Genome map of Campylobacter fetus subsp. fetus ATCC 27374

Abstract A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by Sal I, Sma I and Not I. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4–14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA , rpoB , and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes ( flaAB ) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis , respectively, were used to identify the locations of flaAB , the gyrA and the gyrB genes on the ATCC 27374 chromosome.     

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.     

Antigenic differences among Campylobacter fetus S-layer proteins.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.     

Flagellar apparatus ultrastructure inMesostigma viride (Prasinophyceae)

The ultrastructure of the flagellar apparatus ofMesostigma viride Lauterborn (Prasinophyceae) has been studied in detail with particular reference to absolute configurations, numbering of basal bodies, basal body triplets and flagellar roots. The two basal bodies are interconnected by three connecting fibers (one distal fiber = synistosome, and two proximal fibers). The flagellar apparatus shows 180° rotational symmetry; four microtubular flagellar roots and two system II fibers are present. The microtubular roots represent a 4-6-4-6-system. The left roots (1s, 2s) consist of 4 microtubules, each with the usual 3 over 1 root tubule pattern. Each right root (1d, 2d) is proximally associated with a small, but typical multi-layered structure (MLS). The latter displays several layers corresponding to the S₁ (the spline microtubules: 5–7), and presumably the S₂—S₄ (the lamellate layers) of the MLS of theCharophyceae. At its proximal origin (near the basal bodies) each right root originates with only two microtubules, the other spline microtubules being added more distally. The structural and positional information obtained in this study strongly suggest that one of the right roots (1d) ofMesostigma is homologous to the MLS-root of theCharophyceae and sperm cells of archegoniate land plants. Thus the typical cruciate flagellar root system of the green algae and the unilateral flagellar root system of theCharophyceae and archegoniates share a common ancestry. Some functional and phylogenetic aspects of MLS-roots are discussed.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Morphological Differences in Flagella in Campylobacter fetus Subsp. intestinalis and C. fetus Subsp. jejuni

Intact flagella were isolated from human pathogenic strains of Campylobacter, C. fetus subsp. intestinalis and C. fetus subsp. jejuni, by the method of DePamphilis and Adler and examined by electron microscopy. The isolated flagella were composed of a filament, a hook, a basal body, and a large disk associated with the end of the hook region covering the basal body. The width of the hook was approximately 28 nm, somewhat greater than that of the filament (20 nm in diameter). The hook region of C. fetus subsp. intestinalis was curved, but it was straight in C. fetus subsp. jejuni. The structure of the basal body of the two subspecies was similar to that reported for other gram-negative bacteria. The large disk detached from the flagella showed concentrically arranged circular structures. This structure was more clearly observed in the disk of C. fetus subsp. jejuni than in C. fetus subsp. intestinalis. Observations of thin-sectioned profiles at the attachment site of the flagellum revealed that the large disk is located on the inner side of the outer membrane. The role of the large disk in bacterial movement is not clear, but it is assumed that it acts as an organ to protect the flagellar insertion site from vigorous rotation of the polar end inflicted during bacterial movement.     

Respiratory systems and cytochromes in Campylobacter fetus subsp. intestinalis.

Cell suspensions of Campylobacter fetus subsp. intestinalis grown microaerophilically in complex media consumed oxygen in the presence of formate, succinate, and DL-lactate, and membranes had the corresponding dehydrogenase activities. The cells and membranes also had ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity which was cyanide sensitive. The fumarate reductase activity in the membranes was inhibited by p-chloromercuriphenylsulfonate, and this enzyme was probably responsible for the succinate dehydrogenase activity. Cytochrome c was predominant in the membranes, and a major proportion of this pigment exhibited a carbon monoxide-binding spectrum. Approximately 60% of the total membrane cytochrome c, measured with dithionite as the reductant, was also reduced by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. A similar proportion of the membrane cytochrome c was reduced by succinate under anaerobic conditions, whereas formate reduced more than 90% of the total cytochrome under these conditions. 2-Heptyl-4-hydroxyquinoline-N-oxide inhibited reduction of cytochrome c with succinate, and the reduced spectrum of cytochrome b became evident. The inhibitor delayed reduction of cytochrome c with formate, but the final level of reduction was unaffected. We conclude that the respiratory chain includes low- and high-potential forms of cytochromes c and b; the carbon monoxide-binding form of cytochrome c might function as a terminal oxidase.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Contamination of red-meat carcasses by Campylobacter fetus subsp. jejuni.

Campylobacter fetus subsp. jejuni was commonly present in the feces of unweaned calves (2 to 3 weeks old) and from two of four groups of sheep. One new season lamb (12 to 16 weeks old) carried the organism, but the bacteria were not isolated from cattle. With unweaned calves, the fractions of animals infected and carcasses contaminated were similar. Contamination of carcasses usually involved low densities of C. fetus subsp. jejuni (ca. 1 to 10/cm2), which were isolated from flank but not rump areas. The organism was recovered less frequently from chilled carcasses and deboned veal. Small numbers of C. fetus subsp. jejuni could be recovered from equipment during the processing of unweaned calves but not after routine cleaning.     

Reattachment of surface array proteins to Campylobacter fetus cells.

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 septic arthritis: report of a case.

We report a case of septic arthritis caused by the fastidious gram-negative rod Campylobacter fetus. We suggest that the organism may be part of the endogenous flora and that the clinical infections tend to occur in compromised hosts. Our patient is the first to be described with multiple myeloma and C. fetus septic arthritis. The documented cases of culture-proven C. fetus septic arthritis reported to date have occurred in three men and one woman, all in the seventh and eighth decades of life, with a mono-articular large joint distribution. The septic arthritis always occurred in previously injured joints and curiously enough need not be associated with a toxic-appearing patient. C. fetus infections are also associated with the signs and symptoms of clinical thrombophlebitis. We stress caution in establishing this diagnosis of phlebitis on clinical evaluation only and urge differentiation of true deep vein thrombophlebitis from pseudothrombophlebitis or dissected popliteal synovial cyst. This latter diagnosis may be made non-invasively by ultrasound techniques.     

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.