer expressed SA component which is the normal terminal structure of LPS in wild strain. The capability of the wild strain to induce increased titers of anti-GM(1) antibody and immune-mediated nerve damage was simultaneously lost for the mutant strain. It could be a strong immunopathologic evidence to identify the molecular mimicry hypothesis between CJ LPS and ganglioside epitope in nerve on the pathogenesis of CJ related GBS. The terminal residues containing SA should be as the basic GM1-like structure in CJ LPS.

Animals ; Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; genetics ; immunology ; Campylobacter jejuni ; genetics ; immunology ; G(M1) Ganglioside ; immunology ; Guinea Pigs ; Lipopolysaccharides ; chemistry ; immunology ; Molecular Mimicry ; Mutagenesis ; N-Acetylneuraminic Acid ; chemistry ; immunology ; Peripheral Nervous System Diseases ; immunology ; microbiology

OBJECTIVEUsing 16S rDNA and 23S rDNA genes as the target sequences to develop a system based on oligonucleotide microarray and to detect the seven clinical pathogenic bacteria, commonly seen.

METHODSDouble polymerase chain reaction (PCR) was applied to amplify the segments of 16S rDNA and 23S rDNA genes of the target bacteria. An oligonucleotide microarray was constructed to simultaneously detect EHEC O157:H7, Vibrio parahaemolyticus, Salmonella sp., Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni and Shigella sp. Specificity, sensitivity and reproducibility of the microarray during detection were checked. And then microarray was used to detect the microbes in stool specimens of 81 patients with diarrhea and vomiting.

RESULTSThe double PCR method could simultaneously amplify the target sequences of 16S rDNA and 23S rDNA genes of the seven pathogens. The sensitivity of the developed oligonucleotide microarray could reach 10(3) cfu/ml but no positive results were presented for non-targeted bacteria. The coefficients of differentiation in one lot or among different lots of the microarray slices were 3.89% - 5.81%. The positive detection rate of the stool specimens by oligonucleotide microarray was 39.5% (32/81), with a coincidence of 96.3% (78/81) for the patients and another coincidence of 96.8% (31/32) for bacterial genus or species identification, when comparing to the results by routine bacteriological examinations.

CONCLUSIONThe established assay in this study based on oligonucleotide microarray to detect the seven pathogenic bacteria has many advantages such as convenient, rapid, accurate, stable and high flux, which is suitable for clinical specimen examination and epidemiological field investigation.

Bacteria ; isolation & purification ; Campylobacter jejuni ; isolation & purification ; DNA, Bacterial ; genetics ; DNA, Ribosomal ; genetics ; Escherichia coli O157 ; isolation & purification ; Humans ; Listeria monocytogenes ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Reproducibility of Results ; Salmonella ; isolation & purification ; Sensitivity and Specificity ; Shigella ; isolation & purification ; Vibrio cholerae ; isolation & purification ; Vibrio parahaemolyticus ; isolation & purification

OBJECTIVETo explore the pathogenesis of the damage to peripheral nerves induced by Campylobacter jejuni exotoxin (CJT).

METHODS(1) Animal models: (1) The CJT was extracted from PEN 19-CJ and injected perineurally and intravenously to Wistar rats. (2) The sera and the supernatants of peripheral blood mononuclear cells (PBMCs), taken from the rats immunized with the CJT, were injected perineurally at sciatic nerves of experimental rats and intravenously, respectively. (2) Histopathologic study of sciatic nerves: the animals were sacrificed and their sciatic nerves were examined for tease fibers, transverse section with toluidine blues staining and electron microscopy. (3) Immunohistochemistry: sections of sciatic nerves of either normal rats or human which were incubated with CJT and the sciatic nerves with pathological changes induced by CJT were obtained for observation of the binding capability of CJT with peripheral nerves by SABC and FITC-immunofluorescence methods, and nucleic acid hybridization techniques for detection of TNF-alpha mRNA expression in pathological sciatic nerves samples.

RESULTS(1) Remarkable peripheral neuropathies with axon degeneration and/or demyelination were found in the nerves induced by both CJT injection perineurally and intravenously. The axon degeneration was more obvious. Pathological changes were identified in 76.8% (2,763/3,600) of teasing fibers after perineural injection, but only 9.6% (230/2,400) of fibers were damaged in control group (P < 0.01). The peak severity of fiber damage was found on the 3rd day after CJT intravenous injection with the incidence of abnormal fibers was 19.5% (390/2,000), and abnormalities of 15.5% (310/2000) on the 14th day. However, no abnormal changes were demonstrated in control group (P < 0.01). So was in the groups injected with anti-CJT sera and the supernatants of PBMCs compared with control (P > 0.05). (2) Binding of CJT to the nerve was found dominant in the sciatic nerves taken from normal rats or human either incubated with CJT or in the pathological sciatic nerves induced by CJT to various degrees. The binding of CJT to all these nerves was determined. (3) After intravenous injection with CJT, no histopathologic change could be found in the other viscera of the rats, with the exception of remarkable pathological change in peripheral nerves.

CONCLUSIONS(1) CJT could remarkably damage the peripheral nerves in rats. Specific pathogenicity of CJT to peripheral nerves was well shown, because no histopathologic abnormalities could be found in the other viscera, such as brain, liver and kidney etc. although there was remarkable pathological change along the peripheral nerve in the animals. (2) No immunological pathogenicity of CJT could be demonstrated in the nerves of rats after immunization with CJT.

Animals ; Antibodies, Anti-Idiotypic ; blood ; Bacterial Toxins ; immunology ; toxicity ; Campylobacter jejuni ; immunology ; Exotoxins ; immunology ; toxicity ; Gene Expression ; drug effects ; Peripheral Nerves ; drug effects ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; genetics