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.     

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.     

Evaluation of a Competitive Exclusion Culture and Megan Vac 1 on Salmonella Typhimurium Colonization in Neonatal Broiler Chickens

The present study was designed to evaluate the effect of individual or simultaneous application of 2 products, a competitive exclusion culture (CEC) or Megan Vac 1 (MV), for bioefficacy in reducing Salmonella Typhimurium (ST) cecal colonization in broiler chicks following experimental challenge. In experiment 1, CEC and MV were applied to day-of-hatch broiler chicks, and chicks were experimentally challenged with ST approximately 48 h later. In control chicks, ST was recovered at a level of 3.84 log₁₀ cfu/g following direct plating of cecal contents, and 12 of 19 (63.15%) cultured individual ceca were positive following selective enrichment. In chicks receiving CEC by spray application on day of hatch, a numerical reduction in ST recovered from cecal contents (2.63 log₁₀ cfu/g) and a significant reduction (P < 0.05) in recovery of ST from cultured ceca following selective enrichment (4 of 18, or 22.2%) was observed when compared with controls. Significant reductions in ST cecal colonization in chicks treated with MV alone were not observed in experiment 1. In experiment 2, chicks received day-of-hatch spray application of CEC alone, MV alone, or CEC and MV as a combined application immediately prior to or within 24 h of chick placement. When chicks were experimentally challenged with ST 48 h posthatch, significant reductions (P < 0.05) in cecal colonization by ST were observed with each experimental group when compared with nontreated controls. These data suggest that both commercially available products, alone or in combination, are efficacious in reducing cecal colonization in broiler chicks challenged with ST.     

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.     

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.     

Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks

One-day-old broiler chicks were inoculated with volatile fatty acid producing cecal flora from adult chickens. The chicks were divided into four groups and provided 1) no lactose, 2) 2.5% lactose in water, 3) 5% lactose in feed, or 4) 10% lactose in feed, until 10 days of age. All groups were challenged at 3 days of age with 10(6) or 10(8) S. typhimurium. At 10 days, the number of Salmonella in the ceca of the chicks challenged with 10(6) Salmonella was significantly decreased (P less than 0.01) in the groups provided lactose as compared with the controls. A significant decrease (P less than 0.01) in Salmonella numbers occurred in the chicks challenged with 10(8) Salmonella and provided 10% lactose. Providing 2.5% lactose or 5% lactose failed to inhibit Salmonella growth in chicks challenged with 10(8) Salmonella. The pH of the ceca of the groups provided lactose decreased significantly (P less than 0.05) and was accompanied by significant increases (P less than 0.01) in the concentrations of bacteriostatic acetic and propionic acids. Results showed that providing dietary lactose to broiler chicks and inoculation with normal cecal flora decreased cecal pH, increased the concentrations of bacteriostatic volatile fatty acids, and inhibited Salmonella colonization.     

Effect of dietary lactose on Salmonella colonization of market-age broiler chickens

The effect of providing lactose in feed and inoculation with volatile fatty acid-producing anaerobic cultures (AC) of cecal flora on Salmonella typhimurium colonization was evaluated in broilers. One-day-old chicks were divided into four groups and provided 1) no lactose, no AC; 2) AC, no lactose; 3) AC and lactose on days 1-10; or 4) AC and lactose on days 1-40. All groups were challenged per os with 10(6) Salmonella on day 3 and with 10(8) Salmonella on day 33. Salmonella growth in the cecal contents was significantly decreased (P less than 0.01) on day 10 in the chicks provided lactose from day 1-10. However, after the removal of lactose from the diet, the chicks were susceptible to Salmonella colonization. The number of Salmonella in the ceca was significantly reduced (P less than 0.05) in the chicks provided lactose throughout the 40-day growing period. Dietary lactose decreased the pH of the cecal contents and was accompanied by marked increases in the concentrations of undissociated bacteriostatic volatile fatty acids in the cecal contents.     

鸡空肠弯曲菌对SPF雏鸡致病性研究

用鸡空肠弯曲菌（Ｃａｍｐｙｔｏｂａｃｅｃｒｉｅｊｕｎ：）对１日龄ＳＰＦ（Ｓｐｃｉｉｃ－ｐａｔｈｏｇｅｎｆｒｃｅ）雏鸡进行感染实验。随机将６０只１日龄ＳＰＦ雏鸡分为４组，那和对照组，每组１５只。分别用含菌７．５Ｘ１０８ＣＦＵ／ｍｌ、７．５ｘ１０４ＣＦＵ／ｍｌ，７．５×１０２ＣＦＵ／ｍｌ布氏肉汤菌液经口时ｔ、Ｊ、Ｉ组接种。接种后分别于２、４、６，８、１０日踏踏机取３只／组、经剖构观察病理变化；从心血、肝、胆汁、脾、肠内容物分离病原菌，测量肠道内物空肠弯曲菌菌数。得出以下结论；经口接种３．７５ｘ１０２ＣＦＵ空场弯曲菌即可造成１日龄ＳＰＦ雏鸡感染发病；感染鸡的主要病理变化为肝脏表面有点状，条索状或片状出血，同时伴有形状不规则大小不等的黄白色坏死灶，盲肠膨大充满气泡，十二指肠、空肠、直肠粘膜有散在的小出血点，首选检苗材料为胆汁、其次是脾、肝等器宫。     

Biological control of Salmonella typhimurium in young chickens

The effect of dietary lactose and anaerobic cultures of cecal microflora of mature chickens on the colonization of young broiler chickens by Salmonella typhimurium was evaluated. Newly hatched chicks were given either no treatment (controls), anaerobic cecal cultures, lactose (2.5%) in the drinking water, or both anaerobic cultures and lactose. Chicks were challenged per os at 3 days of age with either 10(6) or 10(8) S. typhimurium resistant to nalidixic acid and novobiocin. On day 10, the cecal contents of the chicks were examined for S. typhimurium, pH, short-chained volatile fatty acids (VFAs), undissociated VFAs, and lactic acid. Chicks given either lactose alone or cecal anaerobes alone had significantly (P less than 0.05) fewer S. typhimurium recovered from their ceca than the controls. Chicks given the combination of dietary lactose and cecal anaerobes had significantly fewer S. typhimurium recovered from their ceca than the chicks given dietary lactose or cecal anaerobes alone. Chicks given lactose had significant (P less than 0.05) increases in the lactic acid concentration of their cecal contents. Increased lactic acid concentrations were directly correlated to decreased cecal pH values and caused a reduction in the total concentration of VFAs but a significant (P less than 0.05) increase in the undissociated form of some VFAs.     

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.     

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.     

Effects of diet formulations containing proteins from different sources on intestinal colonization by Campylobacter jejuni in broiler chickens

The objective of this study was to compare the effects of 3 diet formulations containing different protein sources (animal, plant, and a combination of animal and plant) on the colonization of Campylobacter jejuni in the gastrointestinal tract of broiler chickens. A freshly isolated strain of C. jejuni (biotype IV, serotype HS O:21, O:29, HL untypable) from a broiler chicken was used to infect 3-day-old chicks that had been free of C. jejuni; 0.5 mL of an inoculum containing 10⁸ colony-forming units was administered orally. Shedding of the organism was studied, and C. jejuni in the ceca, jejuni, and crop were enumerated by quantitative culture. The isolates recovered from the birds during the study period of 35 d were characterized and confirmed as C. jejuni by the use of standard methods and underwent biotyping, serotyping, antimicrobial susceptibility testing by disk diffusion and the E-test, and flagellin gene typing. A cyclical pattern of shedding of C. jejuni was observed in all the birds. Colonization was highest in the ceca. The ceca of birds receiving plant-protein-based feed had significantly less colonization then the ceca of birds receiving the other types of feed, whereas the differences in colonization of the jejuni and crops were not significant. Characterization by biotyping, serotyping, and flagellin gene typing showed that 95% of the recovered isolates were identical to the strain used for infecting the chicks. However, with the Lior-HL typing scheme, 74% of the recovered isolates were HL untypable. Antimicrobial resistance testing did not reveal significant differences between the infecting strain and the recovered isolates among the different feed groups.     

Effects of probiotics and maternal vaccination on Salmonella enteritidis infection in broiler chicks

The effects of probiotics and maternal vaccination with an inactivated Salmonella Enteritidis (SE) vaccine on day-old chicks challenged with SE were evaluated. A 2 X 3 factorial arrangement was used (with or without probiotics; breeders nonvaccinated, vaccinated intramuscularly, or vaccinated intraperitoneally). Three trials were conducted in isolation cabinets and SE challenge was different between trials. The number of SE organisms per chick and the time interval between housing and introduction of seeder birds (hereafter called challenge) were 1.6 X 10(8) and 1 hr (Trial I), 1.8 X 10(6) and 12 hr (Trial II), and 1.2 X 10(4) and 24 hr (Trial III). SE recovery was assessed in ceca and liver at 3, 5, and 7 days postchallenge, and the number of colony-forming units (CFU) in ceca was evaluated at 5 and 7 days postchallenge. The number of SE (log CFU) in the ceca reduced 0.56 log (from 7.59 to 7.03) and 1.45 log (7.62 to 6.17) because of the treatment with probiotics in Trials II and III, respectively. The greater reduction in Trial III indicates the importance of the early use of probiotics on the prevention of SE infection. Treatment with probiotics resulted in a smaller number of SE-positive livers after 5 days postchallenge on Trial III. Although there was no significant effect of maternal vaccination on the number of SE CFU in the ceca, a significant effect of maternal vaccination on the SE CFU was observed in the liver, but not in the ceca at 5 days after challenge.     

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.     

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.     

Effect of a commercial competitive exclusion culture on reduction of colonization of an antibiotic-resistant pathogenic Escherichia coli in day-old broiler chickens

One-day-of-age broiler chickens were administered a commercial competitive exclusion (CE) product and then challenged by three different methods with an Escherichia coli O78:K80 that was pathogenic for poultry and resistant to six antibiotics. Three challenge methods were used on 2-day-old broilers: direct challenge, precolonized seeder, and instant seeder. Direct challenge was accomplished by administering the challenge E. coli per os. The precolonized seeder challenge had two chicks that had received the challenge E. coli 24 hr previously, whereas the instant seeder challenge had two chicks given the challenge E. coli per os with immediate placement with the experimental birds. One oral dose of the commercial CE product significantly reduced the colonization of the small intestine, large intestine, and ceca by the highly antimicrobial resistant poultry pathogenic E. coli O78:K80 at 7 and 14 days postchallenge by all three challenge methods. The overall mean reductions in colonization were 3.0 log10 for the large intestine, 3.0 log10 for the small intestine, and 4.0 log10 for the cecum. The most severe challenge method, on the basis of the least amount of reduction of colonization of the challenge E. coli by the CE, was by the direct oral gavage at 2 days of age.     

Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces

Chicks were treated orally on the day of hatch with either fresh or frozen competitive exclusion (CE) cultures (native gut microflora). Chicks were fed either unmedicated feed or one of five commercial broiler starter rations or nine experimental feed mixtures containing varying amounts and combinations of anticoccidial and antimicrobial medicaments. After 2 days, they were challenged with approximately 10(6) colony-forming units of a nalidixic-acid-resistant strain of Salmonella typhimurium. Six days later, chicks were sacrificed and ceca were analyzed for S. typhimurium. Colonization of 2-day-old chicks was prevented or at least greatly reduced in most instances by treatment of chicks with a CE culture, but the efficacy of CE broth cultures stored at -70 C diminished over time. Not all CE cultures tested gave equal protection against Salmonella colonization, and CE cultures were more susceptible to some feed additives than others. Of the commercial or experimental feed tested, only the feed containing the combination of nicarbazin and bacitracin interfered with the protective effect of the CE culture.     

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.     

Cecal colonization of chicks by bovine-derived strains of Campylobacter

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.     

Influence of Campylobacter jejuni cecal colonization on immunoglobulin response in chickens

The immunoglobulin response of chickens to colonization by Campylobacter jejuni isolates B-540 and Clin-1 was monitored. Chicken humoral IgG and biliary secretory IgA (sIgA) responses were assessed by enzyme-linked immunosorbent assay (ELISA). Samples were taken from 128 C. jejuni-colonized chickens and 104 uncolonized chickens housed in a controlled environment. An indirect ELISA was performed using the homologous isolate of C. jejuni as the capture antigen and was developed with the specific goat anti-chicken IgG or IgA alkaline phosphatase conjugates. The ELISA absorbance values of the test samples at 405 nm (serum diluted 1:32 and bile diluted 1:10) were normalized in direct proportion to standard sera and bile sample values. In the colonized chickens, humoral IgG activities were highest at hatch, dropped to their lowest level after 2 weeks, and increased by 8 weeks to levels similar to those detected at hatch. The sIgA activity was lowest at hatch and increased by 4 weeks in colonized chickens while remaining lower in the control chickens. Chickens colonized with isolate B-540 showed a primary sIgA response during the first 4 weeks and reached a plateau over the final 4 weeks. In spite of these limited humoral and secretory immunoglobulin responses, once the chicken ceca was colonized by C. jejuni, the organism persisted throughout the 8-week experiment.