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.     

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 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.     

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.     

Enhancement of Campylobacter jejuni virulence by serial passage in chicks

Four isolates of Campylobacter jejuni were studied to determine changes in virulence following six serial passages in chicks. Chicks that received invasive isolates exhibited diarrhea and depressed weight gain. Immature mice were used to assess virulence of the passaged isolates of C. jejuni. Nine-day-old mice infected with passaged isolates showed lethargy, dehydration, depression, decreased weight gain, and occult blood in feces. Mouse pups inoculated with the third and sixth chick passage levels of an invasive isolate showed significant depression in mean daily weight gain and elevated mortality compared with controls and subjects inoculated with unpassaged isolates. This study demonstrated enhancement of virulence in a C. jejuni isolate following chick passage. In contrast, three other passaged isolates failed to show any consistent increase in virulence.     

Cecal colonization of chicks by porcine strains of Campylobacter coli

Ten genotypically distinct strains of Campylobacter coli were isolated from a swine production facility. These porcine isolates were then orally inoculated into day-of-hatch leghorn chicks and were excellent colonizers of the chick cecum. Campylobacter coli recovered from inoculated chickens were genotypically identical to the challenge strain. The absence of host specificity suggests a possible movement of strains among swine, field animals and birds, and poultry houses.     

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.     

Evaluation of Campylobacter jejuni colonization of the domestic ferret intestine as a model of proliferative colitis

Forty 3- to 17-week old domestic ferrets, including 2 gnotobiotes, were inoculated orally and/or rectally with 10(6) to 10(9) colony-forming units of 1 or more of 4 strains of Campylobacter jejuni, 3 of mink and 1 of human origin. Feeding or gavage of any of the 4 strains, in milk or broth, with or without preinoculation sodium bicarbonate treatment to neutralize stomach acid, induced colonization in 38/40 ferrets; diarrhea lasted 2 to 4 days in conventional kits, 6 days in gnotobiotes. Bacteremia was detected in 4 of 18 tested, 2 to 5 days after inoculation. Two strains caused no more severe disease or prolonged colonization after 3 serial IV passages in kits than they did before passage. Multiple inoculations with a given strain resulted in progressively briefer colonization and milder disease, but subsequent inoculation with a different strain induced colonization and gastrointestinal disease similar to a primary infection. Five kits inoculated rectally after 4 previous homologous inoculations were resistant to colonization as well as to disease. Agglutinin titers of ferrets inoculated orally or rectally once were low or undetectable, but increased in response to repeated inoculation. Pretreatment with a 1% formalin enema caused mild colon irritation without clinical or histologic evidence of proliferative colitis in ferrets concurrently inoculated orally and/or rectally, whether or not they had preexisting antibodies to any strain of C jejuni. Histologic examination of tissues revealed leukocytic infiltration of intestinal lamina propria in 29 of 35 infected kits and 5 of 8 noninfected controls, and cryptosporidiosis in 5 infected kits plus 1 control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of anaerobic cecal microflora and dietary lactose on colonization resistance of layer chicks to invasive Salmonella enteritidis.

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.     

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.     

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.     

The importance of Campylobacter jejuni to the meat industry: a review

Campylobacter jejuni is a microorganism that only recently has been implicated in gastroenteritis in humans. As appropriate methods used for detection of the bacterium have been developed, the rates of illness caused by the pathogen were found to approach or surpass those attributed to Salmonella. Substantial evidence has been gathered to document that the route for human infection is through the ingestion of adulterated food and drink. Some slaughter animals harbor this potential pathogen among the intestinal flora and, consequently, transfer of the organism to carcasses and to the resulting meat products does occur. The most frequently implicated meat is poultry, with an incidence of recovery of C. jejuni from the store-bought poultry meat reported to be at least 50%. Red meat from slaughter animals have also yielded this bacterium from carcasses, but at lower incidence levels. Foodborne disease has been associated most frequently with the ingestion of raw milk, but poultry, hamburger, and other foods have all been implicated as potential sources. However, cause and effect relating the presence of C. jejuni in meat and human gastroenteritis has not been demonstrated. Additional research is needed to determine whether C. jejuni isolated from meat causes gastroenteritis and whether all strains of the organism are virulent. Recognition of C. jejuni as a potential meatborne pathogen by the meat industry is necessary, and appropriate sanitary practices to prevent passage of the organism through meat products should be implemented.     

Intestinal carriage of Campylobacter jejuni and Salmonella by chicken flocks at slaughter.

Campylobacter jejuni were isolated in large numbers from the majority of birds sampled in colonic swabs from 28 of 60 flocks at slaughter. By contrast only small numbers of birds from 11 of the same 60 flocks yielded Salmonella enteritidis serotypes. Three C. jejuni isolates from each flock were serotyped on the basis of their heat-stable antigens, using antisera prepared against 16 serotypes common in Campylobacter diarrhea in man. The majority (72 of 83) of the chicken isolates could be serotyped.     

Phagocytosis and intracellular killing of Campylobacter jejuni by elicited chicken peritoneal macrophages.

In vitro phagocytosis and intracellular survival of Campylobacter jejuni (strains B540 and Clin 1) in chicken peritoneal macrophages were studied. Macrophages were induced with Sephadex G-50 and harvested 48 hr later by peritoneal lavage. The extent of phagocytosis over time was determined by enumerating the intracellular C. jejuni after removal of extracellular C. jejuni with gentamicin. Pre-incubation of C. jejuni with antiserum generally enabled the macrophages to ingest greater numbers of cells than when the organism was pre-incubated in phosphate-buffered saline. C. jejuni were exposed to macrophage uptake for 30 minutes in a 5% CO2 incubator at 42 C. This suspension was then exposed to 12.5 micrograms gentamicin/ml to eliminate extracellular bacteria. Subsequently, the intracellular survival of C. jejuni was examined by monitoring its number within the macrophage at 30 minutes, 3 hr, and 6 hr after phagocytosis. Macrophages from C. jejuni-colonized chickens and from uncolonized control chickens were able to almost destroy the organism within the experimental period.     

Evaluation of efficacy of several disinfectants against Campylobacter jejuni strains by a suspension test

The objective of this study was to determine the efficacy of 16 active compounds and 11 commercial disinfectants against Campylobacter jejuni. Two reference strains (one of avian origin and the other isolated from bovine) and two avian field strains were tested in suspension test in the presence and absence of serum. Chloramine-T, povidone–iodine (1% available iodine), cetylpiridinium chloride, ethanol, isopropanol, chlorhexidine digluconate, formaldehyde, phenol, and 10 of the 11 commercial formulations (eight of them based on quaternary ammonium compounds) showed an excellent disinfectant capability, resulting in the highest level of reduction (>6-log₁₀) in colony-forming units of the four C. jejuni strains compared regardless of the presence or absence of organic material. These compounds might be helpful in the adoption of environmental control measures against C. jejuni.     

Evaluation of efficacy of several disinfectants against Campylobacter jejuni strains by a suspension test

The objective of this study was to determine the efficacy of 16 active compounds and 11 commercial disinfectants against Campylobacter jejuni. Two reference strains (one of avian origin and the other isolated from bovine) and two avian field strains were tested in suspension test in the presence and absence of serum. Chloramine-T, povidone–iodine (1% available iodine), cetylpiridinium chloride, ethanol, isopropanol, chlorhexidine digluconate, formaldehyde, phenol, and 10 of the 11 commercial formulations (eight of them based on quaternary ammonium compounds) showed an excellent disinfectant capability, resulting in the highest level of reduction (>6-log₁₀) in colony-forming units of the four C. jejuni strains compared regardless of the presence or absence of organic material. These compounds might be helpful in the adoption of environmental control measures against C. jejuni.     

The effect of lactic acid sprays on Campylobacter jejuni inoculated onto poultry carcasses.

Spraying poultry carcasses with 1% lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4 degrees C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1% or 2% lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4 degrees C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2% lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.