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Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff) and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1-5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft, 14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution.

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.

Campylobacter fetus (C. fetus) bacteremia is rarely reported. In this article, a 51-year-old Chinese woman with severe hepatitis B virus infection complicated with alcoholic liver cirrhosis was admitted with a 3-month history of fatigue, jaundice, and edema of lower extremity, as well as presenting a high fever. Blood cultures were positive for Campylobacter fetus. The patient was started on treatment with cefuroxime, which was switched after 3 days to meropenem for an additional 2 weeks. The patient was successfully treated with the 3-week course of intravenous antibiotic therapy.

Campylobacter fetus often causes systemic infection in immunocompromised or older patients, and prenatal infection, but Campylobacter has rarely been reported as a cause of adnexitis in healthy young women. Here we report two cases of endometriotic cysts infected by C. fetus for the first time. In case 1, a 28-year-old woman with a left adnexal cyst was hospitalized for left tubo-ovarian abscess and underwent left salpingo-oophorectomy. In case 2, a 22-year-old woman with a right adnexal cyst was hospitalized for a bilateral tubo-ovarian abscess and underwent right salpingo-oophorectomy and left salpingectomy. In both cases, C. fetus was detected on culture, and histopathology indicated a purulent endometriotic cyst. The present findings suggest that endometriotic cyst can be a focus of C. fetus infection., (© 2016 Japan Society of Obstetrics and Gynecology.)

Campylobacter fetus currently comprises three recognized subspecies, which display distinct host association. Campylobacter fetus subsp. fetus and C fetus subsp. venerealis are both associated with endothermic mammals, primarily ruminants, whereas C fetus subsp. testudinum is primarily associated with ectothermic reptiles. Both C. fetus subsp. testudinum and C. fetus subsp. fetus have been associated with severe infections, often with a systemic component, in immunocompromised humans. To study the genetic factors associated with the distinct host dichotomy in C. fetus, whole-genome sequencing and comparison of mammal- and reptile-associated C fetus was performed. The genomes of C fetus subsp. testudinum isolated from either reptiles or humans were compared with elucidate the genetic factors associated with pathogenicity in humans. Genomic comparisons showed conservation of gene content and organization among C fetus subspecies, but a clear distinction between mammal- and reptile-associated C fetus was observed. Several genomic regions appeared to be subspecies specific, including a putative tricarballylate catabolism pathway, exclusively present in C fetus subsp. testudinum strains. Within C fetus subsp. testudinum, sapA, sapB, and sapAB type strains were observed. The recombinant locus iamABC (mlaFED) was exclusively associated with invasive C fetus subsp. testudinum strains isolated from humans. A phylogenetic reconstruction was consistent with divergent evolution in host-associated strains and the existence of a barrier to lateral gene transfer between mammal- and reptile-associated C fetus Overall, this study shows that reptile-associated C fetus subsp. testudinum is genetically divergent from mammal-associated C fetus subspecies., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: Campylobacter fetus subspecies fetus (CFF) is an important pathogen for both cattle and humans. We performed a systematic epidemiological and clinical study of patients and evaluated the genetic relatedness of 17 human and 17 bovine CFF isolates by using different genotyping methods. In addition, the serotype, the dissemination of the genomic island containing a type IV secretion system (T4SS) and resistance determinants for tetracycline and streptomycin were also evaluated., Methods: The isolates from patients diagnosed with CFF infection as well as those from faecal samples of healthy calves were genotyped using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), as well as single locus sequence typing (SLST) targeting cmp1 and cmp2 genes encoding two major outer membrane proteins in CFF. The presence of the genomic island and identification of serotype was determined by PCRs targeting genes of the T4SS and the sap locus, respectively. Tetracycline and streptomycin resistance phenotypes were determined by minimal inhibitory concentration. Clinical data obtained from medical records and laboratory data were supplemented by data obtained via telephone interviews with the patients and treating physicians., Results: PFGE analysis defined two major clusters; cluster A containing 16 bovine (80 %) isolates and cluster B containing 13 human (92 %) isolates, suggesting a host preference. Further genotypic analysis using MLST, SLST as well as sap and T4SS PCR showed the presence of genotypically identical isolates in cattle and humans. The low diversity observed within the cmp alleles of CFF corroborates the clonal nature of this pathogen. The genomic island containing the tetracycline and streptomycin resistance determinants was found in 55 % of the isolates in cluster A and correlated with phenotypic antibiotic resistance., Conclusions: Most human and bovine isolates were separated on two phylogenetic clusters. However, several human and bovine isolates were identical by diverse genotyping methods, indicating a possible link between strains from these two hosts.

The objective is to discuss sexually transmitted diseases caused by Tritrichomonas foetus (T foetus) and Campylobacter fetus (C fetus) subsp. venerealis, with a focus on prevalence, pathogenesis, and diagnosis in cows and bulls. Diagnosis and control are problematic because these diseases cause severe reproductive losses in cows, but in bulls are clinically asymptomatic, which allows the disease to flourish, especially in the absence of legislated control programs. We review research regarding prophylactic systemic immunization of bulls and cows with antigens of T foetus and C fetus venerealis and their efficacy in preventing or clearing preexisting infections in the genital tract. Current diagnostic methods of C fetus venerealis and T foetus (microbial culture and PCR) should be improved. Review of the latest advances in bovine trichomoniasis and campylobacteriosis should promote knowledge and provide an impetus to pursue further efforts to control bovine sexually transmitted diseases., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100% of the heifers and 80% of the bulls were infected, while in the Cfv group, 50% of the heifers and 60% of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6% increase in the Cff group and 7.4% in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40% less for the Cff group and 5.2% for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls., (Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0% to 100% and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.

Control of Campylobacter in the food chain requires a better understanding of the behaviour of the bacteria in relevant environments. Campylobacter species are largely non-pathogenic in poultry, the body temperature of which is 42 °C. However, the bacteria are highly pathogenic in humans whose body temperature is 37 °C. The aim of this study was to examine if switching from commensal to pathogenic behaviour was related to temperature. We examined the growth, motility and invasion of T84 cells by three species of Campylobacter: C. jejuni 81116, C. jejuni M1, C. coli 1669, C. coli RM2228 and C. fetus fetus NC10842 grown at 37 and 42 °C. Our results suggest that C. jejuni isolates grow similarly at both temperatures but some are more motile at 42 °C and some are more invasive at 37 °C, which may account for its rapid spread in poultry flocks and for infection in humans, respectively. C. coli, which are infrequent causes of Campylobacter infections in humans, is less able to grow and move at 37 °C compared to 42 °C but was significantly more invasive at the lower temperature. C. fetus fetus, which is infrequently found in poultry, is less able to grow and invade at 42 °C.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Campylobacter fetus infection is a substantial problem in herds of domestic cattle worldwide and a rising threat in human disease. Application of comparative and functional genomics approaches will be essential to understand the molecular basis of this pathogen's interactions with various hosts. Here we report recent progress in genome analyses of C. fetus ssp. fetus and C. fetus ssp. venerealis, and the development of molecular tools to determine the genetic basis of niche-specific adaptations. Campylobacter research has been strengthened by the rapid advancements in imaging technology occurring throughout microbiology. To move forward in understanding the mechanisms underlying C. fetus virulence, current efforts focus on developing suitable in vitro models to reflect host- and tissue-specific aspects of infection.

Campylobacter fetus is a recognized pathogen of cattle and sheep, though human infection has also been reported. Ingestion of contaminated food or water is a proposed route of transmission for both humans and animals. The subsequent detection of the organism from extra-intestinal and systemic locations implies an ability to translocate across epithelial barriers. To determine how C. fetus disseminates from the intestine, Caco-2 cells cultured on porous membrane supports, were used as model intestinal epithelial cell monolayers. C. fetus was found to translocate equally well in both apical-to-basolateral and basolateral-to-apical directions for up to 24 h without altering Caco-2 cell monolayer permeability as assessed by transepithelial resistance and absence of paracellular diffusion of FITC-inulin. Using modified antibiotic protection assays, C. fetus was also observed to invade and subsequently egress from Caco-2 cells. Caco-2 cell invasion and translocation occurred independently of C. fetus S layer expression. Scanning and transmission electron microscopy revealed the presence of C. fetus associated with both apical and basal surfaces as well as in intracellular locations. C. fetus was, however, never observed in paracellular locations nor associated with Caco-2 cells junctions. Neither C. fetus invasion nor translocation across Caco-2 cell monolayers was impacted by latrunculin A, though translocation was enhanced in the presence of cytochalasin D which disrupted tight junctions. Tubulin cytoskeleton disrupting agents, colchicine and vinblastine, did inhibit C. fetus translocation though entry into Caco-2 cells remained unaffected. Together, translocation without disrupting monolayer integrity, invasion and egression from Caco-2 cells, electron microscopy observations and the requirement of a functional tubulin cytoskeleton for translocation, support a transcellular mechanism of C. fetus translocation across Caco-2 cell monolayers. The ability to invade and subsequently egress would contribute to establishment of an infecting C. fetus population in the host, while the demonstrated ability to translocate across model intestinal epithelial barriers accounts for the observed in vivo recovery of C. fetus from extra-intestinal locations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The pathogen Campylobacter fetus comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis. Although these taxa are highly related on the genome level, they are adapted to distinct hosts and tissues. C. fetus subsp. fetus infects a diversity of hosts, including humans, and colonizes the gastrointestinal tract. In contrast, C. fetus subsp. venerealis is largely restricted to the bovine genital tract, causing epidemic abortion in these animals. In light of their close genetic relatedness, the specific niche preferences make the C. fetus subspecies an ideal model system to investigate the molecular basis of host adaptation. In this study, a subtractive-hybridization approach was applied to the genomes of the subspecies to identify different genes potentially underlying this specificity. The comparison revealed a genomic island uniquely present in C. fetus subsp. venerealis that harbors several genes indicative of horizontal transfer and that encodes the core components necessary for bacterial type IV secretion. Macromolecular transporters of this type deliver effector molecules to host cells, thereby contributing to virulence in various pathogens. Mutational inactivation of the putative secretion system confirmed its involvement in the pathogenicity of C. fetus subsp. venerealis.

Background: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (approximately 75-80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes., Results: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp., Conclusion: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

Campylobacter fetus subsp. fetus infection can occur in female sheep, causing infertility or abortion. Despite extensive research on the effect of these bacteria on female fertility, little research has been done on the influence of C. fetus subsp. fetus on the male factor. Our objective was to examine the influence of C. fetus subsp. fetus on ram sperm. Motility index, percentage of live spermatozoa, mean alphat value (an indication of the chromatin stability of the sperm cell) and percentage of sperm cells expressing the FAS receptor were measured in sperm incubated in the presence or absence of C. fetus subsp. fetus. The motility index and viability of sperm incubated with the bacteria were lower than those of untreated sperm samples after 5 h. In bacteria-incubated sperm cells, the percentage expressing FAS receptor was already significantly elevated at 15 min. Bacteria-incubated sperm showed a greater prevalence of morphological damage. The bacteria were attached to tail and acrosome regions, and the sperm damage was concentrated in both the motility and chromatin regions. Bacteria-infected sperm cells showed a decrease in motility, increase in early acrosome reaction and chromatin damage. Similar effects were induced by incubation of the sperm with supernatants from C. fetus subsp. fetus cultures. Thus this study demonstrates that C. fetus subsp. fetus has a detrimental effect on the quality of ram sperm.

Campylobacter species are well-recognized common causes of gastrointestinal infections. While Campylobacter jejuni is probably the most common Campylobacter isolated in humans, Campylobacter fetus is rather infrequent and mostly related with bacteraemia. Even on such occasions, it seems that immunocompetent individuals are spared. We report a case of C. fetus bacteraemia in a healthy-except for impaired fasting glucose (IFG) levels-farmer, presenting as an acute febrile syndrome and treated successfully as brucellosis.

Campylobacter fetus causes gastrointestinal and systemic infections in humans. Although relapse is common despite antibiotic treatment, the mechanisms are not well understood. The surface-layer proteins (SLPs) of C. fetus, which are critical in virulence, undergo high-frequency phenotypic switching due to recombination of sap homologues, resulting in antigenic variation. To investigate the mechanisms involved in relapsing C. fetus infections, we compared SLP variation in 4 pairs of C. fetus strains that infect humans; initial and follow-up isolations were performed 20 days to 34 months apart. Of the 4 pairs of strains, 2 had antigenic variation, and another provided evidence for selection for SLP-positive populations. Southern hybridization indicated recombination underlying the SLP variation and up-regulation. The fourth pair had the same SLP antigenic profile and sap homologue hybridization pattern, which is consistent with latency of the original strain in a privileged locus. In total, these findings indicate that relapse may reflect at least 3 differing pathogenetic pathways.

A Campylobacter species was isolated from blood from a febrile patient with precursor T-cell acute lymphoblastic leukemia, and after antibiotic treatment, a similar bacterium was isolated from blood 37 days later. Although phenotypic testing did not definitively identify the organisms, molecular analysis indicated that they were the same strain of Campylobacter fetus subsp. fetus and were of reptile origin.

Campylobacter fetus surface layer proteins (SLPs), encoded by sapA homologues, are important in virulence. In wild-type C. fetus strain 23D, all eight sapA homologues are located in the 54-kb sap island, and SLP expression reflects the position of a unique sapA promoter in relation to the sapA homologues. The extensive homologies in the sap island include both direct and inverted repeats, which allow DNA rearrangements, deletion, or duplication; these elements confer substantial potential for genomic plasticity. To better understand C. fetus sap island diversity and variation mechanisms, we investigated the organization and distribution of sapA homologues among 18 C. fetus strains of different subspecies, serotypes, and origins. For all type A strains, the boundaries of the sap island were relatively consistent. A 187-bp noncoding DNA insertion near the upstream boundary of the sap island was found in two of three reptile strains studied. The sapA homologue profiles were strain specific, and six new sapA homologues were recognized. Several homologues from reptile strains are remarkably conserved in relation to their corresponding mammalian homologues. In total, the observed differences suggest that the sap island has evolved differing genotypes that are plastic, perhaps enabling colonization of varied niches, in addition to antigenic variation.

Campylobacter fetus is a rare cause of bone and joint infection. We report a new case in a patient who also had endocarditis due to the same organism. This type of infection usually occurs in immunocompromised hosts. Investigations in our patient found a cancer of the prostate.

Campylobacter fetus subsp. fetus is a zoonotic pathogen and cause opportunistic systemic infection in human who either are pregnant or have an underlying illness. Crystalline surface layer (S-layer), an external structure on the outer membrane is the prime virulence factor of this organism. S-layer is responsible for resistance to both leukocyte phagocytosis and the bactericidal effects of human serum. Antigenic variation of C. fetus surface antigen during infection also help the organism to escape from host immune-system and it is due to variation in the expression of the S-layer proteins (SLPs). C. fetus has multiple SLP-encoding genes and control the expression with inversion of a DNA segment containing the SLP promoter and/or homologous recombination.

Many pathogenic bacteria have evolved mechanisms for evading host immune systems. One evasion mechanism is manifest by the surface layer (S-layer), a paracrystalline protein structure composed of S-layer proteins (SLPs). The S-layer, possessed by 2 Campylobacter species (C. fetus and C. rectus), is external to the bacterial outer membrane and can have multiple functions in immune avoidance. C. fetus is a pathogen of ungulates and immunocompromised humans, in whom it causes disseminated bloodstream disease. In C. fetus, the S-layer is required for dissemination and is involved in 2 mechanisms of evasion. First, the S-layer confers resistance to complement-mediated killing in non-immune serum by preventing the binding of complement factor C3b to the C. fetus cell surface. S-layer expressing C. fetus strains remain susceptible to complement-independent killing, utilizing opsonic antibodies directed against the S-layer. However, C. fetus has also evolved a mechanism for avoiding antibody-mediated killing by high-frequency antigenic variation of SLPs. Antigenic variation is accomplished by complex DNA inversion events involving a family of multiple SLP-encoding genes and a single SLP promoter. Inversion events result in the expression of antigenically variant S-layers, which require distinct antibody responses for killing. C. rectus is implicated in the pathogenesis of periodontal disease and also possesses an S-layer that appears to be involved in evading the human system. Although studied less extensively than its C. fetus counterpart, the C. rectus S-layer appears to confer resistance to complement-mediated killing and to cause the down-regulation of proinflammatory cytokines.

Campylobacter fetus is a Gram-negative bacterial pathogen of humans and ungulates and is normally transmitted via ingestion of contaminated food or water with infection resulting in mild to severe enteritis. However, despite clinical evidence that C. fetus infection often involves transient bacteremic states from which systemic infection may develop and the frequent isolation of C. fetus from extra-intestinal sites, this organism displays very poor invasiveness in in vitro models of infection. In this study, immunofluorescence microscopy and gentamicin protection assays were used to investigate the ability of six clinical isolates and one reference strain of C. fetus to adhere to and invade the human intestinal epithelial cell line, INT 407. During an initial 4-h infection period, all C. fetus strains were detected intracellularly using both techniques, though adherence and internalization levels were very low when determined from gentamicin protection assays. Microscopy results indicated that during a 4-h infection period, four of the five clinical strains tested were adherent to 41.3-87.3% of INT 407 cells observed and that 25.2-34.6% of INT 407 cells contained intracellular C. fetus. The C. fetus reference strain displayed the lowest levels of adherence and internalization. A modified infection assay revealed that C. fetus adherence did not necessarily culminate in internalization. Despite the large percentage of INT 407 cells with adherent bacteria, the percentage of INT 407 cells with intracellular bacteria remained unchanged when incubation was extended from 4 h to 20 h. However, microscopy of INT 407 cells 24 h postinfection (p.i.) revealed that infected host cells contained clusters of densely packed C. fetus cells. Gentamicin protection assays revealed that intracellular C. fetus cells were not only viable 24 h p.i. but also that C. fetus had increased in number approximately three- to fourfold between 4 and 24 h p.i., indicative of intracellular replication. Investigation of the role of the host cell cytoskeleton revealed that pretreatment of host cells with cytochalasin D, colchicine, vinblastine, taxol, or dimethyl sulfoxide (DMSO) did not impact upon C. fetus adherence or internalization of INT 407 cells. Microscopy indicated neither rearrangement nor colocalization of either microtubules or microfilaments in INT 407 cells in response to C. fetus adherence or internalization. Together, these data indicate that clinical isolates of C. fetus are capable of adhering, entering, and surviving within the nonphagocytic epithelial cell line, INT 407.

The role of the surface (S)-layer proteins of Campylobacter fetus subsp. fetus has been investigated using an ovine model of abortion. Wild-type strain 23D induced abortion in up to 90% of pregnant ewes challenged subcutaneously. Isolates recovered from both dams and fetuses expressed S-layer proteins with variable molecular masses. The spontaneous S-layer-negative variant, strain 23B, neither colonized nor caused abortions in pregnant ewes. A series of isogenic sapA and recA mutants, derived from 23D, also were investigated in this model. A mutant (501 [sapA recA(+)]) caused abortion in one of five challenged animals and was recovered from the placenta of a second animal. Another mutant (502 [sapA recA]) with no S-layer protein expression caused no colonization or abortions in challenged animals but caused abortion when administered intraplacentally. Mutants 600(2) and 600(4), both recA, had fixed expression of 97- and 127-kDa S-layer proteins, respectively. Two of the six animals challenged with mutant 600(4) were colonized, but there were no abortions. As expected, all five strains recovered expressed a 127-kDa S-layer protein. In contrast, mutant 600(2) was recovered from the placentas of all five challenged animals and caused abortion in two. Unexpectedly, one of the 16 isolates expressed a 127-kDa rather than a 97-kDa S-layer protein. Thus, these studies indicate that S-layer proteins appear essential for colonization and/or translocation to the placenta but are not required to mediate fetal injury and that S-layer variation may occur in a recA strain.

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.

In vitro adherence assays were used to determine whether the S layer mediated interactions between Campylobacter fetus subsp. venerealis strains and HEp-2 cells. At multiplicity of infection ratios ranging from 0.1:1 through 100:1, quantitation of bacterial adherence by light microscopy revealed that S layer deficient isogenic C. fetus 809K and C. fetus 810K were not less efficient in their attachment to HEp-2 cells; either S layer deficient C. fetus strains interacted with HEp-2 cells in greater numbers than the corresponding wild-type parent strains 809 and 810 or there was no significant difference in adherence levels between wild-type and mutant strains. Adherence of C. fetus strains to HEp-2 cells increased most during the first 2 h of a 22-h incubation period with only a slight increase in C. fetus cell numbers occurring subsequent to 2 h. At each assay point throughout this 22-h time period, equivalent numbers of wild-type and S layer deficient C. fetus strains were observed associated with HEp-2 cells. Prior to 2 h, adherence levels of all C. fetus strains exceeded those of Escherichia coli AB264 and Salmonella typhimurium SL1344. And, unlike S. typhimurium, C. fetus did not undergo significant replication following initial adherence to HEp-2 cells. Campylobacter fetus did not adhere to HEp-2 cells in a localized or aggregative pattern but were randomly distributed over individual HEp-2 cells and at no time during the assay with C. fetus were changes in HEp-2 cell morphology apparent. These data suggest that the S layer is not essential for mediating initial interactions between C. fetus and HEp-2 cells.

Cells of the Gram-negative bacteria Campylobacter fetus are covered by monomolecular arrays of surface layer proteins (SLPs) critical for both persistence in their natural hosts and for virulence. For C. fetus cells, expression of SLPs essentially eliminates C3b binding and their antigenic variation thwarts host immunological defences. Each cell possesses multiple partially homologous and highly conserved SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97-149 kDa. These attach non-covalently via a conserved N-terminus to the cell wall lipopolysaccharide. Recent studies indicate that C. fetus reassorts a single promoter, controlling SLP expression, and one, or more, complete open reading frame strictly by DNA inversion, and that rearrangement is independent of the distance between sites of inversion. In contrast to previously reported programmed DNA inversion systems, inversion in C. fetus is recA-dependent. These rearrangements permit variation in protein expression from the family of SLP genes and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.

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.

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 x 10(-2). Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.

Campylobacter fetus are microaerophilic gram-negative bacteria that are pathogens of animals and humans. These organisms possess paracrystalline surface (S-) layers, composed of acidic high molecular weight proteins. C. fetus strains possessing S-layers are resistant to C3b binding, which explains both serum and phagocytosis-resistance. C. fetus strains also can vary the subunit protein size, crystalline structure, and antigenicity of the S-layer it expresses. Therefore, its S-layer permits C. fetus to resist complement and antibodies, two of the key defenses against extracellular pathogens. C. fetus possesses several full-length genes encoding S-layer proteins with both conserved and divergent sequences, which permits gene rearrangement and antigenic variation.

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.

A HEp-2 cell culture model was used to investigate the antigens required for epithelial cell penetration by Campylobacter jejuni. Penetration of HEp-2 epithelial cells by C. jejuni was significantly inhibited (P less than .05) with C. jejuni lysate and a monoclonal antibody (MAb 1B4) in competitive inhibition assays. Immunogold electron microscopy revealed that MAb 1B4 bound to the flagella and cell surface of low-passage (invasive) C. jejuni M 96, whereas only the flagella of high-passage (noninvasive) C. jejuni M 96 were labeled. Western blot analysis revealed that MAb 1B4 identified an epitope on antigens of 64-44 kDa in lysates prepared from invasive and noninvasive isolates. In addition, antigens of 42-38 kDa were recognized in lysates prepared from only invasive C. jejuni strains. Proteinase K and sodium meta-periodate chemical treatment of C. jejuni M 96 lysate changed the mobility of antigens recognized by MAb 1B4. The increase in mobility demonstrated a decrease in size of molecules and suggested that antigens required for HEp-2 cell invasion by Campylobacter species may be glycoprotein in nature.

The high molecular mass (97-149 kDa) surface array proteins (SAP) of Campylobacter fetus are critical to virulence. We created a bank of 160,000 random 1.0-6.5-kilobase (kb) chromosomal DNA fragments of C. fetus strain 84-32 (23D) using lambda gt11. Screening this bank in Escherichia coli Y1090 with antibody raised against purified SAP permitted isolation and purification of a clone with a 4.0-kb insert. Subcloning this insert in the E. coli vector, pUC9, permitted expression of a protein of approximately 100 kDa, not fused with beta-galactoside or inducible by isopropyl-beta-D-thiogalactopyranoside. Digestion with restriction endonucleases, and construction of deletion mutations indicated that the gene extended over 2.8 kb, proceeding toward the start of the beta-galactosidase gene. Taking advantage of a unique PstI site at 1.7 kb, we subcloned PstI-EcoRI fragments in both orientations into M13 vectors, then generated and sequenced 48 deletion mutants. In the 3974-base insert, an open reading frame, beginning at nucleotide 24 and terminating at 2825, was found to encode a 933-amino acid polypeptide having a calculated molecular mass of 96,758 daltons. The first 20 amino acids exactly match those determined from amino-terminal sequencing, indicating that this protein is secreted without a leader sequence. The deduced amino acid composition matches that of the purified SAP. We identified a ribosomal binding site 9 bases upstream, and a putative transcription terminator (delta G = -12.4) 21 bases downstream. There is partial homology of primary and secondary structure with five other bacterial S-layer proteins.

Two variants of Campylobacter jejuni IN1 differing in the expression of flagella, IN1 (Fla+ Mot+, wild type) and IN1-NM (Fla- Mot-), were tested for their ability to establish infection in adult hamsters. Animals were challenged intracecally with 2 X 10(9) to 5 X 10(10) CFU and monitored for evidence of infection. None of the challenged animals developed illness. There was a significant difference, however, in the ability of IN1 to infect hamsters (35 of 43) compared with that of IN1-NM (1 of 42) (P less than 0.01). Additionally, eight animals challenged with IN1-NM excreted only the campylobacters of Fla+ phenotype, which were shown to be identical with the parental Fla+ Mot+ type. Both groups of animals developed serum immunoglobulin G antibodies to outer membrane proteins of IN1 as measured by an enzyme-linked immunosorbent assay; however, only animals that excreted Fla+ organisms developed antiflagellin antibodies. In vitro reversibility from Fla+ to Fla- occurred with a frequency of 9.2 X 10(-6) per cell per generation; however, reversion from Fla- to Fla+ could not be detected in vitro. Further characterization of IN1-NM showed that it produced a cytoplasmic 62K flagellin subunit protein, but this protein lacked six epitopes detected in IN1 and also differed in its two-dimensional gel electrophoresis pattern. The results of these studies support the concept that an intact flagellum is necessary for intestinal infection with C. jejuni and that this model may be useful for studying the immune response to C. jejuni.

A case of Campylobacter jejuni peritonitis in a patient being treated with continuous ambulatory peritoneal dialysis is described. The significance of the organism isolated from our patient was initially doubtful because of a rapid defervescence of symptoms with minimal specific antibiotic therapy, lack of a preceding diarrhoeal illness and the time taken to isolate and identify the organism. Serial serum IgM, IgA and IgG antibody estimations clearly showed an acute seroconversion confirming that the strain of C. jejuni isolated was the causative organism in this case.

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.

We developed a mouse model to compare the virulence of Campylobacter fetus strains with (S-plus) and without (S-minus) surface array protein (S-protein) capsules. In adult HA/ICR mice pretreated with ferric chloride, the LD50 for S-plus strain 84-32 was 43.3 times lower than its spontaneous S-minus mutant 84-54. Seven strains of inbred mice were no more susceptible than the outbred strain. In contrast to the findings with Salmonella typhimurium by others, 3 X 10(7) CFU of strain 84-32 caused 90% mortality in C3H/HeN (LPSn) mice and 40% mortality in C3H/HeJ (LPSd) mice. High-grade bacteremia in HA/ICR mice occurred after oral challenge with S-plus C. fetus strains and continued for at least 2 d, but was not present in any mice challenged with S-minus strains. Bacteremia at 30 min after challenge was 51.6-fold lower in mice pretreated with 10 microliters of rabbit antiserum to purified S-protein than after pretreatment with normal rabbit serum. Challenge of mice with a mixture of S-minus strain 84-54 and free S-proteins at a concentration 31.1-fold higher than found in wild-type strain 84-32 caused 30% mortality, compared with 0% with strain 84-54 or S-protein alone. These findings in a mouse model point toward the central role of the S-protein in the pathogenesis of C. fetus infection. The S-protein is not toxic per se, but enhances virulence when present on the bacterial cell surface as a capsule.

Sensitivity of 179 strains of thermophilic Campylobacter to 21 chemotherapeutic drugs was studied. Activity of the antibacterial agents against the pathogens was estimated by the MICs. The MIC50 and MIC90 were also determined. All the Campylobacter strains were sensitive to gentamicin, chloramphenicol, neomycin, furazolidone and furagin and resistant to cefazolin, polymyxin E, rifampicin, vancomycin and bacitracin. Differences in the attitude of the Campylobacter isolates from various sources and patients of various age groups to the chemotherapeutic drugs were detected. Possible consideration of the results of comparison of R spectra of Campylobacter strains and the levels of their resistance to antimicrobial drugs as epidemiological markers is discussed.

Two hundred Campylobacter jejuni and Campylobacter coli strains from the same number of adult Swedish patients with acute enterocolitis were tested regarding adherence to and invasiveness in HEp-2 cells and for enterotoxigenicity by the CHO-cell assay. The serogroup characteristics, heat-stable and heat-labile, for each strain were also investigated. Eighty-four percent of the strains were classified as C. jejuni and 16 percent as C. coli. All of the strains were adherent to HEp-2 cells, 39% were invasive and 31.5% enterotoxigenic. We found significantly more invasive strains in the non-enterotoxigenic group than in the enterotoxigenic one. There would seem to be no correlation between enterotoxigenicity or invasiveness and serogroup. The results of this study suggest the existence of multiple mechanisms for C. jejuni- and C. coli-induced diarrhoea and that the mechanisms may differ from one strain to another.

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.