Role of Campylobacter jejuni potential virulence genes in cecal colonization

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.     

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.     

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.     

Colonisation studies using Campylobacter jejuni in chicks

White Leghorn chicks used in this study were hatched from specific pathogen-free eggs. The colonizing capability of Campylobacter (C.) jejuni strains was investigated in 6 experiments. The formation of specific antibodies associated to colonization was also detected. In each experiment, day of hatch chicks were randomly separated into three groups of 24 birds each: two groups colonized experimentally and one control group. Chicks were reared on the floor in three separated, adjacent rooms with sterilized wood shavings as litter. At 2 or 8 days of age, respectively, the chicks in the experimentally colonized groups received between 3.3 x 10(7) and 2.0 x 10(8) colony-forming units (CFU) of C. jejuni via oesophageal gavage. Furthermore, 7, 14, 21, 28, 42 and 56 days after inoculation, 4 chicks of each group were sacrificed by cervical dislocation, at which time blood, liver and faeces were collected for processing. Serum was centrifuged and Campylobacter-specific IgG, IgA and IgM antibodies were measured by an indirect enzyme-linked immunosorbent assay (ELISA). Altogether, the colonizing capability of 11 C. jejuni strains was examined. Surprisingly, there were large differences between the C. jejuni isolates. After these experiments, we could divide the isolates into three groups. 4 out of 11 isolates could not be reisolated, 2 isolates caused weak or delayed colonization and 5 C. jejuni produced strong, long-lasting colonization. In the first days of life (9 days), the C. jejuni-free SPF chicks (control animals) had high IgG titres in sera, which decreased markedly up to the age of 15 days. During the experiments the IgM and IgA titres remained nearly at the same level, i.e., the amounts of maternal antibodies were low and there was no evidence for antibody formation in the chicks themselves. Two- and 8-day-old chicks were inoculated with C. jejuni strain Penner 1. Two-day-old chicks were colonized 3 weeks after inoculation. In comparison with these animals, 8-day-old chicks were colonized already 2 weeks after inoculation. There is the assumption, that the higher maternal antibodies in 2-day-old chicks could be responsible for this delay. In chicks the C. jejuni colonization resulted in a marked IgG (but not IgM and IgA) increase. Apparently, there is a positive relationship between the counts of this pathogen in caeca and the IgG increase.     

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.     

Role of litter in the transmission of Campylobacter jejuni

Autoclaved or non-autoclaved used broiler litter that was experimentally contaminated with Campylobacter jejuni was capable of infecting specific-pathogen-free chicks maintained in modified Horsfall isolators. Artificially infected chicks became fecal shedders of C. jejuni, resulting in contamination of both autoclaved and non-autoclaved used broiler litter. Fecal shedding of C. jejuni by litter-reared, artificially infected chicks persisted for at least 63 days after chicks were transferred to an isolation unit with a wire floor, which prevented coprophagy. C. jejuni was consistently recovered from water and litter in units housing directly and indirectly infected birds, indicating environmental contamination. These experiments demonstrate the potential role of litter in the perpetuation and transmission of C. jejuni infection in commercial chickens.     

Colonization strategy of Campylobacter jejuni results in persistent infection of the chicken gut

Although poultry meat is now recognized as the main source of Campylobacter jejuni gastroenteritis, little is known about the strategy used by the bacterium to colonize the chicken intestinal tract. In this study, the mechanism of C. jejuni colonization in chickens was studied using four human and four poultry isolates of C. jejuni. The C. jejuni strains were able to invade chicken primary cecal epithelial crypt cells in a predominantly microtubule-dependent way (five out of eight strains). Invasion of cecal epithelial cells was not accompanied by necrosis or apoptosis in the cell cultures, nor by intestinal inflammation in a cecal loop model. C. jejuni from human origin displayed a similar invasive profile compared to the poultry isolates. Invasiveness of the strains in vitro correlated with the magnitude of spleen colonization in C. jejuni inoculated chicks. The C. jejuni bacteria that invaded the epithelial cells were not able to proliferate intracellularly, but quickly evaded from the cells. In contrast, the C. jejuni strains were capable of replication in chicken intestinal mucus. These findings suggest a novel colonization mechanism by escaping rapid mucosal clearance through short-term epithelial invasion and evasion, combined with fast replication in the mucus.     

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).     

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.     

Comparative studies on competitive exclusion of three isolates of Campylobacter fetus subsp. jejuni in chickens by native gut microflora

Resistance of young chicks to Campylobacter fetus subsp. jejuni was substantially increased by early exposure to native gut microflora. Protection was demonstrated against two human isolates and a chicken isolate of C. fetus subsp. jejuni. Significant protection against the chicken isolate was observed throughout a 91-day test period. Infection reached 100% (25/25) in the untreated group at 56 days of age and only 4% (1/25) in the group treated with native gut microflora. Campylobacter fetus subsp. jejuni was isolated from the ceca and less frequently from the gall bladder and liver of chicks that actively shed the bacteria. Cultures of feces from chicks reared on wood-shavings litter were often negative, suggesting that culturing litter as an indicator of infection has limited value.     

Detection of Campylobacter jejuni strains in the water lines of a commercial broiler house and their relationship to the strains that colonized the chickens

Campylobacter jejuni is frequently present in the intestinal tract of commercial broiler chickens, and their drinking water has been proposed to be an initial source of bacteria for newly hatched chicks. We studied three sequential commercial broiler flocks raised in a house from which we had cultured C. jejuni from the nipple waters prior to placement of the first flock. Campylobacter cells were detected by immunofluorescence in the biofilm of the drinking nipples during the weeks when the flock was colonized with C. jejuni but not during weeks when the birds were negative. Campylobacter jejuni was isolated from the drinking water during the growth of the first flock and was present in the birds from all three flocks. Randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing with primer OPA11 indicated that seven distinct strains were present within the broiler house. One strain found in drinking water was similar to a strain found in birds in the second flock; however, RAPD-PCR with primer HLW85 showed that the strains were not identical. These results suggest that although the watering system is a potential source of C. jejuni in broiler flocks, the waterborne strain in this study was not detected in the birds.     

Inability of cecal microflora to promote reversion of viable nonculturable Campylobacter jejuni

Campylobacter jejuni cells are able to enter a viable but nonculturable (VBNC) state when they are suspended in water. In the present experiments we inoculated day-of-hatch leghorn and broiler chicks with normal gut microflora and subsequently challenged these with high doses of VBNC C. jejuni. The objective was to determine if the pre-establishment of a normal gut flora would enable VBNC Campylobacter to recover, revert to the vibrionic form, and colonize the cecum. Day-of-hatch leghorn and broiler chicks were gavaged through the esophagus with 0.75 ml of a continuous-flow culture of normal cecal organisms. Two days after gavage, the same chicks were gavaged with 0.75 ml (greater than 10(9) colony-forming units) of a VBNC suspension of C. jejuni. Seven days later, cecal contents were collected, serially diluted, and examined for the presence of viable culturable C. jejuni. Our results demonstrated that the VBNC C. jejuni cells were unable to revert to a vibrionic culturable form capable of colonizing the cecum.     

Characterization of two putative mechanosensitive channel proteins of Campylobacter jejuni involved in protection against osmotic downshock

Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission.     

Factors responsible for the introduction and spread of Campylobacter jejuni infection in commercial poultry production

Campylobacter jejuni and related thermophilic campylobacters were not found in a hatchery or in chicks aged less than seven days. However, an increasing proportion of chicks aged two weeks and older shed campylobacters in their droppings. It was shown that a likely source of C jejuni for young chicks was the environment in the immediate vicinity of the rearing houses, and that infection could readily be introduced on the footwear and clothing of farm staff. Thermophilic campylobacters were found in the air, litter and drinking water containers in the rearing and finishing houses.     

Pathogenicity of Campylobacter jejuni for turkeys and chickens.

A Campylobacter jejuni isolate obtained from a turkey liver, designated C101, and a C. jejuni isolate obtained from the feces of a chicken, designated C111, were used to inoculate their respective hosts. Isolate C101 depressed weight gain by 20% when inoculated into newly hatched poults or 4-day-old poults. It also caused death, hepatic necrosis, and generalized hemorrhages in turkey embryos. The chicken-derived isolate, C111, did not reduce weight gain in newly hatched chicks, but it did induce mortality in chicken embryos. The supernatant of the cultures of both C. jejuni isolates also caused mortality in embryos.     

The effect of apramycin on colonization of pathogenic Escherichia coli in the intestinal tract of chicks

The purpose of the present study was to examine the effect of apramycin sulphate on the colonization of pathogenic E. coli in the intestines of chicks. Apramycin treatment (0.5g/l in the drinking water) of 3-to 5-week-old Leghorn chicks for 24 or 48 hours resulted in a reduction, to an undetectable level, in the number of coliforms in the digestive tract for at least the first 24 h. Per os inoculation of E. coli (O2:K1) after 24 to 48 h of treatment resulted in a significant decrease in colony forming units (cfu) in the digestive tract of the treated chicks. Food deprivation from the time of inoculation did not significantly change the results. However, food and water deprivation caused bacteraemia in a number of the control chicks but not in the treated chicks. Comparison of the level of protection between Leghorn and broiler (Anak strain) chicks revealed that there was a significantly higher (P<0.05) level of bacteraemia in the broiler than in the Leghorn chicks. Chicks treated with 0.25 g/l or 0.125 g/l apramycin for 24 or 48 h before E. coli inoculation showed significantly lower cfu in the colon and caecum than untreated control chicks, but significantly higher cfu were found in the colon than in chicks treated with 0.5 g/l apramycin. Although in vitro preincubation of apramycin with ileum cells did not decrease the percentage of cells to which the bacteria adhered, the number of bacteria adhered per cell decreased significantly. Taken together, our in vitro and in vivo results show that apramycin is effective against E. coli by preventing colonization of the gut by the bacteria, which could lead to a reduction of colibacillosis in poultry.     

Effect of egg storage upon the survival of Campylobacter jejuni in laboratory-infected fertile poultry eggs

Fertile eggs were infected by Campylobacter jejuni in the laboratory by a temperature differential method of inoculation, which resulted in up to 10% of the hatched birds carrying C. jejuni in the intestine. When infected eggs were stored for 5 1/2 days before incubation, the infection rate of the eggs had decreased to 20% or less when set, and no infected chicks were hatched. Inoculation of eggs after 8 days in storage also failed to yield infected chicks. In all cases, the hatch ratio was no different from that of uninfected control eggs.     

Infectious bursal disease virus-induced immunosuppression exacerbates Campylobacter jejuni colonization and shedding in chickens

Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in humans in the United States. Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. To analyze a possible role of IBDV-induced immunosuppression in colonization and shedding of C. jejuni, two experiments were conducted. In both experiments, group 1 consisted of noninoculated control chickens, groups 2 and 3 were inoculated with varying doses of C. jejuni, and groups 4 and 5 were inoculated initially with IBDV followed by doses of C. jejuni similar to groups 2 and 3. Campylobacter jejuni was recovered from the cloaca and cecum, but not the small intestines, from all chickens in groups 2 and 3. In groups 4 and 5, C. jejuni was recovered from the small intestines, cecum, and cloaca from all chickens. The amount (colony-forming units/sample) of C. jejuni recovered from chickens in groups 4 and 5 was significantly greater (P < 0.05) than the amount recovered from chickens in groups 2 and 3; and C. jejuni was also present sooner in these groups than in groups 2 and 3. Bursa samples from chickens in groups 4 and 5 were significantly smaller (P < 0.05) than in the other groups. Additionally, real-time polymerase chain reaction results for IBDV were positive in groups 4 and 5 and negative in all other groups. This study indicated that IBDV infection exacerbated colonization and shedding of C. jejuni, presumably through the immune suppression this virus causes in chickens. It highlights the need for further investigation into the role of immunosuppression in preharvest control strategies for food-borne disease-causing agents.     

Protection of neonatal chicks against a lethal challenge of Escherichia coli using DNA containing cytosine-phosphodiester-guanine motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. We recently have shown that CpG ODN protects against extracellular bacterial infections in mature chickens. The objective of this study was to investigate the effect of CpG ODN on Escherichia coli septicemia in neonatal broiler chicks. Two-day-old chicks, or embryonated eggs that had been incubated for 18 or 19 days, received 50 microg CpG ODN. Three days after exposure to CpG ODN, a virulent isolate of E. coli was inoculated subcutaneously in the neck of each bird. Birds were examined for 7 days post-E. coli challenge and dinical, pathologic, and bacteriologic assessments were conducted. The control group of birds that received no CpG ODN had a survival rate of 0% to 20%. In contrast, groups that received CpG ODN, either by intramuscular or in ovo routes, had significantly higher survival rates (P < 0.0001). Bacterial counts in air sacs were significantly lower when birds or embryos were treated with CpG ODN as compared with controls. A dose as low as 10 microg of CpG ODN, administered intramuscularly, was able to protect birds significantly against E. coli challenge. Formulation of CpG ODN with 30% Emulsigen did not enhance the protection. This study demonstrates that CpG ODN has systemic protective effects in broiler chicks against E. coli infections. This is the first time that CpG ODN has been demonstrated to have an immunoprotective effect against a bacterial infection in chicks following in ovo delivery.     

Phage therapy reduces Campylobacter jejuni colonization in broilers

The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established. Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. Counts were compared with control birds not receiving phage therapy. A clear 3 log decline in C. jejuni counts was initially observed in the therapeutic group, however, after 5 days bacterial counts stabilized at a level 1 log lower than that of the control group. Colonization of C. jejuni in the prevention group was delayed by the treatment and after an initial 2 log reduction, colonization stabilized within a week at levels comparable to the therapeutic group. The CFU and PFU counts displayed opposing highs and lows over time, indicative of alternating shifts in amplification of bacteria and phages. There were no adverse health effects from the phage treatment. Two different phages were combined as therapeutic treatment of Campylobacter positive chickens challenged at the age approaching broiler harvest. This again resulted in a significant decrease in Campylobacter colonization. We conclude that phage treatment is a promising alternative for reducing C. jejuni colonization in broilers.