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.

Development and stability of bacteriocin resistance in Campylobacter spp.

AIMS: Several bacteriocins (BCNs) that were identified from chicken commensal bacteria dramatically reduced Campylobacter colonization in poultry and are being directed toward on-farm control of this important foodborne human pathogen. A recent study has shown that BCN resistance in Campylobacter jejuni is very difficult to develop in vitro. In this study, in vivo development and stability of BCN resistance in Campylobacter was examined. METHODS AND RESULTS: Chickens infected with Camp. jejuni NCTC 11168 were treated with BCN E-760 at the dose of 5 mg kg(-1) body weight day(-1) via oral gavages for three consecutive days, which selected BCN-resistant (BCN(r)) mutants in the treated birds. However, all the in vivo-selected mutants only displayed low levels of resistance to BCN (MIC = 2-8 mg l(-1)) when compared to parent strain (MIC = 0.5 mg l(-1)). Inactivation of CmeABC efflux pump of the BCN(r) mutants led to increased susceptibility to BCN (8-32 fold MIC reduction). Three different BCN(r) Campylobacter strains (in vitro- or in vivo-derived) were examined for the stability of BCN resistance using both in vitro and in vivo systems. The low level of BCN resistance in these strains was not stable in vitro or in vivo in the absence of BCN selection pressure. CONCLUSIONS: Usage of BCN E-760 only selected low-level BCN(r) Camp. jejuni mutants in vivo, and the low-level BCN resistance was not stable in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides helpful information for risk assessment of the future practical application of the anti-Campylobacter BCNs in animals.

Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms.

The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.

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.

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.

Antimicrobial Activities of Bacteriocins E 50-52 and B 602 Against Antibiotic-Resistant Strains Involved in Nosocomial Infections.

The antimicrobial spectra of previously published bacteriocin E 50-52 (39 a.a.; 3,932 Da; pI = 8.5) and bacteriocin B 602 (29 a.a.; 3,864 Da; pI = 7.2) were determined. Named peptides were related to class IIa (pediocin-like) bacteriocins. Minimal inhibitory concentrations (MICs) of bacteriocins have been determined for bacterial isolates that were causative agents of nosocomial infections collected from Russian hospitals in 2003-2007, namely methicillin-resistant Staphylococcus aureus (MRSA) (n = 10); Acinetobacter baumannii (n = 11); Citrobacter freundii (n = 8); Escherichia coli (n = 9); Klebsiella pneumoniae (n = 10); Proteus spp. (n = 6); and Pseudomonas aeruginosa (n = 10). The majority of these tested isolates have been shown to be multidrug resistant and carry genetic determinants of antimicrobial resistance that were detected using polymerase chain reaction (PCR). The MICs of bacteriocin B 602 ranged from ≤0.025-1.56 µg/ml, and for bacteriocin E 50-52 from 0.05 to 6.25 µg/ml for all of 64 bacterial clinical isolates tested. Interestingly, the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E 50-52 and B 602 show good activity against nosocomial bacterial agents resistant to many classes of modern antibacterials used in clinical practice. These bacteriocins should be examined as an alternative in treating infections caused by such agents.

Salmonella species and Campylobacter jejuni cecal colonization model in broilers.

Salmonella and Campylobacter are of concern to the poultry industry because of the continuing association of poultry-borne transmission of these diseases to humans. Live, mature bird interventions can be demonstrated only by comparing colonization in nontreated groups of control birds with treated bird groups. This study attempted to create a reproducible broiler chicken colonization model. When chicks were challenged 2 d posthatch with both Salmonella and Campylobacter, cecal colonization was achieved. By 4 wk posthatch, Salmonella counts per gram of cecal content diminished to very low or nondetectable levels. Campylobacter counts remained high throughout the test period. To achieve the goal of creating a mature bird Salmonella intestinal colonization model, oral treatment of 10 to 25 mg of vancomycin was given to 4-wk-old broilers, and 3 h later a composite of 3 Salmonella isolates were gavaged into the chickens. Birds were sampled 1 and 2 wk later. The data indicated that colonization was achieved at levels of 10(6-7) cfu g(-1) of cecal materials (at wk 5) and >10(2) to 10(4) cfu g(-1) of cecal materials (at wk 6).

The paternal effect of Campylobacter jejuni colonization in ceca in broilers.

Campylobacter jejuni is one of the most common causes of acute enteritis worldwide. Chickens are believed to be the main reservoir of C. jejuni. The role that host genetics play in resistance/susceptibility to C. jejuni colonization in broilers is still not clear. Day-old broilers from 2 parental lines (A and B) and their F(1) reciprocal crosses (C and D) were challenged orally with 10(5) cfu of C. jejuni to address the role of genetics in determining resistance/susceptibility to C. jejuni colonization in broilers. Cloacal swabs were collected on 6, 10, and 13 d postinoculation (dpi), and cecal contents cultured for C. jejuni on 7 and 14 dpi. The number of C. jejuni colonies in the cloacal swabs and cecal contents of each bird were recorded at each time point. Significantly fewer bacteria were found in the cecal contents from line A than B (P < 0.05) and cross D (A male x B female) when compared with cross C (A female x B male) at both 7 and 14 dpi. There was a significant correlation between C. jejuni counts in cloacal swabs and those in cecal contents. The results indicated that a paternal effect might be one of the important genetic factors influencing resistance to C. jejuni colonization in broilers.

Comparison of poultry exudate and carcass rinse sampling methods for the recovery of Campylobacter spp. subtypes demonstrates unique subtypes recovered from exudate.

The carcass rinse procedure is a method commonly used for the detection of Campylobacter spp. on processed poultry products. Alternatively, carcass exudate (weep or drip), a viscous fluid comprised of blood and water that leaks into packaging, can also be sampled. It is unknown however if direct carcass rinse or exudate/weep can be utilized to preferentially recover different Campylobacter spp. subtypes. If there is a difference in subtypes recovered, the Campylobacter spp. subtypes from carcass rinse analysis may not be indicative of consumer exposure, as the exudate is the fluid to which consumers are potentially exposed to due to kitchen cross-contamination. Experiments were conducted to determine if there are differences in recovery of Campylobacter spp. subtypes between the two methodologies. The experiment was performed in triplicate using three flocks located on different farms. For each flock, 50 fecal samples were obtained on the farm, 25 carcass rinses during pre-chill processing, 25 carcass rinses during post-chill processing, and 50 samples from exudate from carcasses stored at 4 degrees C (25 after 2-day storage and 25 after 6-day storage). Each sample type was cultured for Campylobacter spp. Isolates recovered from positive samples were subtyped using flaA SVR (flagellin A-short variable region) DNA sequence typing and compared for relatedness. The data demonstrated that multiple subtypes of Campylobacter jejuni were present in a flock, and that subtypes present in a flock during production were also present on the final processed product. Subtypes recovered by the two recovery methodologies were similar based on flaA SVR classification. Combining the totals from all 3 flocks a total of 10 flaA SVR subtypes were recovered from post-chill carcass rinses and 9 subtypes recovered from 6-day exudate samples.

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.

Frequency and enumeration of Campylobacter species from processed broiler carcasses by weep and rinse samples.

Frequency and numbers of Campylobacter spp. were assessed per freshly processed, contaminated broiler carcass. Campylobacter-positive flocks were identified by cecal sample analysis at slaughter. These flocks had been tested as Campylobacter negative at 4.1 +/- 0.9 d prior to slaughter. Levels of contamination were estimated using 2 sampling approaches per carcass: (1) free weep fluids and (2) whole-carcass, 100 mL of distilled water rinses. Estimations of counts were determined by directly plating dilutions of weeps and rinses onto Campy-Cefex agar and incubating the plates at 41.5 degrees C under microaerobic atmosphere. Confirmation was provided by latex agglutination to quantify levels per milliliter of weep and per 100 mL of rinse. Thirty-two slaughter groups ( approximately 20 carcasses per group) were compared from 2003 to 2004. The Campylobacter-positive weep frequency was 84.8%, whereas the frequency for rinse samples was 74.4% (P < 0.001). Enumeration of Campylobacter spp. on positive samples ranged from 0.70 to 6.13 log(10) cfu/mL of weep (geometric mean of 2.84) and from 2.30 to 7.72 log(10) cfu/100 mL of rinse (geometric mean of 4.38). The correlations between weep and rinse were 0.814 with 0.5 mL of rinse and 0.6294 when applying 0.1 mL of rinse The quantitative regression analyses for these 2 corresponding tests were log(10) rinse (for 0.5 mL of inoculum) = 1.1965 log(10) weep + 0.4979, and log(10) rinse (for 0.1 mL of inoculum) = 1.322 log(10) weep - 0.1521. FlaA SVR sequencing of isolates indicated that the same genotypes were found in weep and rinse samples. Weep and rinse sampling led to different proportions of Campylobacter-positive carcasses detection, but we demonstrated that this difference was reduced by increasing the amount of rinse fluid used for plating.

Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Enumeration and identity of Campylobacter spp. in Italian broilers.

Transmission of Campylobacter to humans has been prominently associated with mishandling or improperly preparing contaminated poultry carcasses. The number of organisms per carcass represents an important measure of human exposure to the agent. Therefore, we wished to estimate this public exposure over 1 yr among Italian broiler carcasses. We sampled 213 broiler carcasses from rinse water samples collected from a single slaughterhouse. Groups of carcasses had mean processed weights ranging from 1.2 to 2.7 kg. These were produced from 22 commercial broiler chicken flocks collected from 12 different farms, 3 of which were seasonally tested. Carcasses were rinsed with sterile water, and the rinse suspension was then serially diluted and spread-plated directly onto Campy-Cefex agar plates. One to 5 typical Campylobacter colonies per plate were identified by polymerase chain reaction as Campylobacter thermo-tolerant species. The overall estimated mean count per carcass in our study was 5.16 +/- 0.80 log10 cfu. This value increased in summer and autumn, as well as on carcasses collected from farms located > 100 km far from the slaughterhouse. A total of 678 Campylobacter colonies were identified by polymerase chain reaction. The majority of isolates were classified as Campylobacter jejuni (49.2%) or Campylobacter coli (47.5%). The overall number of C. jejuni was significantly higher on 1) carcasses weighing > 2 kg, 2) carcasses belonging to flocks with > 10,000 birds, and 3) carcasses collected from farms located > 100 km from the slaughterhouse. Moreover, among farms tested seasonally, C. jejuni was significantly greater than C. coli in winter. These data provide the first results of a continuing survey on Campylobacter loads and species identification from Italian broiler carcasses and represents an important baseline to estimate the human exposure to Campylobacter in Italy.

Isolation of DNA for PCR assays from noncultivable Campylobacter jejuni isolates.

Isolates of Campylobacter jejuni shipped internationally often arrive in a noncultivable state. We describe a PCR-based methodology whereby phylogenetic information can be recovered from noncultivable C. jejuni stored in Wang's transport medium. The robustness of this methodology was initially tested using 5 previously characterized strains of C. jejuni isolated from various sources associated with poultry production. These isolates were stored in Wang's transport medium before being subjected to 1 of 5 treatments designed to render the stored cells noncultivable: prolonged storage at room temperature, prolonged incubation at 42 degrees C, multiple rounds of freezing and thawing, boiling, or contamination with Pseudomonas aeruginosa (ATCC 27853). This method resulted in DNA appropriate for PCR. An approximately 400-nucleotide amplicon from the flaA gene and an approximately 800-nucleotide amplicon from 16S rDNA were readily obtained, and a 1.5-kb section of the flaA locus was amplified from about half of the samples. These results indicate that this method may be useful for isolate typing schemes based on PCR amplification of Campylobacter DNA, including flaA short variable region (flaA SVR) sequencing, multilocus sequence typing (MLST), and flaA PCR-RFLP. By using this method, isolates unrecoverable from transport medium can still be used to provide phylogenetic information for epidemiological studies.

Occurrence of Campylobacter spp. in cecal contents among commercial broilers in iceland.

Campylobacter jejuni remains the most frequently reported bacterial cause of human gastroenteritis in Nordic countries. The primary source of transmission to humans is suggested as mishandled raw poultry or consuming improperly prepared chicken. The focus of this report was to characterize the prevalence and cell numbers of the organism within the commercial Icelandic poultry industry. Commercial broiler flocks were sampled from May 2001 through 2003 in a total population study. At the slaughter plant, 40 randomly selected ceca were obtained from each flock, pooled into four samples containing 10 ceca each, and analyzed. Cell numbers and prevalence of Campylobacter spp. were estimated by direct plating of dilutions onto Campy-Cefex agar and incubating the plates at 42 degrees C under microaerobic atmosphere; colonies were confirmed as Campylobacter spp. by microscopy and latex agglutination to provide quantification of cell numbers per gm of cecal material. A total of 15.4% of the flocks carried the organism at at a maximum cell number of 8.1 x 10(7) cfu/g, having a mean raw count of colonized birds at 1.3 x 10(7) cfu/g (geometric mean of 1.5 x 10(6)). During the 3 years of sampling, the prevalence ranged from 17.6% to 17.3% to 12.7% for slaughter years 2001, 2002, and 2003, respectively. Isolation rates varied with numbers of catch lots (groups of birds taken for slaughter)/flock; with one catch lot/flock, the prevalence was 13.7%, with two 17.5%, and with three 33.3%. With increased flock size, isolation rates also increased; flocks of greater than 5,000 birds had a prevalence of 12.0% positive, 14.0% of flocks with 5,000-10,000 birds were positive, and 25.5% of flocks with more than 10,000 birds were positive for Campylobacter spp. Isolation rates varied with the processing lines: M was positive at 17.3%, B was positive at 10.1%, and G at 17.2%. Flocks were more frequently colonized in the warmer months, and younger birds were less frequently colonized than were older slaughtered birds. This study provides descriptive microbiology pertaining to Iceland broilers in a total population study.

Relationship of messenger RNA reverse transcriptase-polymerase chain reaction signal to Campylobacter spp. viability.

SUMMARY. Discriminating viable from dead cells is of importance in the development of bacterial detection methods. A positive reverse transcriptase-polymerase chain reaction (RT-PCR) amplification signal was tested as a potential predictor of chick colonization. Some researchers have suggested that the presence of messenger RNA (mRNA) may not correlate with cell viability. Chicken colonization by cells that have positive mRNA signal but that are noncultivable would provide a correlation in cell viability and persistence of mRNA. The role of a viable but noncultivable (VBNC) form of Campylobacter spp. for colonization of poultry could be verified by such an mRNA signal. The levels of four strains of Campylobacter spp., previously isolated from poultry feces, declined progressively over time, and loss of cultivability occurred after 6 to 7 wk incubation in phosphate-buffered saline (PBS) at 4 C. Cold-stored, noncultivable and heat-inactivated (60 C for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay, depending on the target transcripts and strains used, although all fresh cultures showed mRNA signals. For the most part, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2, and CH-3 persisted. RT-PCR amplification of transcripts originating from the tkt and cmp genes and a 256-base pair amplicon (from a previously described putative haem-copper oxidase) provided consistent signals, whereas transcripts from the flaA gene did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not cultivable by conventional culture-based methods, did not establish colonization in the intestine of chicks 7 days after challenge. These results lead us to question the correlation between mRNA durability with cell viability as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.

Relationship of Campylobacter isolated from poultry and from darkling beetles in New Zealand.

Campylobacter, a foodborne pathogen closely associated with poultry, is considered to be an important agent of human gastroenteritis in New Zealand. The pathways involved in the contamination of poultry flocks remain unclear; however, many vectors, such as insects, rodents, and wild birds, have been implicated. Infestation of poultry houses by insects, particularly darkling beetles (Alphitobius diaperinus), is difficult to control. Furthermore, darkling beetles are known vectors for a variety of pathogens that include Salmonella, infectious bursal disease virus, Aspergillus, Escherichia coli, and Marek's disease virus. In this investigation, the relationship between darkling beetles and Campylobacter contamination of poultry flocks was investigated. A New Zealand breeder flock and four of its progeny broiler flocks were included in the study. Samples of beetles and of intestinal excreta of the birds were cultured for the presence of Campylobacter spp. A subset of the recovered isolates was subsequently genotyped using flaA short variable region (SVR) DNA sequence analysis. A large number of Campylobacter subtypes were isolated, indicating that Campylobacter colonization of poultry is likely to arise from a number of different reservoirs. However, a set of genetically distinct isolates were found to be common to the broiler flocks and to the beetles. This research provides data that indicates that Alphitobius diaperinus may serve as a source of Campylobacter contamination of poultry. A more thorough understanding of the relationship between beetle infestation and the Campylobacter status of poultry flocks should enable progress in further development of biosecurity control measures.