Identification of Campylobacter fetus by PCR-DNA probe method.

A PCR method for rapid identification of Campylobacter fetus subsp. fetus was evaluated. A fragment of the gene coding for 16S rRNA was amplified from crude cell lysates of 18 C. fetus strains and 30 strains representing other Campylobacter species and subspecies. The amplicons were probed by dot blot hybridization with a digoxigenin-labeled C. fetus-specific oligonucleotide probe. The probe reacted only with C. fetus subsp. fetus and C. fetus subsp. venerealis and may be useful for rapid identification in clinical laboratories.     

Application of different chromosomal DNA restriction digest fingerprints to specific and subspecific identification of Campylobacter isolates.

Eleven strains comprising representatives of different subspecies, biotypes, and serotypes of Campylobacter jejuni and reference strains of C. coli, C. fetus subsp. fetus, C. hyointestinalis, and C. sputorum subsp. sputorum were studied to assess the utility of different DNA profiles for measuring fine differences between allied bacteria. Strains were compared by analyses of HaeIII and XhoI digest patterns of chromosomal DNA and Southern blot hybridization patterns of XhoI digests obtained with an Escherichia coli 16S + 23S rRNA gene probe. Visual comparisons and numerical analyses of the HaeIII and XhoI digest patterns both revealed clear differences between the five Campylobacter species and between representatives of C. jejuni subspecies and biotypes. Only strains with the same Penner serotype gave identical total digest polymorphisms. The advantages of XhoI total digests and Southern blot hybridization patterns were that they were less complex than the HaeIII patterns and easier to compare visually. However, numerical analysis of XhoI data resulted in reduced discrimination. We conclude that DNA fingerprinting using either HaeIII or XhoI fragment polymorphisms has considerable potential as a generally applicable method for identification of Campylobacter isolates, especially at the infrasubspecific level.     

Genetic divergence of Campylobacter fetus strains of mammal and reptile origins

Campylobacter fetus is a gram-negative bacterial pathogen of both humans and animals. Two subspecies have been identified, Campylobacter fetus subsp. fetus and Campylobacter fetus subsp. venerealis, and there are two serotypes, A and B. To further investigate the genetic diversity among C. fetus strains of different origins, subspecies, and serotypes, we performed multiple genetic analyses by utilizing random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and DNA-DNA hybridization. All 10 primers used for the RAPD analyses can distinguish C. fetus strains of reptile and mammal origin, five can differentiate between C. fetus subsp. fetus and C. fetus subsp. venerealis strains, and four showed differences between type A and type B isolates from mammals. PFGE with SmaI and SalI digestion showed varied genome patterns among different C. fetus strains, but for mammalian C. fetus isolates, genome size was well conserved (mean, 1.52 +/- 0.06 Mb for SmaI and 1.52 +/- 0.05 Mb for SalI). DNA-DNA hybridization demonstrated substantial genomic-homology differences between strains of mammal and reptile origin. In total, these data suggest that C. fetus subsp fetus strains of reptile and mammal origin have genetic divergence more extensive than that between the two subspecies and that between the type A and type B strains. Combining these studies with sequence data, we conclude that there has been substantial genetic divergence between Campylobacter fetus of reptile and mammal origin. Diagnostic tools have been developed to differentiate among C. fetus isolates for taxonomic and epidemiologic uses.     

30 years of campylobacters: biochemical characteristics and a biotyping proposal for Campylobacter jejuni.

Several biochemical test systems were studied for their potential usefulness for the examination of strains of Campylobacter species. Most (81%) of the C. jejuni strains hydrolyzed sodium hippurate, but strains of C. fetus, C. sputorum, and C. fecalis did not. Some (46%) of the C. jejuni strains and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains hydrolyzed DNA, but the C. fetus and C. sputorum subsp. mucosalis strains did not. Strains of all species of Campylobacter grew on charcoal-yeast extract agar, but 47% of the C. jejuni strains did not. Alkaline phosphatase activity was recorded for some strains of C. jejuni, but all other species were negative for this activity. Aryl sulfatase activity was detected in 7% of the C. jejuni, 15% of the C. fetus subsp. fetus, and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains, but it was not detected in the C. fetus subsp. venerealis and C. sputorum subsp. mucosalis strains. Most (93%) of the C. jejuni but none of the other Campylobacter strains contained lactobacillic acid when examined for cellular fatty acids. On the basis of results from three of these tests (hippurate hydrolysis, DNA hydrolysis, and growth on charcoal-yeast extract agar), clinical strains of C. jejuni were placed in eight biotypes.     

Hydrogenase activity in catalase-positive strains of Campylobacter spp.

A rapid hydrogenase assay has been developed which may be useful in separating the species Campylobacter jejuni and C. coli from the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. This assay employs the impermeant redox dye benzyl viologen, and positive determinations can be made within 20 min. All strains of C. jejuni and C. coli were found to be strongly hydrogenase positive. All strains of C. fetus subsp. fetus and C. fetus subsp. venerealis were negative for hydrogenase when the assay was performed at a benzyl viologen concentration of 2 mM and an incubation temperature of 30 degrees C. Some strains of C. fetus had low levels of hydrogenase as determined with cell extracts but were hydrogenase negative by the benzyl viologen assay. Since there are few rapid diagnostic tests available for screening Campylobacter isolates, we hope that the rapid hydrogenase assay will prove useful.     

Uncommon Campylobacter species in infant Macaca nemestrina monkeys housed in a nursery.

Studies were conducted to characterize 18 isolates of Campylobacter spp. that could not be identified as either Campylobacter jejuni or C. coli. The isolates were cultured from specimens from 13 of 18 infant nonhuman primates during a prospective epidemiologic study reported previously. Phenotypic tests, DNA hybridization, and analysis of DNA coding for rRNA identified the isolates as C. butzleri (seven isolates), C. hyointestinalis (seven isolates), and C. fetus subsp. fetus or C. fetus subsp. fetus-like organisms (four isolates). Ribotype and polyacrylamide gel electrophoresis patterns indicated that there was heterogeneity among the isolates of C. butzleri and C. fetus subsp. fetus-like organisms.     

Evidence that the Campylobacter fetus sap locus is an ancient genomic constituent with origins before mammals and reptiles diverged

Campylobacter fetus bacteria, isolated from both mammals and reptiles, may be either subsp. fetus or subsp. venerealis and either serotype A or serotype B. Surface layer proteins, expressed and secreted by genes in the sap locus, play an important role in C. fetus virulence. To assess whether the sap locus represents a pathogenicity island and to gain further insights into C. fetus evolution, we examined several C. fetus genes in 18 isolates. All of the isolates had 5 to 9 sapA or sapB homologs. One strain (85-387) possessed both sapA and sapB homologs, suggesting a recombinational event in the sap locus between sapA and sapB strains. When we amplified and analyzed nucleotide sequences from portions of housekeeping gene recA (501 bp) and sapD (450 bp), a part of the 6-kb sap invertible element, the phylogenies of the genes were highly parallel. Among the 15 isolates from mammals, serotype A and serotype B strains generally had consistent positions. The fact that the serotype A C. fetus subsp. fetus and subsp. venerealis strains were on the same branch suggests that their differentiation occurred after the type A-type B split. Isolates from mammals and reptiles formed two distinct tight phylogenetic clusters that were well separated. Sequence analysis of 16S rRNA showed that the reptile strains form a distinct phylotype between mammalian C. fetus and Campylobacter hyointestinalis. The phylogenies and sequence results showing that sapD and recA have similar G + C contents and substitution rates suggest that the sap locus is not a pathogenicity island but rather is an ancient constituent of the C. fetus genome, integral to its biology.     

Discrimination among thermophilic Campylobacter species by polymerase chain reaction amplification of 23S rRNA gene fragments.

By comparing nucleic acid sequences determined for one of the most variable areas of 23S rRNA genes of 23 Campylobacter strains, we were able to identify regions specific for thermophilic Campylobacter strains. Oligonucleotide primers corresponding to these unique regions were synthesized and used in the polymerase chain reaction. One primer pair selectively detected all thermophilic Campylobacter species, while four other primer pairs allowed discrimination among the thermophilic species Campylobacter coli, Campylobacter jejuni subsp. jejuni, Campylobacter lari, and Campylobacter upsaliensis. All primer sets were tested successfully on a large number of clinical isolates.     

Hippurate hydrolysis by Campylobacter fetus.

An additional method for differentiating between Campylobacter fetus subsp. jejuni and C. fetus subsp. intestinalis is reported. Strains of C. fetus subsp. jejuni (18/20) were shown to hydrolyze hippurate in the 2-h rapid test, whereas strains of C. fetus subsp. intestinalis did not.     

Cellular fatty acid composition of Campylobacter fetus.

The cellular fatty acid composition of 29 human and animal isolates of Campylobacter fetus was determined with gas-liquid chromatography. All strains contained small amounts of 3-hydroxytetradecanoic acid, suggesting the presence of lipid A. Each of 13 different blood or stool isolates of C. fetus subsp. jejuni obtained from humans or fowl contained a 19-carbon cyclopropane acid which was not present in C. fetus subsp. fetus (6 strains from cattle) or C. fetus subsp. intestinalis (10 strains from humans and cattle). These findings indicate that C. fetus subsp. jejuni can be rapidly differentiated from other C. fetus by gas-liquid chromatography analysis of cellular fatty acids.     

Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals

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.     

Probable Campylobacter fetus subsp. fetus gastroenteritis.

Three strains of Campylobacter fetus subsp. fetus isolated from cases of gastroenteritis are reported. DNA-DNA hybridizations in addition to biochemical tests were used to confirm the identification of the isolates as C. fetus since all strains grew at 42 degrees C. These isolates, like other C. fetus strains, are susceptible to cephalothin and thus would not have been detected in laboratories with Campylobacter isolation media containing this component.     

Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans.

To determine whether lipopolysaccharide (LPS) structures of Campylobacter fetus are related to the three known heat-stable serogroups, proteinase K-treated whole cell lysates obtained from strains of each serogroup were electrophoresed in polyacrylamide gels. All strains had smooth-type LPS with multiple high-molecular-weight repeating units. The profiles of serogroup A from C. fetus subsp. fetus and from C. fetus subsp. venerealis were identical, but they were different from those of C. fetus subsp. fetus serogroups B and AB. When we immunoblotted the LPS of these serogroups with normal or immune rabbit serum we found homologous recognition between serogroups A from C. fetus subsp. fetus and C. fetus subsp. venerealis. Similarly, serogroups AB and B from C. fetus subsp. fetus showed homologous recognition. However, antiserum against serogroup A did not recognize serogroups B and AB and vice versa. Absorption studies confirmed the identity of LPS from all serogroup A C. fetus strains and cross-reactivity of the serogroup B and AB strains with one another. Serogroup A strains were resistant to the bactericidal activity in normal human serum, whereas serogroup B and AB strains generally were susceptible; isolates from humans predominantly belonged to serogroup A. Results of these studies suggest that the LPS composition forms the basis for the heat-stable serotyping system for C. fetus and that the structural and antigenic variants are associated with differential serum susceptibility.     

Search for Sendai 6/94 viral RNA in the antigen-free cell line Cl-C-2 isolated from human multiple sclerosis brain tissue.

The technique of immunoblotting was used to identify the surface protein antigens of Campylobacter jejuni. Polyclonal antisera were raised in rabbits to formalinized cells of a typical human fecal isolate, C. jejuni VC74. Surface components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractions analyzed included whole cell lysates, sarcosinate-extracted outer membranes, released outer membrane blebs (fragments), isolated flagella, 0.2 M glycine-hydrochloride (pH 2.2) extract, saline extract, and material released by osmotic shocking. The ability of the antisera to recognize corresponding antigens on other strains of thermophilic campylobacters and Campylobacter fetus was also determined. The results demonstrated that heat-labile antigenic specificity was conferred on C. jejuni VC74 by an outer membrane protein with an approximate molecular weight of 92,500. Both the major outer membrane protein and the flagella were immunogenic but did not confer either strain or species serospecificity on the strains tested. Another major antigen on thermophilic campylobacter cells was a surface protein with an approximate molecular weight of 31,000. This common antigen was preferentially removed by glycine extraction but was not detectable in outer membrane prepared by sarcosinate extraction.     

Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. II. PCR direct sequencing of the Campylobacter DNA]

We have developed a sensitive DNA hybridization assay for the detection and identification of Campylobacter species which are recognized as important pathogens of acute diarrheal disease in humans. This technique utilizes DNA probes complementary to nucleotide sequences present in 16S ribosomal RNA (rRNA) of C. jejuni, C. coli, C. laridis, C. fetus, C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis, and polymerase chain reaction. The partial sequence of DNAs encoding the Campylobacter rRNA was first analyzed by direct solid phase sequencing in order to select suitable DNA probes. Amplified target DNA of 240 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. In identification experiments, one of the 10 probes tested here gave a positive hybridization reaction with C. jejuni, C. coli and C. hyointestinalis but not with other Campylobacter species. The other was specifically reactive with C. fetus, C. fetus subsp. fetus and C. fetus subsp. venerealis. When applied to stool specimens, a good correlation was found between the results obtained by the present assay and by biochemical tests. These findings suggest that the nonradioactive probe assay can be used as the practical criterion for differentiating Campylobacter species.     

Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis

Four isolates of an unclassified microaerophilic bacterium resembling Campylobacter species were characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, and protein profile analysis. The unclassified isolates were differentiated from Campylobacter jejuni, Campylobacter coli, Campylobacter fetus subsp. fetus, Campylobacter laridis, Campylobacter pylori, and an ovine isolate. The bacterium was fusiform shaped with a corrugated surface due to the presence of periplasmic fibers and had multiple bipolar flagella. Biochemically, the bacterium was separated from the Campylobacter controls by its negative catalase reaction, negative nitrate reduction, and no growth in 1% glycine. It was also resistant to ampicillin. Protein profile analysis demonstrated nine major protein bands present in the unclassified isolates that were absent in the Campylobacter controls. The bacterium also differed from the ovine isolate by its negative catalase reaction, rapid urea hydrolysis, and susceptibility to clindamycin, erythromycin, and tetracycline. Our results showed that the unclassified bacterium was distinct from the recognized Campylobacter species.     

Biochemical and genetic characteristics of atypical Campylobacter fetus subsp. fetus strains isolated from humans in the United States.

During a 2-year period, 14 biochemically atypical Campylobacter fetus subsp. fetus-like strains were received by the Campylobacter Reference Laboratory at the Centers for Disease Control. Sources of the isolates were blood, nine strains; stools, two strains; amniotic fluid, one strain; and abscesses, two strains. Atypical phenotypic characteristics exhibited by one or more strains were growth at 42 degrees C, 10 strains; no H2S by lead acetate paper, 3 strains; resistance to a 30-micrograms cephalothin disk, 2 strains; and nonmotility, 1 strain. By DNA-DNA hybridization, all 14 isolates and the type strain of C. fetus subsp. fetus (ATCC 27374) were 94 to 100% related in reassociation reactions at 50 degrees C, with 0.0 to 0.5% divergence, and were 86 to 100% related in reassociation reactions at 65 degrees C. Thus, all of these atypical strains were C. fetus subsp. fetus. MICs of 11 antimicrobial agents for these 14 strains were variable. All strains were susceptible to chloramphenicol, erythromycin, gentamicin, and tetracycline, and most were susceptible to ampicillin, clindamycin, and penicillin. Eleven strains were resistant to cephalothin (MIC greater than or equal to 16 micrograms/ml), nine were resistant to rifampin (MIC greater than or equal to 8 micrograms/ml), and all were resistant to nalidixic acid (MIC greater than 32 micrograms/ml) and vancomycin (MIC greater than 32 micrograms/ml). One can expect to see biochemical variability in C. fetus subsp. fetus strains and to encounter such strains from a variety of human sources, the most important of which appears to be blood.     

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.     

Identification of Campylobacter fetus subspecies by phenotypic differentiation and PCR

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis and C. fetus subsp. fetus, which differ in their epidemiologies and clinical importance. The differences between these subspecies make accurate distinction between the two essential. First, the value of seven key tests for the traditional differentiation of C. fetus was investigated. Afterwards, the results of the phenotypic differentiation and PCR were compared to address the question of the reliability of this PCR assay. Altogether, 103 C. fetus isolates were investigated, including the type strains of C. fetus subsp. fetus and C. fetus subsp. venerealis. Depending on the result of the glycine tolerance test, the isolates could be separated into 81 C. fetus subsp. venerealis isolates (glycine intolerant) and 22 C. fetus subsp. fetus isolates (glycine tolerant). For all C. fetus subsp. venerealis strains tested, the results of the selenite reduction assay and sensitivity to metronidazole and cefoperazone completely agreed with the results of the glycine tolerance test (correspondence, 100%). Seventy-three C. fetus subsp. venerealis isolates did not grow at 42 degrees C (correspondence, 90.1%), but eight isolates showed a faintly discernible, flat, dark gray growth. For 22 C. fetus subsp. fetus isolates, the results of additional phenotypic tests only partly agreed with the results of the glycine tolerance test. For C. fetus subsp. fetus the results of the glycine tolerance test showed a relatively good correspondence with those of the selenite reduction assay (correspondence, 81.8%), assays for cefoperazone resistance (correspondence, 86.4%), and assays for growth at 42 degrees C (correspondence, 81.8%). The results of the glycine tolerance test and PCR completely agreed for the 103 C. fetus isolates tested. We conclude that at present the traditional phenotypic characterization of C. fetus subspecies under strongly defined conditions remains indispensable, but this PCR assay constitutes a valuable adjunctive technique for the confirmation of phenotypic results.     

Chemotactic response to formate by Campylobacter concisus and its potential role in gingival colonization.

Human clinical strains of Campylobacter jejuni and Campylobacter fetus subsp. fetus colonized the gastrointestinal tracts of both athymic (nu/nu) and euthymic (+/nu) germfree mice (BALB/c). Viable Campylobacter spp. (10(9) to 10(10) CFU/g [dry weight] of cecum and colon contents) were isolated on day 3 after oral challenge, and similar large numbers of viable cells were evident at several intervals during a 10-month experiment. The stomachs and upper small intestines of nu/nu and +/nu mice that were monoassociated for 224 days with C. jejuni 45100 contained 3 to 4 logs fewer viable bacteria than did their ceca or colons. Athymic mice that were monoassociated for 224 days with C. fetus subsp. fetus had 2 to 3 logs more viable Campylobacter spp. in their upper gastrointestinal tracts than did their +/nu littermates. Large viable populations (approximately 10(9)/g of contents) of C. fetus subsp. fetus were in the ceca and colons of both nu/nu and +/nu mice. All C. jejuni strains used in this study chronically infected the mesenteric lymph nodes of both nu/nu and +/nu mice. C. jejuni strains 24 and INN 73-83, which were cytotoxic for Chinese hamster ovary cells in vitro, were also more frequently isolated from the livers, spleens, and kidneys of nu/nu mice than was the weak cytotoxin-producing strain 45100. Additionally, heat-labile-enterotoxin-producing C. jejuni INN 73-83 was recovered more frequently from the internal organs of monoassociated +/nu mice than were any other Campylobacter spp. tested. Natural gastrointestinal colonization of neonatal nu/nu and +/nu mice (born to Campylobacter-colonized mothers) with Campylobacter spp. appeared to be delayed until approximately 1 to 2 weeks after birth. Conventionalization of C. jejuni 45100-monoassociated BALB/c mice with a complex mouse fecal microflora eliminated viable C. jejuni from the mesenteric lymph nodes by day 14 and from the cecum by day 78. These findings show that the gnotobiotic BALB/c mouse is a new model for studying acute and chronic host-Campylobacter sp. interactions.