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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Enumeration of Campylobacter spp. in broiler feces and in corresponding processed carcasses.

Twenty north Georgia commercial flocks of broiler chickens sampled in 1995 and 11 flocks sampled in 2001 were tested for Campylobacter spp. Direct plating on Campy-Cefex agar was carried out to determine levels of Campylobacter colonization within each flock through the enumeration of the organism in 50 fresh fecal samples 1 day prior to slaughter. The next morning, these flocks were the first to be processed, and levels of the organism per carcass before the chilling operation (50 carcasses per flock) in 2001 and after the chilling operation (50 carcasses per flock) in both 1995 and 2001 were estimated. Levels of the organism on freshly processed broiler carcasses were estimated by the same methods in 1995 and 2001, and a significant reduction from an average of 10(4.11) CFU per carcass in 1995 to an average of 10(3.05) CFU per carcass in 2001 was observed. Levels of Campylobacter spp. found in production and in processing were not strongly correlative, indicating the existence of complex parameters involving production factors and variables associated with flock transport and the processing of the broilers. The reduction in Campylobacter levels on processed carcasses may have contributed to the reduction in the frequency of human disease observed by the Centers for Disease Control during the same period. These data characterize the distribution of Campylobacter in north Georgia poultry operations and should assist in the development of risk assessment models for Campylobacter spp. The results obtained in this study suggest that the implementation of antimicrobial interventions by the poultry industry has already reduced consumer exposure to the organism.

Survival of Campylobacter jejuni in biofilms isolated from chicken houses.

Campylobacter jejuni is a thermophilic and microaerophilic enteric pathogen associated with poultry. Biofilms may be a source of C. jejuni in poultry house water systems since they can protect constituent microorganisms from environmental stress. In this study, the viability of C. jejuni in biofilms of gram-positive chicken house isolates (P1, Y1, and W1) and a Pseudomonas sp. was determined using a cultural method (modified brucella agar) and direct viable count (DVC). Two-day biofilms grown on polyvinyl chloride (PVC) coupons in R2A broth at 12 and 23 degrees C were incubated with C. jejuni for a 6-h attachment period. Media were then refreshed every 24 h for 7 days to allow biofilm growth. Two-day biofilms of P1, Y1, and Pseudomonas spp. enhanced attachment (P < 0.01) of C. jejuni (4.74, 4.62, and 4.78 log cells/cm2, respectively) compared to W1 and controls without preexisting biofilm (4.31 and 4.22 log cells/cm2, respectively). On day 7, isolates P1 and Y1 and Pseudomonas biofilms covered 5.4, 7.0, and 21.5% of the surface, respectively, compared to 4.9% by W1. Viable C. jejuni on the surface decreased (P < 0.05) with time, with the greatest reduction occurring on surfaces without a preexisting biofilm. The number of viable C. jejuni determined by DVC was greater than that determined by the cultural method, indicating that C. jejuni may form a viable but nonculturable state within the biofilm. Both DVC and the cultural method indicate that biofilms enhance (P < 0.01) the survival of C. jejuni during incubation at 12 and 23 degrees C over a 7-day period.

Commercial field trial evaluation of mucosal starter culture to reduce Salmonella incidence in processed broiler carcasses.

A series of four paired-house studies was conducted in Arkansas, Alabama, and Georgia (two farms) to determine the efficacy of Mucosal Starter Culture (MSC) in eliminating or reducing salmonellae in broiler chickens. Randomly designated chicks were treated twice with MSC. First they were sprayed with an MSC solution using a spray vaccination cabinet in the hatchery, and then they received MSC in the first drinking water at the growing house. Chicks were grown in identically constructed and equipped paired houses managed by the same grower. At the end of grow-out, broilers were tested for the presence of salmonellae on the farm and during processing. In three trials where no hatchery salmonellae were found, less salmonellae were found on MSC-treated chickens compared to untreated chickens. On the farm at the end of grow-out, salmonellae were detected in 54 of 150 untreated control chickens compared to 40 of 180 MSC-treated chickens. In the processing plant, significantly (P < or = 0.05) more salmonellae were detected on prechill untreated control carcasses (23 of 180) compared to MSC-treated carcasses (12 of 180) and on untreated postchill processed carcasses (9 of 180) compared to MSC-treated carcasses (0 of 180). In one trial where appreciable (28% of egg shell samples) salmonellae was found before treatment with the MSC, more salmonellae were found in the treated birds than in the control birds both on the farm and after processing. These data confirm that when salmonellae levels were controlled in the hatchery, a significant reduction in the salmonellae was found on processed broiler carcasses treated with MSC and that this reduction in salmonellae was carried through processing to the final processed carcass, thus potentially reducing consumer exposure to salmonellae.

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.

Colonization of broiler chicks by Salmonella typhimurium definitive phage type 104.

The prevalence of an antibiotic-resistant strain of Salmonella Typhimurium definitive phage type 104 (DT104) has increased dramatically in recent years resulting in increased morbidity and mortality in both animals and humans. Colonization and shedding of Salmonella Typhimurium DT104 was studied in broiler chickens in two trials. In trial 1, 180 day-of-hatch chicks (n = 60 per group, n = 30 per replicate) were challenged with 10(6) CFU DT104 (wild-type isolate from poultry) or were commingled with a seeder chick challenged with 10(6) CFU DT104. In trial 2, 360 day-of-hatch chicks (n = 120 per treatment, n = 30 per rep) were divided into three groups. Chicks in the susceptible group were commingled with two seeder chicks that were orally challenged with 10(7) CFU/bird of a pan-sensitive strain of Salmonella Typhimurium DT104. Chicks in the resistant group were commingled with two seeder chicks that were orally challenged with 10(7) CFU/bird DT104 used in trial 1. For both trials, a control group was not exposed to DT104, composite fecal samples were evaluated twice weekly for levels of Salmonella shedding and 20 chicks per group were necropsied weekly and their cecal contents were cultured. At hatch all groups were colonized with naturally occurring Salmonella Senftenberg and Salmonella Mbandaka (trial 1) or Salmonella Senftenberg and Salmonella Ohio (trial 2) prior to exposure to DT104. Throughout the study, the level of Salmonella spp. shedding in feces (trial 1 means 3.1, 2.9, and 3.0 log10 CFU per g feces for challenged, seeder, and control groups, respectively) or ceca (trial 2 means 2.9. 2.9. and 2.5 log10 CFU per g ceca for resistant, susceptible, and control groups, respectively) did not differ among groups. In trial 1, colonization of DT104 remained constant at higher levels in the challenged group (mean 87%, P < 0.01), increased over time in the seeder group (10 to 50%, P < 0.02) and was not recovered from the control chicks. Salmonella Mbandaka colonization remained steady within each group with challenge and seeder groups maintaining higher levels of colonization than the control group. Salmonella Senftenberg colonization levels tended to decline (P = .058) over time in the challenged group (20 to 0%) and significantly decreased (P < 0.01) over time for both the seeder (80 to 0%) and control chicks (85 to 10%). In trial 2, the percentage of chicks colonized with susceptible DT104 declined (r = 0.90, P < 0.05) over the course of the trial from 45 to 0%, while recovery of the resistant DT104 persisted at a mean percentage of 27%. DT104 was not recovered from the control chicks. Salmonella Ohio colonization levels tended to decline (r = 0.79, P > 0.05) over time in the control group (75 to 20%) and significantly decreased (P < 0.05) over time in both susceptible and resistant groups (40 to 10%, r = 0.82 and 55 to 5%, r = 0.85, respectively). Salmonella Senftenberg was recovered from the control group at low frequency throughout the trial and was not recovered from the other groups. For either trial, no apparent affect on morbidity or mortality was observed. Introduction of DT104 by commingling may induce colonization resulting in persistent high levels of shedding in flocks simultaneously with other Salmonella species.

Difficulty in recovering inoculated Campylobacter jejuni from dry poultry-associated samples.

We inoculated 5 cm2 of clean chick pads, 5 g of clean pine shavings, and fresh unsanitized broiler breeder eggshell halves with a cell suspension of Campylobacter jejuni in physiological saline. Inoculation levels were 10(2), 10(3), or 10(4) cells per sample. The samples were allowed to remain at room temperature for 15, 30, or 60 min before addition of enrichment broth. When chick pad samples were inoculated with 102 cells, by 15 min 40% of the samples had detectable levels of Campylobacter, and by 30 to 60 min Campylobacter could be detected in only 20% of the samples. With samples of pine shavings, only 25% of those inoculated with 103 cells were positive for Campylobacter after 15 min and only 5% were positive for Campylobacter after 30 min. When 104 cells were inoculated onto litter, Campylobacter was recovered from 20% of the samples at 15 min and 15% of the samples after 30 min. Eggshells were also found to be a harsh environment. When the inoculum was 102 at 15 min, 8 of 10 samples were positive for Campylobacter but at 60 min only 10% of the samples remained positive for Campylobacter. The current cultural methods may not be adequate for recovering low numbers of Campylobacter from dry samples. Campylobacter may be present but culturally undetectable in the commercial hatchery and hatchery environment.

Distribution and characterization of Campylobacter spp. from Russian poultry.

The distribution of Campylobacter spp. on 13 poultry farms (broiler chicken, quail, pheasant, peacock, and turkey) from eight regions (Vladimir, Vologda, Voronezh, Kaluga, Liptsk, Moscow, Orenburg, and Orel) in Russia was surveyed. Intestinal materials were plated onto Campylobacter-selective medium and plates were incubated microaerobically at 42 degrees C for 24 or 48 h. Identification was based on colonial morphology, microscopic examination, and biochemical tests; latex agglutination assays were used for confirmation. In total, 116 isolates were derived from 370 samples. Isolation rates were similar, regardless of whether the birds were from small or large broiler production farms. Susceptibility of 48 representative (from these production sources) strains of Campylobacter spp. to 38 antimicrobial compounds was determined by disk diffusion assays. All strains tested were sensitive to amikacin, gentamycin, sisomycin, chloramphenicol, imipenem, oleandomycin, erythromycin, azitromycin, and ampicillin. The strains were also sensitive to 100 microg/disk of carbenicillin, fluoroquinolones, and to nitrofurans. Fluoroquinolone sensitivity was most notable and may be related to its limited application in poultry production within Russia. Hippurate and ribosomal RNA gene primers were developed and used to distinguish Campylobacter jejuni and Campylobacter coli and to provide a measure of strain discrimination. The combination of PCR analysis and randomly amplified polymorphic DNA (RAPD) typing were conducted for selected isolates. The various poultry species and the different locations yielded Campylobacter isolates with discrete randomly amplified polymorphic DNA patterns. The distribution and substantial diversity of Campylobacter spp. isolates appears similar to that previously reported in other countries.