Differentiation of the subspecies of Campylobacter fetus by genomic sizing.

Campylobacter fetus subsp. fetus and C. fetus subsp. venerealis are currently differentiated by tolerance to glycine and by their epidemiology. Analysis of C. fetus DNA by pulsed-field gel electrophoresis, after digestion with the restriction endonucleases SmaI and SalI, was used to differentiate between the subspecies. All strains presently identified as C. fetus subsp. fetus had a genomic size of 1.1 Mb, whereas the majority of the C. fetus subsp. venerealis strains had a genomic size of 1.3 Mb. An additional group of strains, which were previously described as C. fetus subsp. venerealis biovar "intermedius" and were able to tolerate higher concentrations of glycine than the rest of the C. fetus subsp. venerealis strains, had an average genome size of 1.5 Mb. We suggest that pulsed-field gel electrophoresis may be useful as an additional aid in the differentiation of C. fetus strains at the subspecies level.     

Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods

AIMS: To assess the efficacy of numerical analysis of PFGE-DNA profiles for identification and differentiation of Campylobacter fetus subspecies. METHODS AND RESULTS: 31 Camp. fetus strains were examined by phenotypic, PCR- and PFGE-based methods, and the 16S rDNA sequences of 18 strains compared. Numerical analysis of PFGE-DNA profiles divided strains into two clusters at the 86% similarity level. One cluster contained 19 strains clearly identified as Camp. fetus subsp. venerealis. The other cluster comprised 12 strains, of which 10 were unambiguously identified as Camp. fetus subsp. fetus. The remaining two strains were identified as Camp. fetus subsp. venerealis by either phenotypic or PCR methods, but not both. At higher similarity levels, clusters containing isolates from each of two countries were identified, suggesting that certain clones predominate in certain geographical regions. CONCLUSION: Numerical analysis of PFGE-DNA profiles is an effective method for differentiating Camp. fetus subspecies. SIGNIFICANCE AND IMPACT OF THE STUDY: Critical comparison of PFGE, PCR, 16S rDNA sequencing and phenotypic methods for differentiation of Camp. fetus subspecies was attained. Novel phenotypic markers for distinguishing subspecies were identified. Evidence for dominant clones of each subspecies in certain countries was provided.     

A new antibiotic combination for frozen bovine semen 1. Control of mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus

Systematic evaluations of new combinations of antibiotics for the control of bovine mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus in a bovine frozen semen process were made. These organisms were standardized to 10(5) to 10(6) colony forming unit (CFU) and inoculated into each ml of raw semen. Antibiotics in a final volume of 0.02 ml were added to each ml of the raw semen and were contained at the same concentration in the nonglycerol portion of the extenders (whole milk, 20% egg yolk citrate, 20% egg yolk tris, Plus-X, and 28% egg yolk tris). The combination of gentamicin (500 ug/ml) tylosin (100 ug/ml) and Linco-Spectin (300/600 ug/ml) was more effective for the control of mycoplasmas and ureaplasmas and equally effective for the control of C. fetus subsp. venerealis and Haemophilus somnus than the standard combination of penicillin, dihydrostreptomycin and polymyxin B sulfate.     

Development of experimental genetic tools for Campylobacter fetus

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.     

Campylobacter fetus subspecies: Comparative genomics and prediction of potential virulence targets

The genus Campylobacter contains pathogens causing a wide range of diseases, targeting both humans and animals. Among them, the Campylobacter fetus subspecies fetus and venerealis deserve special attention, as they are the etiological agents of human bacterial gastroenteritis and bovine genital campylobacteriosis, respectively. We compare the whole genomes of both subspecies to get insights into genomic architecture, phylogenetic relationships, genome conservation and core virulence factors. Pan-genomic approach was applied to identify the core- and pan-genome for both C. fetus subspecies and members of the genus. The C. fetus subspecies conserved (76%) proteome were then analyzed for their subcellular localization and protein functions in biological processes. Furthermore, with pathogenomic strategies, unique candidate regions in the genomes and several potential core-virulence factors were identified. The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes in an island specific for C. fetus subsp. venerealis. The genomic variations and potential core and unique virulence factors characterized in this study would lead to better insight into the species virulence and to more efficient use of the candidates for antibiotic, drug and vaccine development.     

Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs.

Campylobacter fetus subsp. venerealis isolated from a case of human vaginosis was inoculated into the uterus of a C. fetus-negative heifer. Isolates obtained weekly from the vaginal mucus exhibited variations in high-molecular-mass-protein profiles from that of the original inoculum, which had a dominant 110-kDa S-layer protein. Immunoblots of the weekly isolates with monoclonal antibody probes against the 110-kDa S-layer protein and other C. fetus S-layer proteins demonstrated antigenic shifts. Genomic digests of the isolates probed with a 75-mer oligonucleotide of the conserved sapA region also indicated that antigenic variation of the S-layer is accompanied by DNA rearrangement.     

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

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.     

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.     

Reversible expression of flagella in Campylobacter spp.

The in vitro phase variation of flagella and the transition rates between flagellate and aflagellate phenotypes in Campylobacter species including C. jejuni, C. coli, C. lari (thermophilic campylobacters), C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis were investigated. The change from the flagellate to aflagellate phenotype was detected in all of the 12 Campylobacter strains studied. When measured in a motility medium, flagellate to aflagellate transition in thermophilic campylobacters, C. fetus and C. hyointestinalis strains occurred at a rate of 1.8 x 10(-3) to 7.5 x 10(-3), 3.0 x 10(-4) to 7.8 x 10(-4) and 1.8 x 10(-5) to 7.7 x 10(-6) per cell per generation, respectively. Transition from aflagellate to flagellate phenotype occurred at a rate of 5.8 x 10(-6) to 9.3 x 10(-6) per cell per generation in thermophilic campylobacters and 1.0 x 10(-6) to 1.5 x 10(-6) in C. fetus strains. No reversion from aflagellate to flagellate phenotype could be detected in C. hyointestinalis strains. It was concluded that the ability to reversibly express flagella was inherent in the wild-type strains and the transition rates for both directions were consistent for each strain.     

Interbacterial macromolecular transfer by the Campylobacter fetus subsp. venerealis type IV secretion system

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.     

Nitrogen oxide reduction in Wolinella succinogenes and Campylobacter species.

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.     

Sensitivity and specificity of culture and PCR of smegma samples of bulls experimentally infected with Tritrichomonas foetus

The sensitivity (Se) and specificity (Sp) of different testing schemes were estimated for detecting Tritrichomonas foetus (T. foetus) in smegma samples from experimentally infected bulls. Culture and polymerase chain reaction (PCR) on smegma samples were evaluated alone and in parallel testing. Mature dairy bulls (n=79) were intrapreputially inoculated with T. foetus (n=19); Campylobacter (C.) fetus venerealis (n=13); both T. foetus and C. fetus venerealis (n=11); Tetratrichomonas spp. (n=9); C. fetus fetus (n=8); or were not inoculated (n=19). For each bull, smegma samples were collected for 6 week post-inoculation and tested for T. foetus by In Pouch TF culture and PCR. Most T. foetus-inoculated bulls became infected, according to culture (86.7%), PCR (90.0%), and both tests together (93.3%). In T. foetus-inoculated bulls, both tests combined in parallel on a single sample had a Se (78.3%) and Sp (98.5%) similar to two cultures (Se 76.0%, Sp 98.5%) or two PCR (Se 78.0%, Sp 96.7%) sampled on consecutive weeks. The PCR on three consecutive weekly samples (Se 85.0%, Sp 95.4%) and both tests applied in parallel on three consecutive weekly samples (Se 87.5%, Sp 95.6%) were similar to the current gold-standard of six weekly cultures (Se 86.7% and Sp 97.5%). Both tests used in parallel six times had the highest Se (93.3%), with similar Sp (92.5%). Tetratrichomonas spp. were only sporadically detected by culture or PCR. In conclusion, we have proposed alternative strategies for T. foetus diagnostics (for the AI industry), including a combination of tests and repeat testing strategies that may reduce time and cost for bull surveillance.     

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.     

Effects of Penicillin and Glycine on Cell Wall Glycopeptides of the Two Varieties of Vibrio fetus

Actively growing strains of Vibrio fetus venerealis and V. fetus intestinalis, none of which produced penicillinase, were treated with inhibitory levels of penicillin or glycine, primarily to gain insight into the differential sensitivities of the two varieties to both of these compounds. Treatments induced the accumulation of uridine nucleotide glycopeptide precursors which contained amino sugars and amino acids in various molar ratios. Penicillin-induced nucleotides all contained muramic acid and sometimes glucosamine; they generally contained alanine, glutamic acid, diaminopimelic acid, and glycine. Approximately equimolar ratios of these components were observed in some compounds, but ratios varied considerably in others. Glycine-induced nucleotides contained muramic acid and, in some instances, glucosamine. Amino acids were detected only infrequently and usually in low molar ratios. The data suggest that penicillinase production, differences in the chemical composition of glycopeptide, and variations in modes of action of penicillin and glycine cannot individually account for the differential sensitivities of venereal and intestinal strains of V. fetus to these substances.     

In vitro survival of campylobacters in human amniotic fluid

Human amniotic fluids supported growth of Campylobacter fetus subsp. fetus, C. jejuni and C. coli. Campylobacters remained viable for up to 11–12 weeks in amniotic fluid.     

The isolation and prevalence of campylobacters from dairy cattle using a variety of methods

Faecal samples from 94 dairy cows and 42 calves in three different herds were examined by a variety of techniques for campylobacters. Cefoperazone amphotericin teicoplanin (CAT) agar, modified cefoperazone charcoal deoxycholate agar (mCCDA), Karmali agar, and membrane filtration onto blood agar, were used with and without enrichment in CAT broth. Seventy-nine percent of cattle in herd A carried campylobacters, compared with 40% and 37·5% of cattle in herds B and C, respectively. Most animals carried only one species of Campylobacter . Campylobacter hyointestinalis was isolated most frequently (32% animals positive) with Camp. fetus subsp. fetus and Camp. jejuni subsp. jejuni detected in 11% and 7% of animals, respectively. In addition, a novel biotype of Camp. sputorum was isolated from 60% of 47 cows tested in herd A. Direct plating detected only two of the total of 40 animals positive for campylobacter. Enrichment in CAT broth before membrane filtration onto blood agar or CAT agar were the most successful methods of plating. Campylobacter sputorum was isolated from CAT agar and blood agar but not from mCCDA or Karmali agar. Karmali agar incubated at 30 °C was especially effective for isolating Camp. fetus subsp. fetus .     

Oxygen tolerance estimates in Campylobacter species depend on the testing medium

J.P. HODGE AND N.R. KRIEG. 1994. Oxygen tolerance of the microaerophile Campylobacter jejuni subsp. jejuni varied with different brands of complex media which were used for plating the dilute cell suspensions. The tryptone component was one factor. With some tryptones growth occurred at 21% oxygen whereas with others there was no growth at oxygen levels of 15% or higher. A chemically-defined, agar-solidified plating medium was used to estimate the oxygen tolerance of Camp. jejuni subsp. jejuni, Camp. coli and Camp. fetus subsp. fetus , and also to assess the effect of added scavengers of reactive oxygen intermediates on the oxygen tolerance. Some scavengers such as allopurinol, azelaic acid, caffeine, cimetidine, TEMPOL and pyruvate enhanced oxygen tolerance markedly whereas others such as carnosine, dimethyl thiourea, spermidine and superoxide dismutase had little effect.     

Lytic Activity of Vibrio Phages on Strains of Vibrio fetus Isolated from Man and Animals

Five phages isolated from lysogenic strains of Vibrio fetus var. venerealis and two from V. fetus var. intestinalis were tested for lytic activity on 95 V. fetus strains from various animal and human hosts. In addition, virion and plaque morphology of the seven phages were compared. Electron micrographs showed that all were the kite-tailed variety with minor variations in head and tail dimensions. Plaques of V45 and V2 were small, clear and irregular; those of V3, V8, and V19 were large, clear and regular at the edge; the plaques of V16 and V20 were intermediate in size, clear, and very irregular at the edge with satellite plaques. The number of strains lysed by one or more phages were as follows: 29 of 30 from cattle; 7 of 11 from sheep; 1 of 5 from pigs; 1 of 1 from a monkey; and 33 of 42 from human hosts. Four natural groups of phages were derived by statistical measures of percentage of similarity in lytic activity. Group III lysed more strains (46 of 95) than any of the others. Twenty-five strains were lysed by group IV, 23 strains by group I, and 19 strains by group II. Results of this study indicate that phage typing should be a practical supplement to other differential tests for V. fetus.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway.

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.