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The Gram-negative pathogen Campylobacter jejuni is the most common cause of bacterial foodborne disease worldwide. The mechanisms that lead to bacterial invasion of eukaryotic cells and massive intestinal inflammation are still unknown. In this study, we report that C. jejuni infection of mouse macrophages induces upregulation of pro-IL-1β transcript and secretion of IL-1β without eliciting cell death. Immunoblotting indicated cleavage of caspase-1 and IL-1β in infected cells. In bone marrow-derived macrophages from different knockout mice, IL-1β secretion was found to require NLRP3, ASC, and caspase-1/11 but not NLRC4. In contrast to NLRP3 activation by ATP, C. jejuni activation did not require priming of these macrophages. C. jejuni also activated the NLRP3 inflammasome in human macrophages as indicated by the presence of ASC foci and caspase-1-positive cells. Analysis of a vast array of C. jejuni mutants with defects in capsule formation, LPS biosynthesis, chemotaxis, flagella synthesis and flagellin (-like) secretion, type 6 secretion system needle protein, or cytolethal distending toxin revealed a direct correlation between the number of intracellular bacteria and NLRP3 inflammasome activation. The C. jejuni invasion-related activation of the NLRP3 inflammasome without cytotoxicity and even in nonprimed cells extends the known repertoire of bacterial inflammasome activation and likely contributes to C. jejuni-induced intestinal inflammation., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The pathogen Campylobacter jejuni is the principal cause of bacterial food-borne infections. The mechanism(s) that contribute to bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SS) are increasingly recognized to contribute to bacterial pathogenesis by toxic effects on host cells or competing bacterial species. Here we report the presence of a functional Type VI secretion system in C. jejuni. Proteome and genetic analyses revealed that C. jejuni strain 108 contains a 17-kb T6SS gene cluster consisting of 13 T6SS-conserved genes, including the T6SS hallmark genes hcp and vgrG. The cluster lacks an ortholog of the ClpV ATPase considered important for T6SS function. The sequence and organization of the C. jejuni T6SS genes resemble those of the T6SS located on the HHGI1 pathogenicity island of Helicobacter hepaticus. The C. jejuni T6SS is integrated into the earlier acquired Campylobacter integrated element CJIE3 and is present in about 10% of C. jejuni isolates including several isolates derived from patients with the rare clinical feature of C. jejuni bacteremia. Targeted mutagenesis of C. jejuni T6SS genes revealed T6SS-dependent secretion of the Hcp needle protein into the culture supernatant. Infection assays provided evidence that the C. jejuni T6SS confers contact-dependent cytotoxicity towards red blood cells but not macrophages. This trait was observed only in a capsule-deficient bacterial phenotype. The unique C. jejuni T6SS phenotype of capsule-sensitive contact-mediated hemolysis represents a novel evolutionary pathway of T6SS in bacteria and expands the repertoire of virulence properties associated with T6SS.

Background: Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni., Methods/principal Findings: Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function., Conclusions/significance: Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.

The bacterial pathogen Campylobacter jejuni invades mucosal cells via largely undefined and rather inefficient (0.01-2 bacteria per cell) mechanisms. Here we report a novel, highly efficient C. jejuni infection pathway resulting in 10-15 intracellular bacteria per cell within 3 h of infection. Electron microscopy, pulse-chase infection assays and time-lapse multiphoton laser confocal microscopy demonstrated that the mechanism involved active and rapid migration of the pathogen into the subcellular space (termed 'subvasion'), followed by bacterial entry ('invasion') at the cell basis. Efficient subvasion was maximal after repeated rounds of selection for the subvasive phenotype. Targeted mutagenesis indicated that the CadF, JlpA or PEB1 adhesins were not required. Dissection of the selected and parental phenotypes by SDS-PAGE yielded comparable capsule polysaccharide and lipooligosaccharide profiles. Proteomics revealed reduced amounts of the chemotaxis protein CheW for the subvasive phenotype. Swarming assays confirmed that the selected phenotype exhibited altered migration behaviour. Introduction of a plasmid carrying chemotaxis genes into the subvasive strain yielded wild-type subvasion levels and migration behaviour. These results indicate that alterations in the bacterial migration machinery enable C. jejuni to actively penetrate the subcellular space and gain access to the cell interior with unprecedented efficiency.

Campylobacter jejuni is the prime cause of foodborne bacterial gastroenteritis. An important complication of C. jejuni enteritis is Guillain-Barré syndrome (GBS), an immune-mediated disorder of the peripheral nerve. The presence of C. jejuni DNA in peripheral blood mononuclear cells (PBMC) of patients with GBS, patients with C. jejuni enteritis, and healthy subjects was studied. Two target genes, the flagellin and the ceuE genes, were used for polymerase chain reaction (PCR) identification of Campylobacter species in DNA extracted from PBMC. Approximately 30% of the healthy subjects and 50% of the patients with GBS had PBMC containing C. jejuni DNA as verified by Southern blot analysis or sequencing of the PCR products. Cell sorting revealed that Campylobacter DNA was present in CD14(+) and CD33(+) populations, indicating that cells from the myelomonocytic lineage are the Campylobacter DNA-carrying cells. These findings show that Campylobacter DNA is present in blood cells of healthy humans, although viable bacteria could not be demonstrated.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.

The prevalence of Borrelia species infection was determined by polymerase chain reaction (PCR) in 138 ticks collected from dogs which were walked regularly in the wooded areas near the city of Eindhoven, the Netherlands. The PCR amplified the spacer region between the 5S and 23 S rRNA genes, and the Borrelia species was identified by hybridization with specific probes. Borrelia burgdorferi sensu lato was present in 20 of 138 (14.5%) ticks. Four species were identified: B. burgdorferi sensu stricto (n = 8), B. afzelii (n = 4), B. garinii (n = 2), and B. valaisiana (n = 2). One PCR product was non-typeable. Three ticks contained more than one species, all including B. burgdoferi sensu stricto, and one tick even contained four species. There was a significant difference (P < 0.05) in prevalence of B. burgdorferi sensu stricto between non-engorged ticks (either questing or attached) and semi-engorged ticks, 12% (10 of 85) and 2% (1 of 53), respectively.

A PCR assay for the detection of small ruminant lentiviral gag DNA (provirus) in the white blood cells of sheep and goats was developed and compared with a serological test (AGIDT). A sample of the DNA prepared from the white blood cells in 3 ml of blood from 208 sheep and goats from 18 different flocks was subjected to PCR assay. One of 85 animals from flocks accredited under the Dutch national MVV/CAEV control programme was positive by PCR while none was positive by AGIDT. In infected flocks, the AGIDT appeared slightly more sensitive, but preliminary results show that the sensitivity of the PCR assay may be further improved by increasing the number of monocytes tested. The PCR assay, however, was clearly more sensitive in detecting animals in the early stages of infection. With the use of a set of mixed primers and probes, the assay was able to detect the variety of CAEV and MVV strains occurring in the field.

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.

The ability of Taylorella equigenitalis, the causative agent of contagious equine metritis, to invade and replicate in equine derm cells was studied. The kinetics of invasion and replication were determined for four T. equigenitalis strains. On the basis of these experiments, a simpler assay in which the invasive as well as the replicative properties of a particular strain could be determined was developed. This assay was used to characterize 32 strains, which had previously been typed by field inversion gel electrophoresis of genomic restriction fragments. The invasiveness of T. equigenitalis strains ranged from 3 to 0.015 bacteria per cell and seemed to be associated with the contagiousness of the infection. The replication index (number of intracellular bacteria per cell at 24 h after inoculation divided by the number of intracellular bacteria per cell at 4 h after inoculation) varied from 1 to 857 and seemed to be associated with the severity of the symptoms of contagious equine metritis. There was no association between the invasiveness and the replication index of the strains, nor was there an association of invasion and replication with field inversion gel electrophoresis grouping.

Campylobacter jejuni 81116 has two genes coding for flagellin, flaA and flaB. Fully motile wild-type C. jejuni bacteria express the flaA gene, with no flaB message being detected. A nonmotile flaA flaB+ mutant, R1, produced detectable levels of flagellin B which was incorporated into truncated flagella. After R1 had invaded INT-407 cells, a variant with increased motility, R1-V2, was isolated. R1-V2 produced full-length flagella and an increased amount of flagellin B. Transcriptional analysis showed that R1-V2 contained more flaB mRNA than its parental strain, R1. The flaB gene promoter sequence and primer extension experiments confirmed that transcription of the flaB gene is initiated from a sigma 54 promoter. Neither the promoter sequence nor the coding sequence of flaB had changed in R1-V2. In contrast to R1, R1-V2 no longer produced (truncated) flaA mRNA. The sigma 28 flaA promoter sequence was not changed in R1-V2. We propose that expression of the two flagellin genes in C. jejuni 81116 is regulated at the transcriptional level, in such a way that predominantly one gene at a time is transcribed. We compared the levels of invasiveness of the wild-type strain, R1, and R1-V2 for INT-407 cells. The shift in expression from flaA to flaB occurred not only during invasion assays but also under different conditions in the absence of eukaryotic cells.

A PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis, was developed and evaluated. A genus-specific primer-probe set was derived from the 16S ribosomal DNA sequences. The PCR was specific and amplified a 585-bp product from all 64 available T. equigenitalis isolates. This PCR product hybridized with a specific probe in a dot spot assay. A variety of microorganisms from the genital tracts of horses or with a close phylogenetic relationship to T. equigenitalis did not yield a visible PCR product and were all negative in the dot spot hybridization assay. The results of the PCR assay were compared with those of culture by using 191 genital swabs from horses of several breeds. We demonstrate that the sensitivity of the PCR assay is superior to that of culture. The assay is most sensitive when DNA from culture plates incubated for at least 2 days is used. Of the tested samples, 1.5% were positive in the culture assay, whereas 35% were positive in the culture PCR assay. PCR-positive samples were obtained from all breeds tested. This means that many T. equigenitalis-carrying horses go unidentified by the current culturing technique. This affects current views about the spread and control of T. equigenitalis.

The 16S ribosomal DNA sequence of Taylorella equigenitalis (formerly Haemophilus equigenitalis), the causative organism of contagious equine metritis, was determined. A phylogenetic analysis of this sequence revealed a phylogenetic position of T. equigenitalis in the beta subclass of the class Proteobacteria apart from the position of Haemophilus influenzae, which belongs to the gamma subclass of Proteobacteria. A close phylogenetic relationship among T. equigenitalis, Alcaligenes xylosoxidans, and Bordetella bronchiseptica was detected; Spirillum volutans and Chromobacterium fluviatile (Iodobacter fluviatile) were in the same group but slightly removed. This relationship is surprising in view of the considerable differences in the G + C contents of the genomes of these bacteria.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.

The chromosome of Campylobacter jejuni is circular and approximately 1700 kb in circumference. The size of the genome was determined by field inversion gel electrophoresis of restriction endonuclease fragments using lambda DNA concatamers and yeast chromosomes to calibrate the size of the fragments. In view of the low (32-35%) G + C content of the campylobacter genome, enzymes that recognizes GC-rich sequences were used. Of the enzymes tested BssHII (G/C(G)CGC), NciI (CC/CGCG) and SalI (G/TCGAC) appeared to be usable. Hybridization of labeled fragments with two or more fragments from digests with a different restriction enzyme gave the information to order the fragments on the C jejuni chromosome. The localization on the genome of the flagellin and ribosomal gene clusters was determined.

Sixteen monoclonal antibodies (Mcabs) were prepared against infectious bronchitis virus strain M41, all of them reacting with the peplomer protein. One of them, Mcab 13, was able to neutralize the virus and to inhibit hemagglutination. Competition binding assays allowed the definition of five epitopes, designated as A, B, C, D, and E, of which epitopes A and B are overlapping. Furthermore, the binding of Mcab 13 (epitope E) could be enhanced by the addition of Mcabs from group B, C, and D. A dot immunoblot assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. Only the binding of Mcab 13 was affected, indicating that the epitope involved in neutralization and hemagglutination is conformation dependent. The epitopes A to D were highly conserved among IBV strains, while epitope E was specific for strains M41 and D3896. In this last strain, however, this epitope was not involved in neutralization.

The amino acid sequence of the gene for the peplomer protein of the vaccine strain M41 and the Beaudette laboratory strain M42-Salk of avian infectious bronchitis virus (IBV) have been derived from cDNA sequences. As found with other coronaviruses, the peplomer protein carries the epitopes eliciting neutralizing antibodies. The gene encodes a primary translation product of 1162 amino acids with a molecular weight of 128,079. The use of a recent algorithm to predict membrane-protein interactions led to the unambiguous localization of the signal peptide and a transmembrane anchor alpha-helix at the C-terminus. At 50 positions amino acid differences were found between M41 and two Beaudette strains (M42-Salk and M42-Houghton). They are partly clustered in two regions of the protein. These two regions are candidates for neutralization epitopes of the protein.

Twelve Dutch isolates and the M41 strain of infectious bronchitis virus (IBV), a coronavirus of chickens, were characterized by cross-neutralization and T1 finger-printing to elucidate their evolutionary relationship. The T1 fingerprinting showed that the Dutch isolates formed two clusters. The first cluster contained strains H52, H120, D387, V1259, V1385 and V1397; the estimated sequence homology is 99%. Cluster two comprised strains D207, D274, D212, D1466, D3128 and D3896, which have about 95% sequence homology. The M41 virus did not belong to either cluster. The four different serotypes which arose in the late 1970s belonged to cluster two and appeared to be different from the vaccine strains (H52 and H120) used at that time. This indicates that the strains were newly introduced and could have arisen from a common virus. On the other hand, three recently isolated field strains were genetically closely related to the vaccine strains H120 and H52 (cluster one), suggesting that these live vaccine strains themselves could have given rise to these serologically altered field isolates. The data are relevant to the development of new vaccine strategies.

Campylobacter jejuni 81116 is able to switch flagellum formation on and off. To study the expression of flagellin, the main component of flagella, an expression library of C. jejuni DNA was constructed in lambda gt11. Screening of this library with a flagellin-specific antiserum resulted in a clone producing a beta-galactosidase-flagellin fusion protein; it contained a DNA insert of 850 base pairs and coded for about 15 kilodaltons of the flagellin, corresponding to 410 base pairs of the flagellin gene. To study the regulation of the on-and-off switch of flagellum production, a nonmotile variant was isolated from semisolid medium. Western blots (immunoblots) showed the absence of flagellin in the nonmotile form. Southern blots of digested DNA of both motile, flagellate bacteria and nonmotile, aflagellate bacteria were identical, while Northern (RNA) blot analysis showed the absence of flagellin mRNA in the aflagellate form. Thus, it is concluded that reversible flagellin expression is regulated at the transcriptional level. Southern blots suggest that more than one flagellin gene is present. The structure and function of campylobacter flagellin can now be further investigated at the DNA level.