Evaluation of different Campylobacter jejuni isolates to colonize the intestinal tract of commercial turkey poults and selective media for enumeration.

Consumption of contaminated poultry products is the main source of human campylobacteriosis, for which Campylobacter jejuni is responsible for 90% of human cases. Although chickens are believed to be a main source of human exposure to C. jejuni, turkeys also contribute to cases of human infection. Little is known about the kinetics of C. jejuni intestinal colonization in turkeys, or best selective media for their recovery. Enumeration of C. jejuni from intestinal samples can be challenging because most selective Campylobacter media support the growth of non-Campylobacter organisms. In this study, we sought to compare a) C. jejuni isolates that persistently colonize different compartments of the poult intestinal tract, and b) selective media to enumerate C. jejuni from turkey intestinal samples. Three-week-old poults were orally colonized with C. jejuni isolates NCTC 11168 or NADC 20827 (isolated from a turkey flock). Mock-colonized poults were orally gavaged with uninoculated media. Poults were euthanized at d 3, 7, and 21 post colonization and direct plated on different selective Campylobacter media [Campy Line agar with sulfamethoxazole (CLA-S), CHROMagar Campylobacter (CAC) and Campy Cefex] for enumeration. Isolates NCTC 11168 and NADC 20827 poorly colonized the distal ileum. Both isolates colonized the colon, but the number of NADC 20827 significantly decreased at d 21. Isolates NCTC 11168 and NADC 20827 persistently colonized the cecum for up to 21 days. There was no significant difference in the Campylobacter amount recovered on CLA-S and CAC. Campy Cefex failed to prevent growth of background microbes to enumerate C. jejuni from turkey samples. Two independent PCR assays (multiplex PCR and qPCR) confirmed that colonies grown on CLA-S or CAC were C. jejuni. Data from this study demonstrated that isolates NCTC 11168 and NADC 20827 persistently colonized the cecum, and CLA-S or CAC were successful to enumerate Campylobacter from intestinal samples. These findings will be useful to evaluate the host response by C. jejuni in turkeys, and test pre-harvest strategies to reduce its colonization and promote food safety.

Seroprevalence in Chickens against Campylobacter jejuni Flagellar Capping Protein (FliD) in Selected Areas of the United States.

Campylobacter jejuni is a causative pathogen of human acute bacterial gastroenteritis. Infected poultry products are regarded as a major source for human C. jejuni infection. The flagellar capping protein (FliD) is highly conserved among C. jejuni strains/isolates and is antigenic as analysed by immunoblot. In this study, we used the FliD protein as a probe to survey the prevalence of C. jejuni antibodies in chickens from two areas in the United States. A total of 394 samples were tested. Sera from layer breeders of 44-52 weeks of age tested 100% positive, while 4- to 6-week broilers from 22 premises showed 7-100% positivity. These results demonstrate that anti-FliD antibodies were prevalent in the poultry population in the areas of serum samples collected.

Variation in Campylobacter Multilocus Sequence Typing Subtypes from Chickens as Detected on Three Plating Media.

The objective of this study was to compare subtypes of Campylobacter jejuni and Campylobacter coli detected on three selective Campylobacter plating media to determine whether each medium selected for different subtypes. Fifty ceca and 50 carcasses (representing 50 flocks) were collected from the evisceration line in a commercial broiler processing plant. Campylobacter was cultured and isolated from cecal contents and carcass rinses on Campy-Cefex, Campy Line, and RF Campylobacter jejuni/coli agars. When a positive result was obtained with all three media, one colony of the most prevalent morphology on each medium was selected for further analysis by full genome sequencing and multilocus sequence typing. Sequence types were assigned according to PubMLST. A total of 49 samples were positive for Campylobacter on all three media. Forty samples contained only C. jejuni , three had only C. coli , and both species were detected in six samples. From 71% of samples, Campylobacter isolates of the same sequence type were recovered on all three media. From 81.6% of samples, isolates were all from the same clonal complex. From significantly fewer samples (26%, P < 0.01), one medium recovered an isolate with a sequence type different from the type recovered on the other two media. When multiple sequence types were detected, six times the medium with the odd sequence type was Campy-Cefex, four times it was Campy-Line, and six times it was RF Campylobacter jejuni/coli . From one sample, three sequence types were detected. In most cases, all three plating media allowed detection of the same type of Campylobacter from complex naturally contaminated chicken samples.

Comparison of cumulative drip sampling with whole carcass rinses for estimation of Campylobacter species and quality indicator organisms associated with processed broiler chickens.

The whole carcass rinse (WCR) procedure is routinely used as a sampling method for determining the presence and number of quality-indicator organisms or pathogens associated with broiler chicken carcasses in processing facilities. Collection of a cumulative drip sample by placing collection vessels under the processing line could potentially capture a more representative sample of bacterial populations associated with an entire flock with less labor than individual bird rinses. The purpose of this study was to evaluate a cumulative drip sampling method for recovery of Campylobacter spp. and 3 types of quality indicator organisms from broiler carcasses. Cumulative drip and WCR samples were collected on 14 d from a commercial broiler processing facility over a 3-mo period. No statistically significant difference was demonstrated between the WCR and cumulative drip sampling methods in recovery of Campylobacter spp., total aerobes, Enterobacteriaceae, or Escherichia coli associated with the postevisceration samples (P > 0.01). Analysis of the pyrosequencing census data demonstrated high interbird variability and indicates cumulative sampling may be required to obtain fully representative sampling of a flock. For most bacterial taxa, the relative abundance in individual WCR was correlated with cumulative drip samples, but some taxa were undercounted or missed entirely by individual WCR. Consequently, individual carcass rinses may not be representative of the flock microbial community. The cumulative drip sampling technique may save labor and provide a more representative summary of process control in poultry processing facilities.

Comparison of an automated most-probable-number technique with traditional plating methods for estimating populations of total aerobes, coliforms, and Escherichia coli associated with freshly processed broiler chickens.

An instrument (TEMPO) has been developed to automate the most-probable-number (MPN) technique and reduce the effort required to estimate some bacterial populations. We compared the automated MPN technique with traditional microbiological plating methods and Petrifilm methods for estimating the total viable count of aerobic microorganisms (TVC), total coliforms (CC), and Escherichia coli populations (EC) on freshly processed broiler chicken carcasses (postchill whole carcass rinse [WCR] samples) and cumulative drip-line samples from a commercial broiler processing facility. Overall, 120 broiler carcasses, 36 prechill drip-line samples, and 40 postchill drip-line samples were collected over 5 days (representing five individual flocks) and analyzed by the automated MPN and direct agar plating and Petrifilm methods. The TVC correlation coefficient between the automated MPN and traditional methods was very high (0.972) for the prechill drip samples, which had mean log-transformed values of 3.09 and 3.02, respectively. The TVC correlation coefficient was lower (0.710) for the postchill WCR samples, which had lower mean log values of 1.53 and 1.31, respectively. Correlations between the methods for the prechill CC and EC samples were 0.812 and 0.880, respectively. The estimated number of total aerobes was generally greater than the total number of coliforms or E. coli recovered for all sample types (P < 2e⁻¹6). Significantly more bacteria were recovered from the prechill samples than from the postchill WCR or cumulative drip samples (P < 9.5e⁻¹² and P < 2e⁻¹6, respectively). When samples below the limit of detection were excluded, 92.1% of the total responses were within a single log difference between the traditional plating or Petrifilm methods and the automated MPN method.

Differential carbon source utilization by Campylobacter jejuni 11168 in response to growth temperature variation.

Campylobacter spp. readily colonize the intestinal tracts of both human and avian species. While most often commensal organisms in birds, campylobacters remain the leading cause of bacterial gastroenteritis in humans. The association of campylobacters with poultry is well established as a primary route for human exposure. The difference in normal core body temperature between chickens (42 degrees C) and humans (37 degrees C) has been suggested to trigger potential colonization or virulence factors and investigators have demonstrated differential gene expression at the two temperatures. Campylobacter spp. exhibit unique nutritional requirements and have been thought to only utilize amino acids and Kreb cycle intermediates as carbon sources for growth. We evaluated the ability of the genome-sequenced strain of Campylobacter jejuni 11168 (GS) to oxidize 190 different substrates as sole carbon sources at 37 degrees C and 42 degrees C using phenotype microarray (PM) technology. Results indicate that the expected amino acids, l-serine, l-aspartic acid, l-asparagine, and l-glutamic acid were utilized in addition to a number of organic acids. In general, oxidation of the substrates was greater at 42 degrees C than at 37 degrees C with a few exceptions. By employing the PM method, we observed a number of potential false-positive reactions for substrates including the triose, dihydroxyacetone; and the pentose sugars, d-xylose, d-ribose, l-lyxose, and d- and l-arabinose. The presence of genes possibly responsible for utilization of pentose sugars is supported by the genomic sequence data, but actual utilization as sole carbon sources for active respiration has not been observed. A better understanding of the metabolic pathways and nutritional requirements of campylobacters could lead to improvements in culture media for detection and isolation of the pathogen and to future intervention methods to reduce human exposure.

Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria.

Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans, Enterococcus faecium, or Enterococcus hirae, was initially identified as antagonistic to Campylobacter jejuni. The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Campylobacter jejuni, and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies. The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically, the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100 degrees C and was stable at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated feed significantly (P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log(10) CFU. Enterocin E-760 also significantly (P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.

Effect of on-farm litter acidification treatments on Campylobacter and Salmonella populations in commercial broiler houses in northeast Georgia.

Two commercially available litter treatments, aluminum sulfate and sodium bisulfate, were tested to determine their effect on Campylobacter and Salmonella levels associated with commercial broilers during a 6-wk grow-out period. A total of 20 broiler houses at 10 different locations were studied; 5 aluminum sulfate-treated houses, 5 sodium bisulfate-treated houses, and 10 paired, untreated control houses. A single application rate was investigated for each treatment. Fecal samples (n=20 per house) were analyzed at wk 2, 4, and 5 and 6 for Campylobacter and Salmonella. The results indicated that, at the application rates investigated, both acidifying litter treatments caused a slight delay in the onset of Campylobacter colonization in broiler chicks. Salmonella levels remained unaffected, with no significant effect seen with either treatment (P > 0.05). Campylobacter populations and Salmonella incidence associated with unprocessed, whole-carcass rinse samples (n=10 per house) analyzed at the end of production (wk 5 and 6) were unaffected by treatment.

Influence of relative humidity on transmission of Campylobacter jejuni in broiler chickens.

Horizontal transmission of Campylobacter jejuni among broiler chickens has been documented; however, the influence of RH on transmission rates is an important factor that has not been extensively studied. The purpose of our experiments was to determine the rate of C. jejuni colonization among groups of broilers raised in microbiological isolation under high (approximately 80%) and low (approximately 30%) RH conditions. Day-of-hatch chicks (n = 100 per group) were placed on wood shavings in high and low humidity-controlled pens and challenged with C. jejuni by introducing 2 seeder birds orally inoculated with C. jejuni into each group. The rate of colonization was monitored by analyzing ceca from 10 chicks from each group at d 1, 2, 3, 4, and 7. After 3 wk, the remaining chickens were removed, and 100 newly hatched chicks were placed on the contaminated litter. A second trial was conducted with the litter as the only inoculum source. Trials were repeated in this manner with the time between removing birds and placing newly hatched chicks on the litter extended to 6 h, 24 h, and 1 wk. Significant differences in Campylobacter colonization rates were observed between chickens raised under the high and low RH conditions. A delay in colonization was observed in birds raised under the low RH conditions, which increased with the increased time between removal of birds and placement of newly hatched chicks. These experiments demonstrate the importance of humidity in the transmission of Campylobacter from litter, and they could lead to practical applications to help reduce Campylobacter colonization in broilers.

Campylobacter and Salmonella populations associated with chickens raised on acidified litter.

Two commercially available acidifying litter treatments, aluminum sulfate (alum) and sodium bisulfate, were tested to determine their effects on Campylobacter and Salmonella colonization frequencies and populations associated with broilers raised on treated pine litter. To produce contaminated litter, broiler chicks were inoculated with two bacterial cocktails (multistrain mixtures of campylobacters and salmonellae) and were allowed to shed on the litter for about 6 wk. Upon bird removal, litter in duplicate pens was immediately treated with two levels of aluminum sulfate [3.63 or 7.26 kg/4.6 m2 (8 or 16 lb/50 ft2)] or sodium bisulfate 1.13 or 1.81 kg/4.6 m2 (2.5 or 4 lb/50 ft2)]; untreated pens served as controls. Immediately after treatment, day-of-hatch chicks were released in the pens. Frequency and populations of Campylobacter and Salmonella associated with ceca and whole carcass rinse (WCR) samples were determined for each duplicate pen at Weeks 1, 4, and 6. Both levels of the aluminum sulfate and sodium bisulfate litter treatments tested significantly (P < 0.05) reduced Campylobacter colonization frequency and populations in the ceca. Significantly, no Campylobacter was recovered from WCR samples associated with high level aluminum sulfate-treated pens at any time; although control pens were 95, 78, and 38% positive at Weeks 1, 4, and 6, respectively. Salmonella colonization frequency and populations in the ceca were not significantly decreased by any of the treatments investigated. Although effective pathogen control will most likely require a combination of interventions, acidifying treatment of litter in poultry production may serve as a means to help control Campylobacter and to reduce horizontal transmission of pathogens in broiler flocks.

Development of a selective differential agar for isolation and enumeration of Campylobacter spp.

Direct plating is an effective technique for isolation and enumeration of Campylobacters from a variety of sample types; however, distinguishing Campylobacters from non-Campylobacter contaminants that frequently grow on many existing agars is difficult. In this study, it was determined that exposing Campylobacters to low levels (200 mg/liter) of triphenyltetrazolium chloride (TTC) was not inhibitory to growth yet was sufficient to give a deep-red to magenta color to the colonies. The new agars (Campy-Line agar [CLAI and Campy-Line blood agar [CLBA]) are translucent. The contrast of deep-red colonies on a translucent background greatly facilitates Campylobacter isolation and makes enumeration on light boxes or by electronic means possible. Direct plating of broiler carcass rinse samples (n = 20) was compared on Campy-Cefex agar and CLA. Recovery of Campylobacter populations was not significantly different between the agars (P < 0.05); however, enumeration was much less labor intensive with the CLA. No contaminants were observed on the CLA, whereas the Cefex agar supported the growth of approximately 14 contaminating (non-Campylobacter) CFU/ml. In a separate trial, recovery of Campylobacters from carcass rinses (n = 25) was similarly compared on Cefex, CLA, and CLBA. Again, recovery of Campylobacters was not significantly different between the agars (Pearson correlation coefficient = 0.988), whereas about nine contaminating (non-Campylobacter) CFU/ml were observed on Cefex agar and none on CLA or CLBA. Although some contaminants can still grow on CLA and CLBA and can present red colonies, most of these contaminants are easily distinguished from Campylobacter by differences in colony morphology.

Comparison of methods for recovery and enumeration of Campylobacter from freshly processed broilers.

Most traditional Campylobacter detection and enumeration procedures are difficult and time consuming. Estimations of Campylobacter populations by the most probable number (MPN) method are especially laborious. The objective of this collaborative study, performed in duplicate in Agricultural Research Service and Food Safety Inspection Service laboratories, was to compare two MPN procedures (utilizing different selective enrichment broths and plating media) to the direct plating technique for enumeration of Campylobacter from freshly processed (postchill, postdrip) broiler chicken carcasses. Results obtained from the direct plating of carcass rinse samples on Campy-cefex agar were not significantly different (P > 0.05) from an MPN procedure employing Hunt's Campylobacter selective enrichment broth followed by recovery on modified Campylobacter charcoal differential agar. However, both of these procedures provided significantly (P < 0.05) better recovery than a second MPN procedure using Rosef's selective enrichment broth followed by plating on Mueller-Hinton blood agar with antibiotics. The direct plating method offers a more simple, less expensive, more rapid alternative to traditional MPN procedures for estimating Campylobacter populations associated with freshly processed broiler carcasses.

Effect of yeast-supplemented feed on Salmonella and Campylobacter populations in broilers.

The effect of the yeast, Saccharomyces boulardii, on experimental cecal colonization of broilers with Salmonella typhimurium and Campylobacter jejuni was investigated. Duplicate pens of broiler chicks were given ad libitum access to a standard feed supplemented with no yeast (control), or 1 g (1x), or 100 g (100x) dried S. boulardii/kg feed. All chicks except negative controls were challenged on Day 4 with 3.2 x 10(8) cfu S. typhimurium and 6.5 x 10(8) cfu C. jejuni by oral gavage. After 3 wk, the broilers were euthanatized and ceca were aseptically removed and analyzed for Salmonella and Campylobacter. Frequency of Salmonella colonization was significantly (P < 0.05) reduced due to yeast treatment. Of the positive control birds, 70% were colonized with Salmonella; whereas only 20 and 5% of the 1x and 100x yeast-treated birds were colonized. Mean number of Salmonella per gram of ceca and contents were log 1.64, 0.35, and 0.15, respectively, for the control, 1x, and 100x yeast-treated birds. Campylobacter colonization was not significantly affected by yeast treatment. Similar results were obtained from a second trial conducted in larger isolation floor pens.

Yeast treatment to reduce Salmonella and Campylobacter populations associated with broiler chickens subjected to transport stress.

The prevalence of Campylobacter and Salmonella on retail poultry carcasses remains a significant public health concern. The stresses associated with transporting poultry prior to slaughter have been shown to increase pathogen populations both in the intestinal tract and on the carcass exterior. The yeast, Saccharomyces boulardii, was evaluated for its ability to reduce populations of Salmonella and Campylobacter in broiler chickens subjected to transport stress. Chicks, inoculated with individual strains of Salmonella and Campylobacter were held for 6 wk and then divided into two groups with half of the chickens receiving 10% dried yeast in the feed for 60 h. The birds were then caged and transported to simulate commercial conditions. After euthanatizing the birds, the ceca were aseptically removed and analyzed for Salmonella and Campylobacter. With no yeast treatment, transport stress caused the Salmonella colonization frequency to increase more than fivefold, from 3.3 to 16.7%. Yeast treatment significantly reduced the frequency of Salmonella colonization to lower than prestress levels, as no Salmonella were recovered from the ceca of these birds (P < 0.05). Similar results were obtained from birds challenged with a mixture of Salmonella and Campylobacter strains. Before transport, 53.3% of these chickens were positive for Salmonella. Transport stress increased the colonization rate to 67.5% in control birds, whereas the colonization of yeast-treated chickens decreased to 40% (P < 0.05). Frequency of Campylobacter isolation from the ceca was not affected by treatment, but Campylobacter populations present in the ceca were significantly reduced in the mixed strain trial (P < 0.05).

Factors Affecting Aflatoxin B1 Removal by Flavobacterium aurantiacum.

This study was conducted to investigate several factors affecting the removal of aflatoxin B1 by Flavobacterium aurantiacum NRRL B-184. A simple spectrophotometric procedure was evaluated and compared to an established high-performance liquid chromatography (HPLC) method and found to be useful for determining aflatoxin concentration in test solutions of phosphate buffer. Using the spectrophotometric method, 72-h cultures of F. aurantiacum were observed to remove more toxin from solution than 24-h cultures. Likewise, populations of 1010cells removed aflatoxin at a faster rate than did 109 cells, although the total amount removed did not differ. Transferring F. aurantiacum cultures in tryptic soy broth every 3 days for over 3 days for over 8 months had no apparent effect on their ability to remove measurable amounts of aflatoxin B1 from solution. Populations of 1 × 109 CFU/ml or less heat-inactivated F. aurantiacum were unable to remove aflatoxin B1 from phosphate buffer.

Role of Toxin Concentration and Second Carbon Source in Microbial Transformation of Aflatoxin B1 by Flavobacterium aurantiacum.

The aflatoxins are toxic and carcinogenic mycotoxins which contaminate a variety of foods and feeds. The bacterium Flavobacterium aurantiacum NRRL B-184 has been previously shown to be effective in degrading aflatoxin B1) in liquid test medium as well as in several food types. The purpose of this study was to investigate the effects of an added nutrient source and added aflatoxin on the ability of F. aurantiacum to degrade aflatoxin B1. Radioactively labeled aflatoxin B1 was added to test solutions containing cells in phosphate buffer or tryptic soy broth. Nonlabeled aflatoxin B1 was also added to similar flasks. Analysis of radioactive CO2 and water- and chloroform-soluble portions of the cell supernatant fluids revealed that neither added nutrients nor added nonlabeled toxin had a significant influence on the microbial transformation of aflatoxin B1 These results suggest that the microbial degradation of aflatoxin by F. aurantiacum is probably a mineralization phenomenon and not a co-metabolism. The ability of this organism to detoxify aflatoxin without the need for exogenous energy sources could be important to future endeavors attempting to use the organism, or the mechanisms responsible, in fermentation reactions.

Evidence for Degradation of Aflatoxin B1 by Flavobacterium aurantiacum.

The fate of [14C]aflatoxin B1 in the presence of Flavobacterium aurantiacum was determined. [14C]aflatoxin B1 was incubated with live or dead F. aurantiacum cells for up to 72 h. Samples were extracted with chloroform and the aqueous and non-polar phases were analyzed for 14C content over time. The normally chloroform-soluble [14C]aflatoxin B1 is rapidly converted to water-soluble degradation products by live cells. Some radioactivity was initially associated with live cells but was lost at about the same rate as the water-soluble degradation products accumulated. The evolution of 14CO2 by the live cells provides further evidence that at least a portion of the [14C]aflatoxin B1 is metabolized. Dead F. aurantiacum cells bind some aflatoxin but are unable to carry out the degradation to water-soluble compounds or carbon dioxide.