Influence of host lineage on cecal colonization by Campylobacter jejuni in chickens

The resistance to cecal colonization by Campylobacter jejuni was assessed by challenging three crossbred stocks of commercially available broiler chickens. These three stocks, designated A, B, and C, were related as follows: Offspring from four pedigreed grandparent flocks were used as progenitors. Stock B was derived by cross-breeding grandparent 1 with grandparent 3. Stocks A and C were crossbreeds from grandparents 1 and 2 and grandparents 3 and 4, respectively. Campylobacter jejuni were gavaged into 48-hour-old chicks, using the same levels of challenge dose for each of the different chicken stocks. Six days post-challenge, the birds were sacrificed, and cecal contents were plated onto Campylobacter-selective media. Results from two replicate trials with three isolates of C. jejuni indicated that chicken stock A was colonized in only two of 60 ceca, stock B in six of 60, and stock C in 19 of 60 chicken ceca. Statistical analysis of these data indicate that resistance to cecal colonization by C. jejuni was significantly (P less than 0.05) influenced through chicken host lineage.     

Interaction of Campylobacter spp. and Human Probiotics in Chicken Intestinal Mucus

Campylobacter is the most common cause of bacterial food‐borne diarrhoeal disease throughout the world. The principal risk of human contamination is handling and consumption of contaminated poultry meat. To colonize poultry, Campylobacter adheres to and persists in the mucus layer that covers the intestinal epithelium. Inhibiting adhesion to the mucus could prevent colonization of the intestine. The aim of this study was to investigate in vitro the protective effect of defined commercial human probiotic strains on the adhesion of Campylobacter spp. to chicken intestinal mucus, in a search for alternatives to antibiotics to control this food‐borne pathogen. The probiotic strains Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS and a starter culture strain Lactococcus lactis ssp. lactis adhered well to chicken intestinal mucus and were able to reduce the binding of Campylobacter spp. when the mucus was colonized with the probiotic strain before contacting the pathogen. Human‐intended probiotics could be useful as prophylactics in poultry feeding for controlling Campylobacter spp. colonization.     

Potential use of characterised hyper-colonising strain(s) of Campylobacter jejuni to reduce circulation of environmental strains in commercial poultry

Sixty-two persistently colonising Campylobacter jejuni strains were tested for their ability to dominate colonisation of the chicken gastrointestinal tract in competition with each other leading to selection of dominant or "hyper-colonising"Campylobacter strains, which are able to displace others in the chicken intestinal tract. One such strain was shown to be a hyper-efficient coloniser of chickens, as it was able to displace other colonising strains, as well as maintain itself in the chicken intestinal tract for the duration of the 56-day broiler production cycle. Once colonisation was established, this hyper-colonising C. jejuni strain, 331, could not be displaced by other colonising or hyper-colonising strains. We proposed that a defined, hyper-colonising strain, or a cocktail of defined strains with a similar phenotype, could form the basis for biological control of unknown/uncharacterised Campylobacter strains from the environment that continuously colonise chicken flocks. To validate this approach, three different chicken infection trials were carried out. These trials demonstrated that the dominant strain of C. jejuni was able to colonise broiler chickens consistently and for the entire life of the birds irrespective of the day of inoculation and antimicrobial agents used in the feed to control other pathogenic micro-organisms. In addition, we have shown that the bio-control strain was able to replace other colonising strains at various points of a 56-day broiler production cycle irrespective of time and type of inoculation. This strain was also capable of re-establishing itself following the challenge with other strains, with and without re-challenge. This work represents a "proof of principle" that a defined C. jejuni strain could be used to biologically control circulation of uncharacterised environmental strains in commercial poultry flocks.     

Colonization characteristics of Campylobacter jejuni in chick ceca

We report our findings on several parameters influencing cecal colonization of chickens by Campylobacter jejuni. Thirty-five colony-forming units (CFU) of a composite culture of C. jejuni colonized the ceca of one-half of the newly hatched chicks challenged by oral gavage. A challenge dose of 3500 CFU/chick consistently colonized the ceca of all chicks challenged. Challenge doses of approximately 10(5) CFU of C. jejuni per chick resulted in consistent cecal colonization, regardless of whether the birds were challenged 1, 2, or 3 days post-hatch. Four isolates showed consistently strong cecal colonization abilities, whereas two isolates colonized the ceca in only 20 of 122 chicks when given levels of 10(5) CFU per chick. One of these poorly colonizing isolates was repeatedly transferred by fecal-oral passage through chicks; subsequently, this isolate was able to consistently colonize chicks. Competitive exclusion (CE) microflora did not diminish the colonization rates for C. jejuni. Birds treated with five different CE cultures were colonized at a rate of 81 of 84 chicks; control chicks were similarly consistently colonized (45 of 46 chicks).     

Genotyping Campylobacter jejuni strains isolated from the gut and oviduct of laying hens

Campylobacter jejuni frequently colonizes the avian intestine. Recent evidence suggests that this organism can also colonize the oviduct of laying hens. However, the source and role of this colonization are unknown. Isolates from the ceca, cloacae, and oviducts of 11 laying hens in three intensive egg-producing flocks were genotyped by Fla typing with the restriction fragment length polymorphism of the polymerase chain reaction product of the flaA and flaB genes (fla typing) and pulsed-field gel electrophoresis (PFGE). A diversity in fla types and PFGE types was observed within and between flocks. Individual birds could be colonized by different genotypes at various intestinal and oviduct sites. However, the oviduct of individual birds appeared to be colonized by only one genotype at the time of sampling. In two birds, matching isolates investigated from the intestinal and reproductive tracts were genotypically identical but different from those oviduct isolates found in other birds in the same flock. Interestingly, not all cecal isolates appeared to be equally able to colonize the oviduct. These results suggest that oviduct colonization may result from ascending infection via the cloaca and that some strains of C. jejuni may be better adapted than others to oviduct colonization.     

Profiles of toxin production by thermophilic Campylobacter of animal origin.

Seventy-five strains of Campylobacter jejuni and C. coli, which were isolated from a variety of animal species, primarily poultry, were examined for production of toxin. Polymyxin extracts were tested in in vitro assays using CHO-KI, FCL (foetal calf lung), Vero, HeLa and CEF (chicken embryo fibroblast) cells. The toxic effects observed were cell rounding and death. Extracts from almost all C. jejuni and C. coli strains were toxic to both CHO-KI and FCL cells and 69.0% of C. jejuni isolates and 75% of C. coli isolates were also toxic to CEF cells. 50.7% of C. jejuni extracts were toxic to Vero cells and 46.5% toxic to HeLa cells. None of the C. coli isolates were toxic to either of these cell lines. None of the strains tested produced cytotonic enterotoxin. No differences in toxigenicity patterns were evident between Campylobacter isolated from different sources.     

Isolation of Campylobacter Fetus Subsp. Jejuni from Faeces of Norwegian Poultry

Pooled faeces samples from 106 poultry flocks in Norway were examined. Campylobacter fetus subsp. jejuni was isolated from 10 of 100 chicken flocks and from 4 of 6 turkey flocks. Eight of the 14 isolates were classified as biotype C. jejuni, which is frequently associated with human campylobacteriosis. Five strains belonged to the biotype C. coli. One strain was resistant to nalidixic acid but differed from the biotype NARTC in its ability to hydrolyse hippurate. The results indicate that C. fetus subsp. jejuni is widespread among Norwegian poultry.     

Novel employment of lactate dehydrogenase release from porcine aortic endothelial cells (PAEC) as a quantitative marker of cytotoxic activity in thermophilic Campylobacter spp. from human faecal isolates, poultry and environmental sources

The aim of this study was to employ a novel cytotoxicity assay based on primary porcine aortic endothelial cells in combination with a lactate dehydrogenase release assay to quantitatively determine differences in cytotoxin production between Campylobacter jejuni, C. coli, C. lari and urease-positive thermophilic campylobacters (UPTC), isolated from human faeces, animals and environmental sources. Campylobacter isolates totalling 34 and comprising of C. jejuni (n = 24) C. coli (n = 5) and UPTC (n = 4) and C. lari (n = 1) were analysed. The cytotoxic response ranged from 32.15 to 64.47% and 33.08 to 59.41%, for C. jejuni from chicken and human isolates, respectively and there was no statistically significant difference (P > 0.05) in cytotoxic response between C. jejuni isolated from humans and chicken isolates (50.78% versus 50.55% cytotoxicity, respectively). However, there was a difference in response between C. jejuni and C. coli isolated from chickens (50.78% versus 33.22% cytotoxicity, respectively). The greatest cytotoxic response was obtained with the UPTC group of organisms examined (n = 4 isolates) (mean cytotoxic response = 57.11% cytotoxicity. Employment of this cytotoxin assay may help identify virulent strains in poultry that could potentially proceed to cause clinical problems for humans and thus intervention measures targeted at the reduction or elimination of such specific strains, may be sought.     

Campylobacter-induced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells

Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.     

Comparative study of colonizing and noncolonizing Campylobacter jejuni

Campylobacter jejuni A74/O and A74/C are congenic strains. An oral dose of 10(5) organisms of strain A74/C colonizes chicken intestines. Strain A74/O, from which A74/C is derived, does not colonize the chicken intestines with an oral dose of 10(5) organisms. In this study, the congenic bacteria were compared to identify possible colonization mechanisms. Differences were not observed in plasmid content or by HindIII, Pst I, Acc I, HincII, Ava I, Ava II, Xba I, and BamHI restriction enzyme digestion of total DNA. Transmission electron microscopy of negatively stained samples revealed no differences between the strains. Sections of cecal tissue from nonfed day-of-hatch chicks were cultured with each strain for 2 hours and then examined by light and electron microscopy. Both strains caused necrosis of villus epithelial cells. Immunofluorescent or silver staining revealed strain A74/C located deep in numerous epithelial crypts, but strain A74/O only was present in one sample mixed with sloughed necrotic cells. Similarly, organisms were detected by transmission electron microscopy deep in crypts in tissues cultured with A74/C, but not A74/O. Cells of A74/C detected in crypts did not appear to associate with epithelial cells. The strains did not differ in chemotactic behavior to mucin or fucose.     

Influence of antibody treatment of Campylobacter jejuni on the dose required to colonize chicks

This study was designed to clarify the role of antibodies in controlling chicken colonization by Campylobacter jejuni. Cecal colonization by C. jejuni was compared after the organism was exposed either to phosphate-buffered saline, normal rabbit serum, rabbit hyperimmune anti-C. jejuni serum, or anti-C. jejuni antibodies extracted from chicken bile. Antibodies from chicken bile were extracted by affinity absorption against outer-membrane proteins from the challenge organism. Sera were heated 1 hour at 56 C to destroy complement activity. Bacterial inoculum levels were enumerated after 1 hour exposure at 4 C to the various treatments. The heated sera and the bile antibodies were not bactericidal, and bacterial agglutination was not evident. Serial dilutions of the antibody-treated C. jejuni were given by gavage into 1-day-old chicks. Six days later, the ceca were removed from the chicks, and samples were cultured on Campylobacter-charcoal differential agar. The colonization dose-50% was increased by twofold to 160-fold when the organism was preincubated with hyperimmune antiserum or the bile antibodies as compared with preincubation with phosphate-buffered saline. We conclude that antibodies inhibit chicken cecal colonization by C. jejuni.     

Effect of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in cultured cells and in colonization of the chicken gut

Campylobacter jejuni produces cytolethal distending toxin (CDT) that causes host cells to arrest during their cell cycle and that is involved in the pathogenesis of inflammatory diarrhea in humans. To assess the role of CDT in adherence and invasion of different cultured host cells (HeLa and HD-11) and in colonization of the chicken intestine, the genes of C jejuni NCTC11168 encoding the toxin subunits (cdtA, cdtB, and cdtC) were inactivated by insertional mutagenesis. No significant difference was found in adhesion of the wild-type C. jejuni and the isogenic mutants to HeLa and HD-11 cells. All of the mutants exhibited a decrease (>10-fold) in the ability to invade HeLa cells, but no significant difference was noticed for HD-11 cells. The ability of mutants to colonize birds either directly or by horizontal transfer was unchanged. These data indicated that although the production of cytotoxin does not play a role in the adherence to either human or avian cells, it may play a role in the invasion, survival, or both of C. jejuni in human cells, which are more susceptible to C. jejuni internalization. The CDT also does not seem to play a role in the colonization of poultry.     

Dose response and organ invasion of day-of-hatch Leghorn chicks by different isolates of Campylobacter jejuni

Colonization of the ceca and organ invasion by different isolates of Campylobacter jejuni were investigated in day-of-hatch leghorn chicks. This model of Campylobacter colonization of the ceca demonstrates that 1) day-of-hatch birds do not naturally contain cecal Campylobacter, 2) ceca can be colonized with C. jejuni by oral gavage and not by cloacal inoculation; 3) C. jejuni can be recovered from the ceca up until at least 7 days postinoculation, 4) cecal colonization occurs when as little as 10(2) colony-forming units is orally inoculated into chicks, and 5) different C. jejuni isolates vary both in their ability to colonize the ceca and in their ability to invade the liver. These studies demonstrate that we have a working animal model for Campylobacter colonization for day-of-hatch chicks. This animal model is being used to examine intervention strategies such as vaccines by which Campylobacter can be reduced or removed from the food animal.     

Campylobacter jejuni strains of human and chicken origin are invasive in chickens after oral challenge

The aim of the study was to evaluate the colonizing ability and the invasive capacity of selected Campylobacter jejuni strains of importance for the epidemiology of C jejuni in Danish broiler chickens. Four C. jejuni strains were selected for experimental colonization studies in day-old and 14-day-old chickens hatched from specific pathogen free (SPF) eggs. Of the four C. jejuni strains tested, three were Penner heat-stable serotype 2, flaA type 1/1, the most common type found among broilers and human cases in Denmark. The fourth strain was Penner heat-stable serotype 19, which has been shown to be associated with the Guillain Barré Syndrome (GBS) in humans. The minimum dose for establishing colonization in the day-old chickens was approximately 2 cfu, whereas two- to threefold higher doses were required for establishing colonization in the 14-day-old chickens. Two of the C. jejuni strains were shown to be invasive in orally challenged chickens as well as in three different human epithelial cell lines.     

PCR-based restriction fragment length polymorphism (RFLP) analysis of Campylobacter jejuni isolates from humans, chickens and dogs in northern Taiwan

Two hundred and twenty strains of Campylobacter jejuni (70 human, 51 canine and 99 chicken strains) were isolated from September 2003 to September 2004 in northern Taiwan. These strains were subtyped by PCR-RFLP analysis of the flagellin (FlaA) gene. On the basis of restrictive digest, six types were identified with AfaI, seven types with MboI and five types with HaeIII. With the combination of these three enzymes, 47 distinct PCR-RFLP patterns were observed-25 each from human and chicken isolates, and 9 from canine isolates. In human strains, the most frequently occurring types were Cj-28 (14.3%), Cj-17 (10%), Cj-16 (8.6%), Cj-37 (7.1%) and Cj-46 (7.1%). In canine strains, the most prevalent types were Cj-1 (33.3%), Cj-26 (19.6%), Cj-3 (15.7%), Cj-2 (9.8%) and Cj-10 (9.8%). In chicken strains, the most frequently occurring types were Cj-46 (40.4%), Cj-29 (9.1%), Cj-45 (7.1%) and Cj-41 (5.1%). The results suggest that poultry is a source, but not the sole source, of C. jejuni infection in humans. Two RFLP types, Cj-17 and Cj-37, frequently occurring in human isolates in this study have also been found to be prevalent in human isolates in Japan, China and the Czech Republic, indicating a possible international clonal spread.     

Colonization factors of Campylobacter jejuni in the chicken gut

ABSTRACT: Campylobacter contaminated broiler chicken meat is an important source of foodborne gastroenteritis and poses a serious health burden in industrialized countries. Broiler chickens are commonly regarded as a natural host for this zoonotic pathogen and infected birds carry a very high C. jejuni load in their gastrointestinal tract, especially the ceca. This eventually results in contaminated carcasses during processing. Current intervention methods fail to reduce the colonization of broiler chicks by C. jejuni due to an incomplete understanding on the interaction between C. jejuni and its avian host. Clearly, C. jejuni developed several survival and colonization mechanisms which are responsible for its highly adapted nature to the chicken host. But how these mechanisms interact with one another, leading to persistent, high-level cecal colonization remains largely obscure. A plethora of mutagenesis studies in the past few years resulted in the identification of several of the genes and proteins of C. jejuni involved in different aspects of the cellular response of this bacterium in the chicken gut. In this review, a thorough, up-to-date overview will be given of the survival mechanisms and colonization factors of C. jejuni identified to date. These factors may contribute to our understanding on how C. jejuni survival and colonization in chicks is mediated, as well as provide potential targets for effective subunit vaccine development.     

Serogroups of Campylobacter jejuni from man and animals

A total of 186 campylobacter strains from aborted calf and sheep fetuses, from scouring dogs, rabbits and man, and from retailed poultry were isolated and examined biochemically and serologically for heat stable antigens. Immune sera were produced in rabbits against Penner reference strains from 1 to 60, and against two field isolates. Out of 186 biochemically tested strains 179 (96.2%) proved C. jejuni and only 6 (3.2%) C. coli. One strain has been identified as C. laridis. In cattle and sheep 3.2 and 21.7% respectively of all campylobacter abortions were due to C. jejuni infection. The same agent caused 12.7% of diarrhoea of dogs. The campylobacter infection rate of freshly slaughtered and dressed chicken varied between 25 and 64.3%. Out of the serologically examined 140 C. jejuni strains 118 (84.3%) could be assigned to 16 Penner serogroups and 13 (9.3%) to 2 further serogroups. Serogroups 8 (31.4%), 1 (19.3%) and 2 (12.1%) occurred most frequently. The human isolates represented the widest serotype distribution, as 32 tested strains belonged to 12 serogroups. All those serogroups which caused abortion or diarrhoea in animals or were isolated from poultry carcases were isolated also from man with diarrhoea, but some serogroups were found only in man.     

In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus

The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.     

Fla-DGGE analysis of Campylobacter jejuni and Campylobacter coli in cecal samples of broilers without cultivation

In a commercial broiler flock during rearing multiple genotypes of Campylobacter jejuni may be present as well as in gastrointestinal tracts of individual birds. The aim of this study was to optimize and apply a denaturing gradient gel electrophoresis assay of the flagellin gene (fla-DGGE) for analysis of C. jejuni and Campylobacter coli in cecal samples of broilers without prior cultivation. One C. coli and 21 C. jejuni strains isolated from broiler flocks, of which 14 typed as unique by restriction fragment length polymorphism of flaA and two undefined strains, were clustered into 9 groups when applying fla-DGGE. Spiking of cecal samples revealed that fla-DGGE is able to detect at least 4.55-5.96logCFUCampylobacter/mlcecal material. The presence of 3 strains spiked in cecal material was demonstrated by fla-DGGE as the corresponding bands were visible on the DGGE gel. Naturally contaminated cecal samples were shown to contain different types of C. jejuni and C. coli. Fla-DGGE has some potential as a cultivation-independent fast primary subtyping method for C. jejuni and C. coli in cecal samples of broilers.