N. J. Stern

A Differential-selective Medium and Dry Ice-generated Atmosphere for Recovery of Campylobacter jejuni

A selective-differential medium for isolation of Campylobacter jejuni from chicken carcasses was developed. The medium, Campy-Cefex, consisted of Brucella agar, 5% lysed horse blood, 0.05% ferrous sulfate (FeSO4.7H2O), 0.05% sodium pyruvate, 0.02% sodium bisulfite, and antibiotic supplements of 33 mg/L sodium cefoperazone and 200 mg/L cycloheximide. A total of 41 chicken carcass samples were plated onto Campy-Cefex, Campylobacter cefoperazone desoxycholate agar, and Campylobacter brucella agar plate media. Campy-Cefex proved as productive and selective as the other media. Campy-Cefex allowed for easier differentiation of C. jejuni from other flora compared to differentiation on Campylobacter cefoperazone desoxycholate agar medium. Differentiation of the non- Campylobacter spp. flora from Campylobacter spp. was the same on both Campy-Cefex and Campy-BAP. The selectivity for the organism on Campy-Cefex was better than on Campy-BAP. Growth of seven isolates of C. jejuni in microaerobic-(5% O2, 10% CO2, 85% N2) and dry ice-generated atmospheres was also assessed. After 24 h of incubation, the mean log10 CFU generated, using the same culture suspensions and medium, was 2.07 and 1.81 for the microaerobic and dry ice atmospheres, respectively. These two developments allow for simplification of materials and methods required to isolate C. jejuni from foods.

Distribution of Campylobacter spp. in selected U.S. poultry production and processing operations.

A study was conducted of 32 broiler flocks on eight different farms, belonging to four major U.S. producers. The farms were studied over I complete calendar year. Overall, 28 (87.5%) of the flocks became Campylobacter positive, and only four (12.5%) remained negative throughout the 6- to 8-week rearing period. In the majority of flocks, sampled every 2 weeks throughout production, Campylobacter-positive fecal and cecal samples were not detected until 4 to 8 weeks of age. In only six of the flocks were environmental samples found to be positive before shedding of Campylobacter was detected in the birds. Even in some of the Campylobacter-negative flocks, contamination of the rearing environment was positive for Campylobacter but did not result in the birds subsequently excreting the organism. These findings are discussed in relation to U.S. husbandry practices and present uncertainty about sources of Campylobacter infection for poultry flocks. Birds were often transported to the processing plant in coops that were already contaminated with Campylobacter, and the organisms were sometimes found in samples of scald water and chill water. After chilling, the proportions of Campylobacter-positive carcasses from different producers ranged from 21.0 to 40.9%, which is lower than in other studies, and possible reasons are considered.

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).

Determination of the Incidence of Salmonella spp., Campylobacter jejuni, and Clostridium perfringens in Wild Birds near Broiler Chicken Houses by Sampling Intestinal Droppings

Several methods were evaluated for collecting fecal and intestinal samples from wild birds found near broiler chicken houses. A few intestinal samples and cloacal swabs were obtained from European starlings and house sparrows. Most of the samples collected consisted of wild bird droppings found on or near the houses. Samples were collected from each of four farms of a broiler integrator during a grow-out cycle: a cycle in the summer for farm A, fall for farm B, and spring, summer, fall, and winter for farms C and D. Of the 25 wild bird intestinal and fecal samples collected from a broiler house on farm A during a grow-out cycle in July-August 1997, 24% were positive for Salmonella spp., 4% for Campylobacter jejuni, and 28% for Clostridium perfringens. Of the nine fecal samples collected from broiler house B in a grow-out cycle in September-November 1997, 33% were positive for Salmonella spp., 11% for C. jejuni, and 22% for C. perfringens. For farms C and D, of the 23 samples collected in March-April 1998, 0 were positive for Salmonella spp., 11% for C. jejuni, and 52% for C. perfringens; of 27 samples collected in June-July 1998, 4% were positive for Salmonella spp., 0 for C. jejuni, and 13% for C. perfringens; of 24 samples collected in August-October 1998, 14% were positive for Salmonella spp., 5% for C. jejuni, and 4% for C. perfringens; of 14 samples collected December 1998-January 1999, 0 were positive for Salmonella, 50% for C. jejuni, and 14% for C. perfringens. The incidence of these bacterial enteropathogens in wild birds near the broiler chicken houses suggests that wild birds that gain entry to poultry grow-out houses have the potential to transmit these pathogens to poultry.

Sources and movement of Salmonella through integrated poultry operations: a multistate epidemiological investigation.

The prevalence of Salmonella from numerous sources in 32 integrated broiler operations of high- and low-performing broiler houses was characterized from four states across four seasons. Previous studies of Salmonella in broilers have been limited in scope, offering only a snapshot of pathogen prevalence as seen on a small number of individual farms. Twenty-six different sample types were collected from the hatchery to the end of processing, and Salmonella was found in all sample types. A total of 10,740 samples were analyzed for Salmonella, and 973 (9.1%) of these samples, including 49 of 798 (6.1%) carcass rinse samples, were Salmonella positive. Hatchery transport pads (389 of 765, 50.8%), flies (28 of 150, 18.7%), drag swabs (57 of 402, 14.2%), and boot swabs (20 of 167, 12%) were samples from which Salmonella was most frequently isolated. Thirty-six different serotypes were identified, and the most frequently encountered serotypes were Salmonella Senftenberg, Salmonella Thompson, and Salmonella Montevideo. Determining critical contaminating sources and following the movement of Salmonella through integrated poultry operations will help researchers and the industry develop practical intervention strategies.

Molecular Subtype Analyses of Campylobacter spp. from Arkansas and California Poultry Operations

ABSTRACT Campylobacter isolates from diverse samples within broiler production and processing environments were typed by using flaA short variable region DNA sequence analysis. Sixteen flocks from four different farms representing two broiler producers in Arkansas and California were analyzed. Fourteen of the flocks (87.5%) were Campylobacter-positive; two remained negative throughout the 6-week rearing period. In general, multiple clones were present within a flock. Additionally, clones found within a flock were also present on the final product, although the diversity of Campylobacter spp. on the final product appeared to be reduced relative to that observed within the flock. Comparison of clones between flocks on the same farm revealed that some clones of Campylobacter persisted in multiple flocks. Furthermore, some clones were identified across the two farms that were under the same management. In two sampling periods, environmental isolates were positive for Campylobacter prior to flock shedding. Environmental samples associated with five additional flocks were positive for Campylobacter concomitantly with recovery of Campylobacter from the birds. Analysis of the environmental isolates that were positive prior to flock shedding demonstrated that in some instances the environmental isolates possessed genotypes identical to those of isolates originating from the flock, while in other cases the environmental isolates possessed genotypes that were distantly related to isolates obtained from the flock. Analyses of environmental isolates that tested positive concurrently with the positive isolates from the flocks demonstrated varied results; in some instances the environmental isolates possessed genotypes identical to those of isolates originating from the flock, while in other cases the environmental isolates possessed genotypes that were distantly related to isolates obtained from the flock. These data suggest that the external environment may contribute to Campylobacter contamination during poultry production and processing. However, environmental contamination with Campylobacter does not appear to be the sole contributing factor.

Comparison of Three Methods for Recovery of Campylobacter spp. from Broiler Carcasses

We compared three enrichment methods, with different sampling times (mid-and endpoint incubation) and various dilutions of enrichment culture for productivity in the isolation of Campylobacter spp. from 50 retail-level chicken carcasses. We subcultured the enrichment cultures onto three Campylobacter spp.-selective media (Campy-BAP, CCDA, Campy-Cefex) to compare yields of the organism. The highest yield (43/50) of Campylobacter spp. from these carcasses was derived by using the 24-h enrichment culture of Hunt & Radle diluted 1:100 before plating onto any of the three selective plating media. When all carcass analyses were combined, Campylobacter spp. was recovered from 49 of 50 broilers. This study indicates the optimum approaches for the recovery of Campylobacter spp., as well as the high incidence of the organism among the broiler carcasses tested.

Bacteriocins to control Campylobacter spp. in poultry--A review.

The unacceptably high frequency of Campylobacter jejuni transmission from poultry to humans encourages scientists to consider and create alternative intervention strategies to control the pathogen in poultry production. Extremely high numbers of Campylobacter (often >10(8) cfu/g of poultry intestinal material) potentiate high numbers of the organism on the processed broiler carcass with increasing consequent human health risk. Many scientists believe interventions during poultry production portend the greatest opportunity for reducing risk of disease. Over the past 10 yr, we have focused our studies on nonantibiotic bacteriocin application to intervene during animal production and this is the subject of the current review. The application of therapeutic bacteriocin treatments to reduce poultry colonization diminishes Campylobacter from >10(8) cfu/g of cecal materials to nondetectable or very low levels in treated birds. Further, the review provides scientists with a useful starting point for the further development of industry-applicable interventions leading to reduced transmission of this agent in human disease.

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.

Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and characterization of its bacteriocin, including the antimicrobial activity spectrum.

ABSTRACT Lactobacillus salivarius 1077 (NRRL B-50053) was isolated from poultry intestinal materials, and in vitro anti-Campylobacter jejuni activity was demonstrated. The isolate was then used for bacteriocin production and its enrichment. The protein content of the cell-free supernatant from the spent medium was precipitated by ammonium sulfate and dialyzed to produce the crude antimicrobial preparation. A typical bacteriocin-like response of sensitivity to proteolytic enzymes and resistance to lysozyme, lipase, and 100°C was observed with this preparation. The polypeptide was further purified by gel filtration, ion-exchange, and hydrophobic-interaction chromatography. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), Edman degradation, and isoelectrofocusing were used to characterize its 3,454-Da molecular mass, the amino acid sequence of its 37 residue components, and the isoelectric point of pI 9.1 of the bacteriocin. Bacteriocin L-1077 contained the class IIa bacteriocin signature N-terminal sequence YGNGV. MICs of bacteriocin L-1077 against 33 bacterial isolates (both Gram negative and Gram positive) ranged from 0.09 to 1.5 μg/ml. Subsequently, the therapeutic benefit of bacteriocin L-1077 was demonstrated in market-age (40- to 43-day-old) broiler chickens colonized with both C. jejuni and Salmonella enterica serovar Enteritidis. Compared with untreated control birds, both C. jejuni and S. Enteritidis counts in colonized ceca were diminished by >4 log10 and S. Enteritidis counts in both the liver and the spleen of treated birds were reduced by 6 to 8 log10/g compared with those in the nontreated control birds. Bacteriocin L-1077 appears to hold promise in controlling C. jejuni/S. Enteritidis among commercial broiler chickens.