Enrichment and Direct Plating for Detection of Campylobacter in Chicken Liver Rinse and Exudate.

ABSTRACT: Foodborne campylobacteriosis has been traced to undercooked chicken liver dishes; thus, it is important to use the best available culture methods when testing for the presence of Campylobacter. We compared two Campylobacter enrichment broths-Bolton formulation and Neogen formulation-in combination with three selective plating media-Campy-Cefex, Campy-Line and RF Campylobacter agars-for detection of Campylobacter from fresh retail chicken livers. In each of three experiments, nine replicate tubs of chicken livers were sampled by drawing exudate and a pooled rinse of five whole liver lobes. Results are reported as number positive and compared by Fisher's exact test. In experiment 1, no combination of enrichment and plating media significantly outperformed another for detection of Campylobacter (P > 0.05); all tubs were found to include Campylobacter in both exudate and liver rinse. In experiment 2, serial dilutions of samples were plated before and after enrichment. Exudate was found to be significantly more likely than rinse to support detection of Campylobacter by direct plating (P < 0.05); most exudate samples included at least 10 CFU Campylobacter per mL. Enrichment improved detection from rinse, but not exudate; all enrichment and plating combinations resulted ≥1,000 CFU/mL from most enriched samples. In experiment 3, samples were diluted before enrichment to determine effect of enrichment on ever lower numbers of Campylobacter. Enrichment did not improve recovery of Campylobacter from exudate or undiluted rinse (P > 0.05). However, when rinse samples were diluted to lower Campylobacter numbers, enrichment improved detection (P < 0.05). Overall, all media combinations tested were equivalent for detection of Campylobacter from chicken livers; sensitivity for detection seemed to be increased by using liver exudate compared with a pooled rinse of liver lobes.

Recovery of Campylobacter from feed using different enrichment media.

Studies were conducted to investigate the recovery of Campylobacter from feed. The impact of feed moisture, water activity, pH, number of background microflora and the use of different antibiotic supplements in Campylobacter enrichment broth (CEB) on Campylobacter recovery were evaluated in five studies. Broiler starter feed was inoculated with 104 -105 cfu of Campylobacter/g and stored at 24 °C and 43% RH. Enrichment culture was conducted on the day of inoculation or 24 h post inoculation and every 48 h of storage thereafter for 14 d. Feed moisture, water activity, pH and level of background microflora were not correlated with Campylobacter recovery. The incubation of feed in CEB with no antibiotic supplement resulted in the number of background microflora increasing to 109 cfu/g and the pH of the media decreasing to pH 4-5 impacting recovery. Addition of certain antimicrobial supplements to CEB reduced background microflora growth and maintained a near neutral pH. Campylobacter was recovered up to 10 days post inoculation when using CEB containing antibiotic supplements compared to 1 day in CEB. These findings suggest that Campylobacter can be recovered from feed and the type of antimicrobial supplement utilized influences recovery by controlling extraneous microbial growth which occurs during enrichment.

Effects of a dry hydrogen peroxide disinfection system used in an egg cooler on hatchability and chick quality.

A sanitation method that could continually clean and disinfect the air and surfaces in a hatchery could provide a second layer of microbial reduction on top of routine cleaning and disinfection. A gaseous dry hydrogen peroxide (DHP) system has been used in other facilities for this purpose and could have potential for use in chicken hatcheries. Because the DHP is a true gas and can permeate through the entire hatchery space, contact with eggs during storage and incubation could potentially interfere with normal hatching processes. Therefore, the aim of this study was to evaluate the effects of the DHP system on hatching parameters and chick quality. A total of 3,960 hatching eggs were collected from an ∼40-week-old Ross 308 broiler breeder flock and distributed in 2 treatments: treated and nontreated. For the treated group, the egg cooler was cleaned, and 1 DHP generator was placed inside. Two other DHP generators were placed in the common area outside as well. Both areas were treated for 7 D before placement of eggs, and then eggs were collected and placed inside the cooler over a 4-day period. Eggs were then stored for an additional 3 D after the last collection. Dry hydrogen peroxide levels were recorded each day during storage. For the nontreated group, all DHP machines were removed from the cooler and external room, and the egg cooler was cleaned. Eggs were collected in the same way for the control group as the treated group. After storage, eggs were placed into a single stage Natureform incubator. The eggs exposed to DHP showed higher (P < 0.05) hatchability of fertile eggs and lower (P < 0.05) early embryonic dead than eggs from the nontreated group. No other parameters evaluated were different between groups. Based on this work, the DHP treatment of fertile eggs had no detrimental effect on any performance parameter, with potential positive effects seen on hatch of fertile eggs and early embryonic dead embryos.

Antimicrobial interventions to reduce Salmonella and Campylobacter populations and improve shelf life of quail carcasses.

Quail (Coturnix japonica) is processed and marketed as fresh meat, with limited shelf life. The objective of this study was to evaluate the efficacy of antimicrobial interventions during slaughter on reducing Salmonella and Campylobacter contamination and to determine the microbiological shelf life of quail during refrigerated (4°C) storage. Three antimicrobials, peracetic acid (400 ppm; PAA), Citrilow (pH 1.2), and Cecure (cetylpyridinium chloride [CPC], 450 ppm), along with a water and no-treatment control were evaluated. Quail carcasses (n = 75) were inoculated with a cocktail of nalidixic acid-resistant Salmonella Typhimurium and gentamicin-resistant Campylobacter coli. After 30 min of attachment time, quail carcasses were submerged in each antimicrobial solution for 20 s with air agitation. Noninoculated quail carcasses (n = 25) were similarly treated, packaged, and stored under refrigeration (4°C). Aerobic plate counts (APC), psychrotroph counts (PC), Enterobacteriaceae counts (ENT), total coliform counts (TCC), and Escherichia coli counts on quail carcasses were determined on 1, 4, 7, and 10 d. Salmonella and Campylobacter populations were determined by plating on Petrifilm APC supplemented with 200-ppm nalidixic acid and Campy Cefex agar supplemented with 200-ppm gentamycin, respectively. No significant reductions in (P > 0.01 log cfu/mL) in APC, PC, ENT, TCC, and E. coli counts were observed on carcasses submerged in water. However, treatments with PAA, Citrilow, and CPC significantly reduced (P ≤ 0.05) Salmonella and Campylobacter coli contamination. Citrilow showed greater (P ≤ 0.05) reduction in Salmonella and Campylobacter population (1.90 and 3.82 log cfu/mL reduction, respectively) to PAA and CPC. Greater (P ≤ 0.05) reductions in APC, PC, ENT, TCC, and E. coli counts (2.22, 1.26, 1.47, 1.52, and 1.59 log cfu/mL, respectively) were obtained with the application of CPC. Application of antimicrobial interventions resulted in a reduction in Campylobacter and Salmonella, APC, PC, and ENT populations after treatments (day 0) and throughout the storage period (day 10). Use of antimicrobial interventions after slaughter can improve the microbiological safety and shelf life of quail.

Efficacy of peroxy acetic acid in reducing Salmonella and Campylobacter spp. populations on chicken breast fillets.

Poultry processors use antimicrobials to reduce the risk of pathogens on poultry and poultry products. The efficacy of selective and nonselective plating media to enumerate injured Salmonella (selective media-brilliant green sulfa agar and Petrifilm Enterobacteriaceae Plate Count; nonselective media-tryptic soy agar and Petrifilm Aerobic Plate Count) and Campylobacter (selective medium-Campy cefex agar and nonselective medium-Brucella agar) populations and the efficacy of peroxy acetic acid (PAA) to reduce Salmonella and Campylobacter populations on chicken breast fillets were evaluated. All plating media for Salmonella and Campylobacter contained nalidixic acid (200 ppm) or gentamycin (200 ppm), respectively. Breast fillets were sprayed or immersed in PAA (500 ppm) for 10 min for evaluation of the plating media. Breast fillets inoculated with a mixed Salmonella and Campylobacter cocktail were sprayed (5 or 10 s) or immersed (4-30 s) in PAA (100, 400, 500, or 1,000 ppm) for evaluation of PAA efficacy. Salmonella populations were higher (P ≤ 0.05) when plated on nonselective media compared with the selective media for the non-PAA treated fillets, although the differences in populations were low (<0.32 log CFU/mL). For both the microorganisms, populations on PAA treated (immersion or spray) fillets were similar when enumerated on nonselective or selective media within each treatment (PAA immersion or spray). Both immersion and spray applications reduced (P ≤ 0.05) the Salmonella and Campylobacter populations compared with the control. Increasing the PAA concentration to 250, 500, and 1,000 ppm resulted in greater reductions (P ≤ 0.05) in Salmonella and Campylobacter populations. Immersion of the inoculated breast fillets in 1,000 ppm PAA solution for 30 s resulted in Salmonella and Campylobacter population reductions of 1.92 and 1.87 log CFU/mL, respectively. Method of antimicrobial application (immersion and spray) did not affect the reductions in Salmonella and Campylobacter populations. Either immersion or spray application can be used to improve microbial safety of chicken breast fillets in a poultry processing plant.

Research Note: Evaluation of several inoculation procedures for colonization of day-old broiler chicks with Salmonella Heidelberg.

Before starting a study with many birds, it helps to know the method of chick inoculation. The objective was to compare 3 methods of Salmonella challenge (oral gavage [OR], intracloacal inoculation [IC], and seeder bird [SB]). Day-old broiler chicks (n = 100) were inoculated with 106 colony forming units (CFU) per chick of a marker strain of Salmonella Heidelberg (SH) with each route of inoculation. Chicks (n = 25) inoculated by each route were placed in floor pens on fresh pine shavings litter. For the seeder batch, 5 colonized chicks, each orally gavaged with 106 CFUs, were placed with 20 pen mates. Two weeks after inoculation, 10 birds from each pen and the 5 inoculated seeder birds were euthanized, the ceca were aseptically removed and macerated with a rubber mallet and weighed, and 3 times (w/v) buffered peptone was added and stomached for 60 s. Serial dilutions were made and plated onto Brilliant Green Sulfa plates containing 200 ppm nalidixic acid. Plates were incubated along with the stomached ceca for 24 h at 37°C. If no colonies appeared on the plates, an additional plate was streaked from the preenriched bag and incubated for 24 h at 37°C. In addition to all seeder birds being positive, the number of SH-positive birds out of 20 sampled in each group was 13, 17, and 7 for OR, IC, and SB, respectively. The level of SH per g of ceca and cecal contents was log (SE) 3.0 (0.7), 2.0 (0.4), and 2.6 (0.4) for OR, IC, and SB, respectively. After enrichment, the number of colonized birds out of 20 was 18, 20, and 10 for OR, IC, and SB, respectively. In conclusion, this study suggests that IC is the method to use to ensure most of the challenged birds are colonized. However, if you prefer to have a smaller percentage of the birds colonized with higher levels, then OR might be better.

Campylobacter subtypes detected in broiler ceca and livers collected at slaughter1.

Foodborne campylobacteriosis has been linked to undercooked chicken liver. We have detected Campylobacter in chicken livers available at retail. The objective of the current project was to determine the prevalence and subtype of Campylobacter associated with livers and ceca of the same broiler carcasses at commercial slaughter. Within 2 min of commercial evisceration, we collected liver and ceca of one broiler carcass from each of 70 discreet flocks over a 12-mo period. Liver surface, liver internal tissue, and cecal contents were cultured for Campylobacter using standard methods. One example of the predominant colony type was selected from each positive sample for whole genome sequencing and multilocus sequence typing. We detected Campylobacter in at least one sample from 58 of 70 (83%) carcasses/flocks; 41 ceca, 57 liver surface samples, and 19 liver internal tissue samples were positive. For 11 of 18 carcasses from which all samples were positive, the predominant colony types were indistinguishable. However, some carcasses did have multiple subtypes of Campylobacter. Of carcasses with Campylobacter on the surface of the liver and within the ceca, it was more likely that the subtypes be the same than different (P < 0.01). However, Campylobacter subtypes detected in internal liver tissue were not more likely to be the same as those detected in ceca (P > 0.05). We detected different subtypes of Campylobacter from internal liver tissue and liver surface of seven broiler carcasses/flocks. Livers from a large percentage of broiler carcasses/flocks can have one or more subtypes of Campylobacter.

Effect of probiotics on fecal excretion, colonization in internal organs and immune gene expression in the ileum of laying hens challenged with Salmonella Enteritidis.

A study was conducted to evaluate the supplementation of probiotics on Salmonella colonization in the ceca and various internal organs as well as immune response in laying hens challenged with Salmonella enterica serovar Enteritidis (SE). Thirty-two 46-wk-old White Leghorns (W-36) were housed individually in wired laying cages under 16L:8D lighting schedule. Hens were challenged individually with nalidixic acid resistant Salmonella Enteritidis (SENAR) after which time they were grouped into four treatments: T1 = SENAR unchallenged control, T2 = SENAR challenged control, T3 = SENAR challenged + 0.05% probiotics (Lactobacillus plantarum), and T4 = SENAR challenged + 0.1% probiotics. All hens, including T1, were euthanized and sampled for the liver with gall bladder (L/GB), ileum, ovary, spleen, and ceca on 7-days post-infection (dpi). Fecal screening was performed on individual hens at both 3 and 6 dpi. No difference was detected between the treatments in cecal SENAR enumeration, and the mean log 10 cfu/gm of SENAR in the ceca was 3.7 for all three treatments. The prevalence of SENAR was lowest for ovary in all treatments and was highest in the spleen. However, there were no significant differences among the treatments in the internal organs. There was no significant difference in the fecal shedding among the treatments on either 3 or 6 dpi, with incidence of positive feces higher at 3 dpi compared to 6 dpi (100 vs. 70% to 80%). SENAR challenge resulted in significant upregulation (P < 0.05) of interleukin (IL)-1ß, 6, 10, interferon gamma (IFN-γ), and toll-like receptor (TLR)-4 mRNA expression. Highest level of probiotics resulted in a significant decrease in IFN-γ and elevation of IL-6 and IL-10 gene expression in the ileum. However, IL-1B and TLR-4 gene expression were not different from the SENAR challenge control. This study reveals that there was important regulation of immune genes by probiotics supplementation.

Multilocus Sequence Subtypes of Campylobacter Detected on the Surface and from Internal Tissues of Retail Chicken Livers.

Foodborne campylobacteriosis has been traced to undercooked chicken liver. The objectives of this study were to determine the prevalence of Campylobacter associated with chicken livers at retail and to determine which subtypes are detected on the surface and in the internal tissues of the livers. Fifteen packages of fresh chicken livers, each representing a unique combination of processing plant and sell-by date, were collected at each of three retail grocery stores in Georgia. Three intact, undamaged livers per container ( n = 45) were selected and sampled using each three methods: outside swab, inside swab accessed by pressing through a heat-sterilized outer surface, and whole liver blended in enrichment broth. Each liver sample with 0.1 mL of exudate from packages was cultured for Campylobacter by plating on Campy-Cefex agar. The most prevalent Campylobacter colony type from each positive sample was subjected to whole genome sequencing and multilocus sequence typing. Campylobacter was detected in at least one sample from every package. Surface swabs were positive for 29 of 45 livers, but significantly fewer swabs of internal tissue were positive, 14 of 45 ( P < 0.01). Campylobacter was detected in 30 of 45 blended whole liver samples. Multiple subtypes were detected from eight livers. In four livers, a different subtype was dominant on the surface than was dominant internally. In one liver, three subtypes were detected. Various subtypes of Campylobacter can be readily isolated from fresh retail chicken livers; therefore, undercooked chicken livers pose a food safety risk.

Effect of dietary supplementation of nitrocompounds on Salmonella colonization and ileal immune gene expression in laying hens challenged with Salmonella Enteritidis.

Foodborne disease caused by Salmonella Enteritidis (SE) is one of the important public health and economic concerns. A study was conducted to determine the effect of supplementation with 2-nitroethanol (NE) and 2-nitropropanol (NP) on Salmonella recovery of internal organs as well as on the immune gene expression in the ileum of laying hens. Thirty-six White Leghorns were orally gavaged with nalidixic acid resistant Salmonella Enteritidis (SENR). Hens were housed individually in wire-laying cages and randomly assigned to six dietary treatments: T1 = SENR unchallenged (negative control), T2 = SENR challenged (positive control), T3 = SENR challenged + 100 ppm NE, T4 = SENR challenged + 200 ppm NE, T5 = SENR challenged + 100 ppm NP, and T6 = SENR challenged + 200 ppm NP. Hens were sampled at 7 days post inoculation (dpi). Ceca, liver with gall bladder (L/GB), and ovary samples were collected for bacteriology, and ileum samples were collected for analysis of immune gene expression. T3 and T6 significantly reduced (P < 0.05) cecal SENR count, whereas T4 and T5 were not different from T2, the SENR challenged control. There was no significant difference in SENR reduction in the L/GB or ovary after supplementation of either nitrocompounds. Pro- and anti-inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1B, IL-6, toll-like receptors (TLR)-4, and IL-10 all were significantly upregulated (P < 0.05) after SENR challenge. Supplementation at both levels of NE and NP showed a significant immune gene expression response in the ileum with reduction of IFN-γ, IL-6, TLR-4, and IL-10 mRNA expression. Overall, nitrocompounds such as NE and NP can be used in the intervention strategy to reduce Salmonella infection in hens.

Evaluation of encapsulated sodium butyrate on growth performance, energy digestibility, gut development, and Salmonella colonization in broilers.

Two experiments were conducted to determine the effect of an encapsulated sodium butyrate (Na-B) with targeted releasing times on broiler performance, energy digestibility, intestinal morphology, and ceca Salmonella colonization. In experiment 1, 3 different Na-B products (CMA, CMP, and CMS) were evaluated following a challenge with a nalidixic acid-resistant Salmonella typhimurium (STNAR). Cobb-Cobb male birds were placed 8 per pen into 6 replicates for each treatment. Treatments included 6 Na-B treatments (500 and 1,000 ppm of each product) plus 2 control (non-challenged and challenged). Birds were orally gavaged with 0.1 mL of 107 cfu/mL STNAR on d 4. Ceca and ileal samples were collected on d 11. In experiment 2, CMA and CMP products were evaluated for a full grow-out period without an external challenge. Cobb-Cobb male birds were distributed among 45 floor pens with 24 birds per pen. Treatments included 4 product treatments (500 and 1,000 ppm of each product) plus one control. Feed intake and pen weight were obtained on d 14, 28, and 42. Experiment 1 showed that CMP at 1,000 ppm had the highest value for BW and BWG on d 4 (P = 0.07). Adding CMA and CMP at 500 ppm increased ileal digestibility energy (IDE) compared to the challenged control (P ≤ 0.05). The Salmonella recovery data indicated that the challenge had a significant but mild impact, since it did not affect the performance variables but did result in a significant increase in log10 cfu/g cecal material between the non-challenged and challenged control (1.42 vs 3.72). Experiment 2 showed that both products improved the villus height in the duodenum on d 21 (P = 0.08) and IDE on d 42, relative to the control (P ≤ 0.05). This study demonstrates that Na-B has the potential to improve growth in broilers at an early age. The beneficial effects on intestinal morphology and IDE are affected not only by dosage level, but also by the product's releasing time.

Effect of dietary bacteriophage supplementation on internal organs, fecal excretion, and ileal immune response in laying hens challenged by Salmonella Enteritidis.

With the current researches on replacing antibiotics with different dietary interventions, bacteriophages (BP) are potential antimicrobial intervention because of their ability to affect specific bacteria. A study was conducted to evaluate the role of BP against Salmonella enterica serovar Enteritidis (SE) on SE internal organs colonization and ileum immune response in laying hens. Hens were challenged both orally and intracloacally with 108 cfu/mL cells of nalidixic acid resistant Salmonella Enteritidis (SENAR). Thirty-two Single Comb White Leghorns were randomly allocated to 4 dietary treatments: 1) unchallenged control (negative control; T1), 2) SENAR challenged control (positive control; T2), 3) SENAR challenged + 0.1% BP (T3), and 4) SENAR challenged + 0.2% BP (T4). The number of SENAR in the ceca was significantly reduced by 0.2% BP supplementation (P < 0.05) at 7 d post infection (dpi). The respective number of SENAR was reduced from 2.9 log cfu/gm in T2 and T3 to 2.0 log cfu/gm in T4. There was no significant effect of T3 on reduction of numbers of cecal SENAR. A significant reduction of SENAR was observed in the liver with gall bladder (LGB) from 0.75 in T2 to 0.18 log cfu/gm in T4. In the spleen, T4 significantly reduced (P < 0.05) SENAR to 0.56 log cfu/gm compared to T2 and T3 (0.94 log cfu/gm). There was no significant effect of T3 in reduction of prevalence of spleen SENAR. By supplementing 0.2% BP (T4), the SENAR in the ovary was reduced to 0 log cfu/gm. There was a significant reduction (P < 0.05) in fecal SENAR at 6 dpi by T4 (0.71 log cfu/gm) compared to the positive control (1.57 log cfu/gm). The expression of interferon (IFN)-Γ, interleukin (IL)-6, and IL-10 was significantly increased in the ileum by SENAR challenge compared to the negative control. This study suggests that apart from commonly used prebiotics or probiotics, BP are pathogen-specific and can be used as one of the dietary strategies to reduce SE colonization and induce immune modulation in laying hens.

Treatment with a low pH processing aid to reduce Campylobacter counts on broiler parts.

New regulations and performance standards for Campylobacter have been implemented by the USDA - Food Safety and Inspection Service (FSIS). The objective of this study was to evaluate treatment with a low pH processing aid (CMS1 PoultrypHresh), a formulated low pH processing aid, to reduce numbers of Campylobacter which could help companies meet regulatory requirements. Two experiments (3 replicates each) were conducted. Experiment 1, in each of 3 replicates, skin-on split chicken breasts (n = 15) were obtained from a local grocery and divided into groups of 5. The skin of each part was inoculated with approximately 107 cells of a gentamicin resistant C. coli (CCGR) marker strain in an area of approximately 6.5 cm2. CCGR cells were allowed to attach for 5 min prior to treatment. Ten inoculated breasts were individually placed into separate 6 L plastic storage boxes containing either 3.5 L deionized water or PoultrypHresh solution at a pH of 1.4. Parts were subjected to agitation (bubbled air) for 25 s. After treatment, each part was removed, allowed to drain for 5 s, and placed into a plastic bag prior to mechanical rinsing with 150 mL of buffered peptone water for 60 s. Five inoculated breasts served as controls, were untreated with a dip or agitation and sampled as above. Experiment 2 procedures were repeated using skin-on thighs under the same conditions. Rinsates were collected from each chicken part, serially diluted, and plated onto Campy Cefex agar with 200 ppm gentamicin (CCGen). All plates were incubated microaerobically (5% O2, 10% CO2, 85% N2) for 48 h at 42°C, colonies were counted and the cfu/mL was log transformed. The use of PoultrypHresh on split breast produced a 99.6% reduction compared to untreated controls, while thighs showed a 99.4% reduction. This study demonstrated an approximate 3 log reduction (P < 0.05) using a 25 s air agitation treatment in PoultrypHresh at pH 1.4 with no observable damage, which will help processors meet FSIS regulations.

Influence of commercial laying hen housing systems on the incidence and identification of Salmonella and Campylobacter.

The housing of laying hens is important for social, industrial, and regulatory aspects. Many studies have compared hen housing systems on the research farm, but few have fully examined commercial housing systems and management strategies. The current study compared hens housed in commercial cage-free aviary, conventional cage, and enriched colony cage systems. Environmental and eggshell pool samples were collected from selected cages/segments of the housing systems throughout the production cycle and monitored for Salmonella and Campylobacter prevalence. At 77 wk of age, 120 hens per housing system were examined for Salmonella and Campylobacter colonization in the: adrenal glands, spleen, ceca, follicles, and upper reproductive tract. All isolates detected from environmental swabs, eggshell pools, and tissues were identified for serotype. Two predominant Salmonella were detected in all samples:S.Braenderup andS.Kentucky.Campylobacter coli and C. jejuni were the only Campylobacter detected in the flocks. Across all housing systems, approximately 7% of hens were colonized with Salmonella, whereas >90% were colonized with Campylobacter Salmonella Braenderup was the isolate most frequently detected in environmental swabs (P<0.0001) and housing system impacted Salmonella spp. shedding (P<0.0001).Campylobacter jejuni was the isolate most frequently found in environmental swabs (P<0.01), while housing system impacted the prevalence of C. coli and jejuniin ceca (P<0.0001). The results of this study provide a greater understanding of the impact of hen housing systems on hen health and product safety. Additionally, producers and academia can utilize the findings to make informed decisions on hen housing and management strategies to enhance hen health and food safety.

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.

Prevalence and Serogroup Diversity of Salmonella for Broiler Neck Skin, Whole Carcass Rinse, and Whole Carcass Enrichment Sampling Methodologies following Air or Immersion Chilling.

The purpose of this study was to evaluate neck skin (NS), whole carcass rinse (WCR), and whole carcass enrichment (WCE) sampling procedures for Salmonella isolation and serogroup identification from the same broiler chicken carcass treated with air or immersion chilling. Commercially processed and eviscerated broiler carcasses were collected from a commercial processing plant, individually bagged, and transported to the pilot processing plant. In experiment 1, carcasses were air chilled to 4°C. In experiment 2, carcasses were immersion chilled with or without chlorine. After air chilling, Salmonella was detected on 78% of NS and 89% of WCE samples. Only one Salmonella serogroup was detected from each of 13 Salmonella-positive NS samples, and two serogroups were detected on 1 Salmonella-positive NS sample. Only one Salmonella serogroup was detected from each of 13 Salmonella-positive WCE samples, and two serogroups were detected from 3 Salmonella-positive WCE samples. After immersion chilling without chlorine, Salmonella was detected on 38% of NS, 45% of WCR, and 100% of WCE samples. Without chlorine, the 15 Salmonella-positive NS samples included 14 samples with one serogroup and 1 sample with two serogroups. Only one Salmonella serogroup was detected from WCR samples after immersion chilling. Of 40 Salmonella-positive WCE samples, 23 had a one, 14 had two, and 3 had three Salmonella serogroups. After immersion chilling with chlorine, Salmonella was detected on 35% of NS, 0% of WCR, and 90% of WCE samples. With chlorine, the 14 Salmonella-positive NS samples included 11 samples with one serogroup and 3 samples with two serogroups. No Salmonella serogroups were detected from WCR samples after immersion chilling with 20 mg/liter free chlorine. The 36 Salmonella-positive WCE samples included 21 samples with one serogroup and 15 samples with two serogroups. NS and WCE sampling methodologies yielded similar prevalence and serogroup diversity after air chilling. However, after immersion chilling with or without chlorine, WCE sampling yielded significantly higher (α ≤ 0.05) prevalence and serogroup diversity than either NS or WCR sampling methodologies.

Optimizing buffering chemistry to maintain near neutral pH of broiler feed during pre-enrichment for Salmonella.

Salmonella is a human pathogen that can accompany live broilers to the slaughter plant, contaminating fully processed carcasses. Feed is one potential source of Salmonella to growing broilers. Monitoring feed for the presence of Salmonella is part of good agricultural practice. The first step in culturing feed for Salmonella (which may be at low numbers and sub-lethally stressed) is to add it to a pre-enrichment broth which is incubated for 24 h. During the course of pre-enrichment, extraneous bacteria metabolize carbohydrates in some feed and excrete acidic byproducts, causing the pH to drop dramatically. An acidic pre-enrichment pH can injure or kill Salmonella resulting in a failure to detect, even if it is present and available to infect chickens. The objective of this study was to test an array of buffering chemistries to prevent formation of an injurious acidic environment during pre-enrichment of feed in peptone water. Five grams of feed were added to 45 mL of peptone water buffered with carbonate, Tris pH 8, and phosphate buffering ingredients individually and in combination. Feed was subjected to a pre-enrichment at 35°C for 24 h; pH was measured at 0, 18, and 24 h. Standard phosphate buffering ingredients at concentrations up to 4 times the normal formulation were unable to fully prevent acidic conditions. Likewise, carbonate and Tris pH 8 were not fully effective. The combination of phosphate, carbonate, and Tris pH 8 was the most effective buffer tested. It is recommended that a highly buffered pre-enrichment broth be used to examine feed for the presence of Salmonella.

Microbiological impact of three commercial laying hen housing systems.

Hen housing for commercial egg production continues to be a societal and regulatory concern. Controlled studies have examined various aspects of egg safety, but a comprehensive assessment of commercial hen housing systems in the US has not been conducted. The current study is part of a holistic, multidisciplinary comparison of the diverse aspects of commercial conventional cage, enriched colony cage, and cage-free aviary housing systems and focuses on environmental and egg microbiology. Environmental swabs and eggshell pools were collected from all housing systems during 4 production periods. Total aerobes and coliforms were enumerated, and the prevalence of Salmonella and Campylobacter spp. was determined. Environmental aerobic and coliform counts were highest for aviary drag swabs (7.5 and 4.0 log cfu/mL, respectively) and enriched colony cage scratch pad swabs (6.8 and 3.8 log cfu/mL, respectively). Aviary floor and system wire shell pools had the greatest levels of aerobic contamination for all eggshell pools (4.9 and 4.1 log cfu/mL, respectively). Hens from all housing systems were shedding Salmonella spp. (89-100% of manure belt scraper blade swabs). The dry belt litter removal processes for all housing systems appear to affect Campylobacter spp. detection (0-41% of manure belt scraper blade swabs) considering detection of Campylobacter spp. was much higher for other environmental samples. Aviary forage area drag swabs were 100% contaminated with Campylobacter spp., whereas enriched colony cage scratch pads had a 93% positive rate. There were no differences in pathogen detection in the shell pools from the 3 housing systems. Results indicate egg safety is enhanced when hens in alternative housing systems use nest boxes. Additionally, current outcomes indicate the use of scratch pads in hen housing systems needs to be more thoroughly investigated for effects on hen health and egg safety.

The effect of high-level chlorine carcass drench on the recovery of Salmonella and enumeration of bacteria from broiler carcasses.

A study was conducted to determine the bacteriological effect of exposing processed broiler carcasses to a high (10-fold increase) concentration chlorinated drench. During each of 6 replicate trials, eviscerated prechill carcasses were obtained from a commercial processing plant and chlorine-treated carcasses were subjected to a 1-min drench in 500 mL of a 500 mg/kg chlorine solution (sodium hypochlorite). Water-drenched carcasses were treated the same way except water was used in place of chlorinated water drench. Control carcasses were not drenched. All carcasses were then subjected to a whole carcass rinse (WCR) in 450 mL of buffered peptone water, from which 50 mL of the rinsate was removed for enumeration of total aerobic bacteria (APC), Escherichia coli, and total coliforms (TC). The entire carcass was then incubated 24 h at 37°C (whole carcass enrichment, WCE) for recovery of Salmonella. Levels of bacteria recovered from WCR were lower by 0.6 log10 cfu/mL for APC, 0.8 for E. coli, and 0.9 for TC when carcasses were drenched with water compared with undrenched control levels. Similarly, the levels of bacteria recovered from WCR were further lower by 1.0 log10 cfu/mL for APC, 0.5 for E. coli, and 0.5 for TC, when carcasses were drenched with 500 mg/kg of chlorine compared with water. However, there was no significant difference (P > 0.05) in prevalence of Salmonella among the treatments (29% positive for control, 26% positive for water, 38% positive for chlorinated). These results indicate that drenching eviscerated carcasses with water or chlorinated water at 500 mg/kg significantly, but minimally, reduces the numbers of APC, E. coli, and TC bacteria recovered compared with undrenched carcasses. However, neither drenching carcasses with water or high chlorine had an effect on the prevalence of Salmonella that remain with the carcass as determined by WCE. The results of this study confirms the importance of maintaining and replenishing free chlorine for optimal antimicrobial activity, because chlorine at 500 mg/kg was rapidly used within 1 min of exposure to the carcass to <10 mg/kg.

Recovery of Salmonella serovar Enteritidis from inoculated broiler hatching eggs using shell rinse and shell crush sampling methods.

This study compared the recovery of Salmonella from hatching eggs using 3 sampling methods (eggshell rinsing, eggshell crush following a previous rinse, and eggshell crush without previous rinse). Eggshells were drop-inoculated with approximately 10(1), 10(2), or 10(3) cfu/eggshell of Salmonella Enteritidis and allowed to dry at room temperature for 1 or 24 h. For the shell rinse groups, each inoculated egg was rinsed with buffered peptone water. These rinsed eggs were used for the shell crush with previous rinse groups, and each egg was aseptically cracked, the contents discarded, and the eggshell and membranes crushed with buffered peptone water. This same crush procedure was used for the shell crush without previous shell rinse eggs. The recovery of Salmonella 1 h after inoculation for shell rinse sampled eggs was 16% positive at 10(1), 49% at 10(2), and 93% at 10(3) cfu/eggshell challenge. For the shell crush with previous shell rinse, sampled egg recovery was 0% positive at 10(1), 3% at 10(2), and 17% at 10(3) cfu/eggshell. For the shell crush, sampled eggs had recovery of 23% positive at 10(1), 69% at 10(2), and 96% at 10(3) cfu/eggshell challenge. The recovery of Salmonella 24 h after inoculation for the shell rinse eggs was 3% positive at 10(1), 12% at 10(2), and 22% at 10(3) cfu/eggshell challenge; recovery for shell crush with previous shell rinse sampling was 2% positive at 10(1), 8% at 10(2), and 5% at 10(3) cfu/eggshell challenge; and for the shell crush sampling recovery was 2% at 10(1), 32% at 10(2), and 42% at 10(3) cfu/eggshell challenge. Eggshell crush was a more sensitive (∼10 percentage points) sampling method than eggshell rinse at both 1 and 24 h, but both methods were equally optimal when the inoculum was at 10(3) and samples were collected after 1 h. Waiting 24 h after inoculation to sample significantly lowered the recovery for both the shell rinse and shell crush sampling methods by ∼40 percentage points.