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The antimicrobial activity of metal-based compounds, including metal oxides, has resulted in numerous agricultural, industrial, and medical applications. Zinc oxide nanoparticles are toxic to Gram-positive and Gram-negative bacteria as well as to some fungi. In this study we assess the sensitivity of Campylobacter fetus , a Gram-negative bacterial pathogen of humans and animals, to ZnO nanoparticles and determine whether the S layer protects C. fetus from the antibacterial action of these nanoparticles. Broth and agar dilution assays revealed that ZnO nanoparticles at 100 μg/mL were bacteriocidal for C. fetus . Resazurin reduction assays confirmed the absence of metabolic activity, indicating that C. fetus cells had not entered into a viable but nonculturable state. Photoactivation of ZnO nanoparticles greatly enhanced their antibacterial activity, as evidenced by minimum bacteriocidal concentration (MBC) values decreasing to 16-62.5 μg/mL as a function of strain. MBC assays completed in the presence and absence of catalase revealed that H 2 O 2 , a product of ZnO nanoparticle photoactivation, contributed to C. fetus but not to C. jejuni cell death. S-layer-expressing C. fetus strains were more resistant to H 2 O 2 -mediated cell killing than were isogenic S-layer-deficient strains. These data indicate that C. fetus is sensitive to the antibacterial activity of ZnO nanoparticles and that the C. fetus S layer imparts protection against photoactivated nanoparticles.

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus , such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus . The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus , a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon., Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus , such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus . The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus , a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Background: Campylobacter fetus (C. fetus) can cause disease in both humans and animals. C. fetus has been divided into three subspecies: C. fetus subsp. fetus (Cff), C. fetus subsp. venerealis (Cfv) and C. fetus subsp. testudinum (Cft). Subspecies identification of mammal-associated C. fetus strains is crucial in the control of Bovine Genital Campylobacteriosis (BGC), a syndrome associated with Cfv. The prescribed methods for subspecies identification of the Cff and Cfv isolates are: tolerance to 1 % glycine and H2S production., Results: In this study, we observed the deletion of a putative cysteine transporter in the Cfv strains, which are not able to produce H2S from L-cysteine. Phylogenetic reconstruction of the core genome single nucleotide polymorphisms (SNPs) within Cff and Cfv strains divided these strains into five different clades and showed that the Cfv clade and a Cff clade evolved from a single Cff ancestor., Conclusions: Multiple C. fetus clades were observed, which were not consistent with the biochemical differentiation of the strains. This suggests the need for a closer evaluation of the current C. fetus subspecies differentiation, considering that the phenotypic differentiation is still applied in BGC control programs.

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.

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.)

Classifications of the Campylobacter fetus subspecies fetus and venerealis were first described in 1959 and were based on the source of isolation (intestinal versus genital) and the ability of the strains to proliferate in the genital tract of cows. Two phenotypic assays (1% glycine tolerance and H2S production) were described to differentiate the subspecies. Multiple molecular assays have been applied to differentiate the C. fetus subspecies, but none of these tests is consistent with the phenotypic identification methods. In this study, we defined the core genome and accessory genes of C. fetus, which are based on the closed genomes of five C. fetus strains. Phylogenetic analysis of the core genomes of 23 C. fetus strains of the two subspecies showed a division into two clusters. The phylogenetic core genome clusters were not consistent with the phenotypic classifications of the C. fetus subspecies. However, they were consistent with the molecular characteristics of the strains, which were determined by multilocus sequence typing, sap typing, and the presence/absence of insertion sequences and a type I restriction modification system. The similarity of the genome characteristics of three of the phenotypically defined C. fetus subsp. fetus strains to C. fetus subsp. venerealis strains, when considering the core genome and accessory genes, requires a critical evaluation of the clinical relevance of C. fetus subspecies identification by phenotypic assays., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

Chemoreceptor and chemotaxis signal transduction cascade genes of C. fetus subsp. fetus 82-40 show high level of similarity to that in C. jejuni and appears to include sixteen diverse transducer-like protein (tlp) genes that appear similar to nine of the twelve tlp genes in the C. jejuni NCTC 11168 with a percent identity ranging from 15 to 50%. Sixteen putative C. fetus 82-40 tlp genes belong to three classes: A, B, and C, as well as an aerotaxis gene, based on their predicted structure. C. fetus subsp. fetus 82-40 chemoreceptor and chemotaxis signal transduction pathway genes have close phylogenetic relationship of chemotaxis genes between Campylobacteraceae and Helicobacteraceae.

The biofilm forming abilities of 16 strains representative of 14 of the 16 species comprising the genus Campylobacter were determined on glass, stainless steel, and polystyrene plastic. The formation of biofilms has been suggested as a means by which Campylobacter is able to persist within an inhospitable environment. Of the eight microaerophilic Campylobacter species, including two strains each of Campylobacter jejuni and Campylobacter fetus, only C. jejuni strain 81-176 reliably produced a visible biofilm on multiple surfaces. Alternately, all six strains of the anaerobic Campylobacter species reliably produced visible biofilms on multiple surfaces. Electron micrographs of the individual biofilms showed relatively homogeneous biofilms produced by the anaerobic strains, while the microaerophilic C. jejuni strain 81-176 produced a biofilm containing similar quantities of both the spiral and coccoid forms. This survey suggests a difference in the biofilm forming potentials and the morphologies of the bacteria comprising the biofilms between anaerobic and microaerophilic species of Campylobacter. Additionally, differences observed in the biofilm forming ability of two strains of C. jejuni suggest the need for a further investigation of the biofilm forming potential of this species using a larger number of strains.

Campylobacter fetus is a recognized pathogen of cattle and sheep that can also infect humans. No adhesins specific for C. fetus have to date been identified; however, bacterial attachment is essential to establish an infecting population. Scanning electron microscopy revealed C. fetus attachment to the serosal surface of human colonic biopsy explants, a location consistent with the presence of the extracellular matrix (ECM). To determine whether the ECM mediated C. fetus adherence, 7 C. fetus strains were assessed in a solid-phase binding assay for their ability to bind to immobilized ECM components. Of the ECM components assayed, adherence to fibronectin was noted for all strains. Attachment to ECM components was neither correlated with S-layer expression nor with cell-surface hydrophobicity. Ligand immunoblots, however, identified the S-layer protein as a major site of fibronectin binding, and modified ECM binding assays revealed that soluble fibronectin significantly enhanced the attachment of S-layer-expressing C. fetus strains to other ECM components. Soluble fibronectin also increased C. fetus adherence to INT 407 cells. This adherence was inhibited when INT 407 cells were incubated with synthetic peptides containing an RGD sequence, indicating that integrin receptors were involved in fibronectin-mediated attachment. Together, this data suggests that C. fetus can bind to immobilized fibronectin and use soluble fibronectin to enhance attachment to other ECM components and intestinal epithelial cells. In vivo, fibronectin would promote bacterial adherence, thereby, contributing to the initial interaction of C. fetus with mucosal and submucosal surfaces.

Campylobacter jejuni and Campylobacter coli strains were isolated from feces of dairy cattle at farms with no known problem due to campylobacteria. Farms were located in the northeast, desert southwest, and Pacific west. Twenty isolates were identified by ribotyping with a RiboPrinter. The ability of these bovine isolates to colonize the ceca of chicks was determined by challenge inoculation and reisolation of the challenge strain from the ceca at 1 and 2 wk after challenge. Isolates recovered from chick ceca were examined by ribotyping to assure they matched the challenge strain. One hundred percent of the bovine-derived challenge strains were capable of colonizing chicks. These results indicate that dairy cattle may be asymptomatic Campylobacter carriers and potential sources of campylobacteria contamination of poultry facilities.

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.

Studies were undertaken to assess the bactericidal activity of phagocytes isolated from blood and the uterine lumen of clinically healthy cows after ovulation, and from cows in which endometritis was induced experimentally. Experiments were carried out on 28 clinically healthy cows of the black and white lowland breed. Animals were aged 5 years and were used between the 2nd and 8th day after spontaneous ovulation. Cows were divided into four groups. Group I comprised animals in which cell-mediated type immune reaction was induced in the left uterine horn by intrauterine challenge with tuberculin. Cows in this group were initially vaccinated with M. bovis via the intrauterine route. In group II, Arthus type immune reaction was induced by challenging immunized animals with C. fetus ssp. veneralis through intrauterine instillation. The non-specific inflammatory process was initiated in the uterus of animals in group III by one instillation of lipopolysaccharide from S. abortus equi. Animals in group IV were set as control and received a phosphate buffered saline instillation into the uterine lumen. The cells from the left uterine horn were washed out 6 h after induction. Neutrophils were isolated from blood samples collected from all animals within the same time. The bacterial activity of cells from the uterine lumen and blood was assessed with the nitro-blue tetrazolium reduction test. Results are presented as increase in optical density resulting from a constant number of phagocytizing cells (delta OD/10(6) cells). Induction of cell-mediated immune reaction or Arthus type immune reaction in the uterus significantly boosts the intracellular capability of uterine cells to kill bacteria through the oxidation system. Experimentally induced non-specific endometritis weakens the bactericidal activity of uterine phagocytes, while peripheral blood phagocytes efficiently kill the engulfed bacteria.

Pure cultures of several Campylobacter spp. induced a specific humoral immune response after they colonized and infected gnotobiotic mice; however, Campylobacter-immune mouse serum was not bactericidal (in vitro), manifested a weak agglutination reaction (in vitro), and showed specificity (strain 45100-immune mouse sera) for the homologous (infecting) Campylobacter strain, but was not able to passively protect germfree athymic (nu/nu) BALB/c mice against Campylobacter infection and diarrhea. Active immunization of germfree nu/nu mice with Formalin-killed C. jejuni also did not protect the gnotobiotic mice from Campylobacter infection and diarrhea. It appears from the results of our initial gnotobiotic studies that antibodies in serum against the infecting strain of C. jejuni may not play an essential role in resistance to Campylobacter disease in mice.

Hydrophobicity and surface charge of clinical isolates of Campylobacter jejuni strains were investigated by aqueous two-phase partitioning (one-step and counter-current distribution), ion exchange chromatography and hydrophobic interaction chromatography. There was a good correlation between the different physico-chemical methods reflecting the same bacterial property. All strains were negatively charged and exposed a hydrophobic surface, but to a varying extent. Bacteria with a high negative surface charge and a weak hydrophobic surface adhered better to human intestinal HT-29 cells than strains with less charge and a more hydrophobic surface. Highest adhesion was shown by a strain differing from all the others in charge properties. It was also found that the tendency to aggregate was higher among the strains showing the greatest degree of adherence.

Culture supernatants of four Campylobacter jejuni strains induced a net sodium secretory flux (plasma-lumen) and an impaired glucose transport in perfused jejunal segments of adult rats in vivo.

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.

The adhesion of Campylobacter jejuni and C. coli to isolated porcine intestinal brush border membranes was studied by phase-contrast and electron microscopy. Approximately 45% of the cell population adhered to the brush borders, possibly in a specific manner. Pretreatment of the brush borders with trypsin or pronase, and competitive inhibition with L-rhamnose caused a slight reduction of the adhesion. Different forms of pretreatment of the bacterial cells reduced their ability to adhere, but also their motility.

The amount of crude Campylobacter jejuni enterotoxin present in culture products was quantitated by comparing the response of these preparations with that of pure Escherichia coli heat-labile toxin (LT) in the Chinese hamster ovary assay and in enzyme-linked immunosorbent assays that used GM ganglioside or antisera to LT or both. Maximum C. jejuni enterotoxin production was achieved by growth at 42 degrees C for 24 h under agitation in supplemented GC medium. Adding polymyxin separately to either the broth supernatant or the cells enhanced the recovery of toxin; the yield from cell lysates was much lower. The quantity of C. jejuni enterotoxin produced by clinical isolates obtained locally or provided from Mexico varied widely, over a spectrum from none to large amounts; quantitative values for the amount of C. jejuni enterotoxin determined by the Chinese hamster ovary and enzyme-linked immunosorbent assays correlated with the degree of secretory potency of this material in ligated rat ileal loops. The cytotonic activity of C. jejuni enterotoxin in Chinese hamster ovary cells was abolished by heating at 96 degrees C for 10 min and by preincubation either with GM ganglioside or with LT or cholera toxin antisera. The secretory activity of C. jejuni enterotoxin in ligated rat ileal loops was passively neutralized by antiserum to LT, and immunizing rats with either LT or its B subunit significantly (P less than 0.001) reduced fluid response to active challenge with C. jejuni enterotoxin in ligated ileal loops. These observations indicate that strains of C. jejuni vary in their capacity to elaborate a heat-labile enterotoxin that has close immunological homology with LT and cholera toxin.

The effects of sub-inhibitory concentrations (MIC 50 and MIC 25) of clindamycin, erythromycin and chloramphenicol on the adherence of Campylobacter jejuni to INT 407 cells were studied. The results indicated that the adhesion was inhibited at various extents, mostly by MIC 50, and that the inhibition increased with time. The interpretation of our data is that the antibiotics can interfere with the adhesin(s) synthesis.

Campylobacter jejuni/coli strains were isolated from the faeces of 240 patients suffering from acute enteritis. The following characteristics were investigated: (i) growth at different temperatures, and on different substrates under either microaerophilic conditions or anaerobically, with fumarate or nitrate as terminal electron acceptors; (ii) production of H2S in cysteine-containing broth; (iii) hydrolysis of hippuric acid; (iv) DNase; (v) alkaline phosphatase; (vi) beta-lactamase; (vii) presence of menaquinone; and (viii) reduction of selenite. Based on characteristics (ii)-(v), the strains could be divided in 9 phenotypical groups. Most of the strains represented group 2 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%), and groups 8 (DNase-, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%). The other groups were of minor importance. On the other hand, most of the isolates from the United States (Weaver, 1981) fitted well into group 1 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase+) which might demonstrate geographical variations among C. jejuni/coli.

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.

The heat susceptibility of four bacterial enteropathogens in foods and water was studied to develop effective recommendations for travelers to regions where diarrheal diseases are important health problems. All enteropathogens tested survived well in foods stored at refrigerator temperature (4 degrees C), room temperature (25 degrees C), and 50 degrees C, which is too hot to touch. Tap water had to be heated above 65 degrees C to reliably kill all bacterial enteropathogens. At 13 of the 14 tourist-oriented hotels in four countries, water from the hot water tap did not reach temperatures of 65 degrees C. The implications of this study are that food and water that are too hot to touch may still be contaminated with bacterial enteropathogens. Travelers should be advised that food, water, or beverages are safe only if they have been brought to boiling or near-boiling temperatures prior to consumption.

For the purpose of ascertaining the survival time of Campylobacter jejuni in foodstuffs under differing conditions, milk, ice-cream, meat salad and drinking water were inoculated with a defined quantity of Campylobacter jejuni and subsequently the number of colony-forming units was determined at first at 12-hour intervals and later once a day. It was found that the survival time in milk at +5 degrees C was 15 days, in drinking water 6 days, but only 7 and 4 days respectively at 22 degrees C. In frozen ice-cream (vanilla) C. j. was still identified after 30 days and longer, in frozen water (-20 degrees C) even after several weeks. In sour meat salad (pH 4.2) the survival time of the inoculated C.j. amounted to a few hours only. The conclusions to be drawn from these results are discussed with reference to the epidemiology of the human diseases caused by C.j. and to kitchen hygiene.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.

Investigations of the etiology of diarrhea in patients in South Australia and the Northern Territory showed that Campylobacter spp. other than Campylobacter jejuni and C. coli were common in children. Campylobacters which were hippurate positive, nitrate negative, and susceptible to cephalothin and polymyxins were shown to be closely related to C. jejuni by DNA studies. Thermotolerant catalase-negative campylobacters were also isolated. These were H2S negative and biochemically resembled the catalase-negative or weak strains found in dogs in Sweden. DNA studies showed these campylobacters to be distinct from C. sputorum subsp. sputorum and to form a homogeneous group distinct from the enteropathogenic catalase-positive campylobacters. Preliminary studies suggest that these campylobacters are related to the Swedish catalase-negative or weak strains.

Invasion and intracellular survival of Campylobacter jejuni in HEp-2 cells were analyzed by transmission electron microscopy and by viable counts after killing of extracellular bacteria by gentamicin. During the first 30 min after challenge, no bacteria were seen in association with the host cell. After 1 h, campylobacters apparently attached to the cell membrane, with areas of close appositions. In these areas, an intracellular network of actin-like filaments was seen beneath the plasma membrane. Other bacteria were included into endocytic vacuoles. After 3 h, an intense lysosomal response was observed in the host cells, as determined by the presence of myelinic forms and acid phosphatase activity. After 9 h, bacteria still contained in vacuoles showed signs of degradation with a change from spiral to coccal forms. Morphological evidence of phagosome-lysosome fusion was also seen, and these observations by transmission electron microscopy correlated well with a decrease in bacteria viability 9 h after challenge, as determined from separate kinetics studies. Inhibitors of phagocytosis were observed to reduce markedly the entry of C. jejuni into the cells at concentrations which apparently did not affect bacterial viability. These results suggest that the campylobacters were successively attached to the HEp-2 cell membrane, internalized by a phagocytic-like mechanism, and digested after phagosome-lysosome fusion.

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.

Campylobacters have been isolated in Bangladesh from patients with gastroenteritis, from healthy individuals and animals. A total of 180 Campylobacter isolates, 100 from patients with gastroenteritis reporting to International Centre for Diarrhoeal Disease Research, Bangladesh hospitals, 50 from asymptomatic carriers and the other 30 from domestic animals of the index cases have been characterized following standard procedures available. All isolates tested have been confirmed as Campylobacter jejuni. Irrespective of their source of isolation, Campylobacter isolates showed identical biochemical behaviours. All isolates showed thermophilic characteristics of jejuni/coli group. None of the isolates grew at 25 degrees C. Uniform sensitivity to nalidixic acid, metronidazole, 2, 3, 5-triphenyltetrazolium chloride, resistance to cephalothin was observed; H2S production in all isolates by different methods was confirmed. All isolates from different sources confirmed to be C. jejuni; none of the isolates, even from the animal source could be characterized as C. coli.

Twenty-one isolates were tested for their ability to adhere to and invade HEp-2 cells in vitro. Of the 21 organisms tested, 2 did not invade the HEp-2 cells, and 1 of these did not adhere to the epithelial cells. Campylobacter jejuni clinical isolates were more invasive than the nonclinical strains that were tested. When HEp-2 cells were treated with cytochalasin B, the invasiveness of C. jejuni was reduced, indicating active participation of the host cell in the uptake of these organisms. The number of intracellular C. jejuni isolates decreased when Campylobacter whole-cell lysates were absorbed onto HEp-2 cell monolayers. Experiments were also conducted to identify the functional sites of the antigens responsible for expression of Campylobacter invasion. Oxidation of lysates with sodium meta-periodate significantly affected its inhibitory capacity. This implies that the Campylobacter invasive ligand appears to be dependent upon an intact carbohydrate moiety.

Most Campylobacter jejuni strains are sensitive and most Campylobacter fetus strains are resistant to the bactericidal activity in normal human serum. We purified lipopolysaccharides from Campylobacter strains to determine whether their composition and structure relate to serum susceptibility. The lipopolysaccharide of two serum-sensitive strains was best isolated by the Galanos procedure, but for two serum-resistant strains a cold-ethanol extraction was optimal. For each lipopolysaccharide preparation, the ratio of 2-keto-3-deoxyoctonate to protein was increased by 100 to 1,000-fold over that of whole cells. For serum-resistant strains, total carbohydrates was a high proportion of lipopolysaccharide weight; for serum-sensitive strains, 2-keto-3-deoxyoctanate was a high proportion of total carbohydrates. By polyacrylamide gel electrophoresis, the lipopolysaccharide of serum-sensitive strains appeared rough, but for serum-resistant strains a smooth-type ladder was seen, with a minimal core region and several high-molecular-weight complexes. Proteinase K-treated whole-cell lysates showed polyacrylamide gel electrophoresis profiles similar to that of pure lipopolysaccharide. Proteinase K-treated whole-cell lysates from seven serum-sensitive C. jejuni strains all had rough profiles, and five serum-resistant C. fetus strains all had smooth profiles. These studies indicate that lipopolysaccharide composition may be an important determinant of serum susceptibility among Campylobacter species and that serum resistance is usually associated with a smooth-type lipopolysaccharide.

The adhesion of three strains of Campylobacter jejuni to coverslips and microwells coated with isolated extracellular matrix components, fibronectin, laminin and types I, III, IV and V collagens was studied. Fibronectin mediated the adherence of C. jejuni, but there were differences in the binding capacities of the strains. Type I, III and V collagens mediated very strongly the attachment of two strains of C. jejuni. All three strains attached weakly to basement membrane-specific type IV collagen. Laminin was capable of mediating the adhesion only when present at a higher concentration. The observations indicate that extracellular matrix components may serve as anchor molecules for C. jejuni adhesion and that several attachment mechanisms occur simultaneously.

Auto-agglutinated and non-agglutinated cells of Campylobacter jejuni and C. coli were examined by transmission electronmicroscopy in phosphotungstate negative stain. Agglutination was induced by three factors (1) extracellular DNA, (2) an aggregated protein, probably a bacteriophage precursor, and (3) free phage-tail sheaths. Auto-agglutinated cells were often "leaky," with a mantle of adhering DNA. About 80% of the auto-agglutinated cells could be resuspended after treatment with DNAase. Flagella were loosely embedded in protein aggregates, especially in phage-infected cultures. They were clumped in a side-by-side arrangement by free phage-tail sheaths. These findings suggest that auto-agglutination could be minimised in suspensions of organisms intended for use in agglutination tests by harvesting early logarithmic-phase cells containing no more than a low phage population. The most common C. jejuni phage had a contractile tail, a head diameter of 60-70 nm, and an overall length of 180-210 nm. A phage isolated from C. jejuni strain 1590 was morphologically identical with C. coli phage.

The chemotactic behavior of Campylobacter jejuni was determined in the presence of different amino acids, carbohydrates, organic acids, and preparations and constituents of mucin and bile. L-Fucose was the only carbohydrate and L-aspartate, L-cysteine, L-glutamate, and L-serine were the only amino acids producing a chemotactic (positive) response. Several salts of organic acids, including pyruvate, succinate, fumarate, citrate, malate, and alpha-ketoglutarate, were also chemoattractants, as were bile (beef, chicken, and oxgall) and mucin (bovine gallbladder and hog gastric). Most constituents of bile tested individually were chemorepellents, but the mucin component was chemoattractant. The chemotactic behavior of C. jejuni toward L-fucose, a constituent of both bile and mucin, may be an important factor in the affinity of the organism for the gallbladder and intestinal tract.

Speed and motion patterns of Campylobacter fetus ssp. jejuni, Escherichia coli and Pseudomonas aeruginosa were recorded using a closed circuit television camera attached to a phase contrast microscope. A Sony video analysis system was used to stop frame videotape at 1/7th and 1/15th. Bacterial speeds were: Campylobacter 29.2 micron/s, E. coli 8.9 micron/s and P. aeruginosa 16.8 micron/s.

Campylobacter jejuni was an almost regular finding in chickens and in minced meat from pigs and cattle sold in ordinary food stores. The bacteria survived on the food at 4 degrees C for one week and frozen at -20 degrees C for three months. None of the strains tested survived heat treatment at 60 degrees C for longer than 15 min. C. jejuni is apparently a frequent guest in most kitchens. Correct food handling and heat treatment to at least 60 degrees C for 15 min should be enough to prevent infection.

Nine strains of Campylobacter species other than Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis were isolated from patients with acute diarrhea. All nine strains showed preferred growth at 37 degrees C under microaerophilic conditions. Conventional microbiological tests and DNA-DNA dot blotting were used to identify these strains. Three of the nine Campylobacter strains hydrolyzed hippurate, reduced nitrate, produced catalase, were resistant to cephalothin, and were shown to be highly related to C. jejuni type strains. Two strains had negative or weak catalase activity and were hippurate negative. Three other strains had characteristics similar to those of Campylobacter cinaedi. The ninth strain, isolated from a homosexual man with antibodies to human immunodeficiency virus (human T-cell lymphotropic virus type III), showed unique features different from those of all the known campylobacters used in this study. This strain grew well at 25 and 37 degrees C and was catalase and nitrate positive, hippurate negative, and resistant to cephalothin.

Fertile chicken eggs were infected in our laboratory with Campylobacter jejuni suspensions by using temperature or pressure differential methods of inoculation. After 2 days of incubation, over 90% of the eggs carried C. jejuni when iron was present in the inoculum. This percentage declined rapidly until by day 8, less than 10% of the eggs were detectably infected. However, up to 11% of hatched, healthy chicks carried C. jejuni in their intestinal tracts. The isolated organisms were of the same serotype as the initial inoculum. C. jejuni was recovered without difficulty when the intestinal tracts of chicks were enriched, but recovery from early dead-in-shell or infertile eggs was poor. This poor recovery and the rapid decline of C. jejuni after 2 days of egg incubation suggest that the vibrio is sensitive to some part of the incubating egg or to the temperature of prolonged incubation. It was impossible to predict which eggs would yield infected chicks on the basis of the number of organisms taken up by each egg, and no correlation existed between the number of organisms taken up and the efficiency of the hatch, i.e., the hatch ratio. If iron was omitted from the inoculum broth, the egg infection rate at day 2 was lower.

Cells of Campylobacter jejuni damaged by freezing or mild heat showed increased sensitivity to hydrogen peroxide and also required the addition of catalase for growth on nutrient agar. These phenomena, which could have important practical consequences, were not associated, however, with changes in total catalase activity and may be due to alterations in outer membrane permeability.

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.

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.

Potential measures for reducing the survival of campylobacters during commercial scalding of poultry have been evaluated in a series of laboratory trials. At 50 degrees C, the lower temperature limit of commercial scalding, raising the pH of a buffered heating medium from 6.0 to 9.0 markedly increased the heat sensitivity of Campylobacter jejuni but the effect was largely nullified in the presence of 1 per cent 'organic material' (50:50 horse blood and milk). Either in the presence or absence of organic material a more rapid rate of kill was observed at 60 degrees C and it was again enhanced by raising the pH to 9.0. Use of a mild detergent at a concentration of 1000 ppm had little effect on the survival of C jejuni at 50 degrees C, but the addition of a cationic quaternary ammonium product at 50 to 100 ppm was highly effective in enhancing the rate of kill, even in the presence of organic material. It is suggested that such products should be evaluated in commercial scalding systems as a possible means of preventing the spread of campylobacters and other organisms of significance to public health.

We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.