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.     

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).     

Campylobacter colonization of the crops of newly hatched leghorn chicks

We have studied colonization of crops in newly hatched leghorn chicks (a layer breed) by wild-type and mutant strains of Campylobacter jejuni. We established that the wild-type parent strain forms a stable population level within the crop and that the mutant strains will do likewise. Concentrations of mutant strains in the crop were usually below that of the wild-type parent strain and ranged from 10(3) to 10(5) colony-forming units. These results differ from results we have previously reported concerning cecal colonization, where these same mutant strains lacked colonizing ability. The present results, therefore, indicate that bacterial factors necessary for colonization of the crop are not the same as those needed for colonization of the cecum.     

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.     

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.     

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.     

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.     

Colonization of broilers with Campylobacter in conventional broiler-chicken flocks

Eight of 16 conventional broiler-chicken flocks examined contained Campylobacter. All isolates were identified as C. jejuni except from 1 flock were C. coli was isolated. One herd consisting of 6 different houses where Campylobacter regularly has been isolated was continuously examined. It was not possible to isolate Campylobacter from newly hatched chickens or from environmental samples and cloacal swabs during the 2 first weeks of growth.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

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.     

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.     

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.     

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.     

Campylobacter succession in broiler chickens

The competitive ability of Campylobacter coli OR12 over C. jejuni OR1 has been examined in experimental broiler chickens following the observation that C. coli replaced an established C. jejuni intestinal colonisation within commercial chicken flocks reared outdoors [El-Shibiny, A., Connerton, P.L., Connerton, I.F., 2005. Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens. Appl. Environ. Microbiol. 71, 1259-1266]. Co-cultures of C. coli OR12 with C. jejuni OR1, revealed that the two species were able to grow together at similar growth rates in exponential growth phase but if the disparity of the inoculum ratios were >log(10)4 in favour of C. coli OR12, C. jejuni OR1 was observed to prematurely enter decline phase. Chickens were pre-colonised with C. jejuni OR1 at 21-days-old to examine succession in vivo. The birds were inoculated between 2 and 12 days later with C. coli OR12, to determine if the second isolate could efficiently colonise and compete with an established C. jejuni strain. C. coli OR12 were able to co-colonise before replacing C. jejuni OR1 as the dominant species when the birds were more than 27 days of age at the time of administration over a 4-day period. If these criteria were met C. coli OR12 became the dominant isolate otherwise co-colonisation occurred until they were met. C. coli OR12 was also found to displace three alternative C. jejuni strains from pre-colonised chickens challenged with C. coli OR12 at 30 days of age and tested at 40 days. These data raise the possibility of manipulating populations of Campylobacter colonising chickens through competition.     

Irrelevance between the induction of anti-Campylobacter humoral response by a bacterin and the lack of protection against homologous challenge in Japanese Jidori chickens

On-farm vaccination of chickens against Campylobacter jejuni is considered a potentially effective countermeasure to decrease campylobacteriosis via consumption of contaminated chicken meat, but is not yet available. In this study, 2 groups of Jidori chicks were immunized subcutaneously with a formalin-killed C. jejuni with 2 different adjuvants. Other chicks served as the unvaccinated control group. Both vaccines induced high levels of anti-Campylobacter IgG but did not decrease bacterial excretion in cecal droppings and bacterial load in the liver and spleen after oral challenge with 10(5) CFU of the homologous strain. Further study is needed to address the observed irrelevance and to develop a novel effective vaccine against C. jejuni.     

Effect of coated and non-coated fatty acid supplementation on broiler chickens experimentally infected with Campylobacter jejuni

The aim of this study was to examine whether and to what extent the supplementation of feed with a coated or non-coated mixture of fatty acids (caprylic and capric acid) affects broiler chickens experimentally infected with Campylobacter jejuni. The study was carried out using 48 chickens divided into four experimental groups. Throughout the whole rearing period (1-42 days), the chickens were fed a diet supplemented with 0.25% caprylic and capric acid (1:1), coated or non-coated. At the age of 14 and 28 days, chickens were orally challenged with C. jejuni. At regular time intervals post-inoculation, the shedding of C. jejuni was assayed using quantitative real-time PCR. Both supplements significantly decreased faecal C. jejuni counts by 1.2-4.1 log(10) CFU/g 4 days post-inoculation; after this time period, the effect of medium-chain fatty acids (MCFA) was less pronounced or absent. Campylobacter jejuni counts in excreta samples were significantly lower in chickens fed coated MCFA than in those fed non-coated MCFA. No effect of MCFA on feed intake or growth of chickens was observed. In conclusion, (i) MCFA are active against C. jejuni and (ii) the encapsulation enhanced the efficacy of the acids. These results allow the recommendation of using MCFA as feed additives in chickens, preferably 2-3 days before slaughter.     

禽源空肠弯曲杆菌的分离鉴定及其致病性的初步研究

本研究对广东部分地区不同品种家禽空肠弯曲杆菌的流行状况进行了调查。通过菌落和菌体形态、生化特征、培养特性等生物学特性和多重PCR方法对所分离菌株进行鉴定,结果表明广东地区鸡、鸭和鹅空肠弯曲杆菌的带菌率分别为7.93%、2.46%和4.16%。从分离株中选取10株进行了致病性试验,结果表明禽源空肠弯曲杆菌对雏鸡的主要病理变化是腹泻便血,肝脏出现白色坏死灶,盲肠充血、膨大充满气泡和红色内容物。     

Immunization of chickens with temperature-sensitive mutants of Mycoplasma gallisepticum

Temperature-sensitive (TS) mutants of the S6 strain of Mycoplasma gallisepticum (MG) were used to immunize newly hatched chickens. Immunized chickens developed antibodies to the wild-type (WT) S6 strain as demonstrated by serologic tests. MG was recovered from nasal cavities but not from the lower respiratory tract of the immunized chicks. Three weeks after intranasal immunization, chickens were challenged via the air sacs with the virulent S6 strain. Immunized chickens were significantly better protected from development of air-sac lesions than were controls.     

The specificity of antibody in chickens immunised to reduce intestinal colonisation with Campylobacter jejuni

Poultry consumption has been identified as a major risk factor for human infection with Campylobacter jejuni in developed countries. C. jejuni is present in the gastrointestinal tract of broiler chickens at the time of slaughter, and faecal contamination of carcases during processing results in significant campylobacter loads on carcases. One approach to reducing the level of carcase contamination with C. jejuni is to control campylobacter infection in broiler chickens. To this end, the study described here investigated the specificity of antibody in serum and intestinal secretions of chickens that had been immunised with campylobacter antigens and then challenged with viable bacteria. The immunodominant antigens in the serum of birds that showed a 2-log reduction in caecal colonisation with C. jejuni included flagellin protein (61–63 Kd) and three additional antigens of 67, 73.5 and 77.5 Kd. Only flagellin and the 67 Kd antigen were recognised by IgG antibody in gastrointestinal secretions of the same birds. Antibody from chickens immunised with purified native flagellin protein recognised flagellin protein and the 67 Kd antigen in Western blots probed with serum, but only the flagellin proteins (61–63 Kd) in Westerns probed with gastrointestinal secretions. Analysis of the specificity of the response to flagellin protein using recombinant clones that expressed regions of the flagellin gene suggests that epitopes in each region of the flagellin protein were immunogenic. Of the immunodominant antigens, only flagellin appeared to be surface-exposed on viable C. jejuni, although conformational epitopes of flagellin appeared to be sensitive to the method of antigen purification. The results of this study suggest that flagellin and possibly the 67 Kd antigen may be valuable for immunological control of intestinal infection with C. jejuni in chickens, but that further work is required to purify these as vaccine candidates by using methods that preserve conformational epitopes.     

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.