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.

Recovery of thermophilic Campylobacter by three sampling methods from river sites in Northeast Georgia, USA, and their antimicrobial resistance genes.

Sixteen sites in the watershed of the South Fork of the Broad River (SFBR) in Northeastern Georgia, USA, were sampled in two seasons to detect Campylobacter. Sites were classified as mostly influenced by forest, pasture, wastewater pollution control plants (WPC) or mixed use. Sampling was repeated in the late spring and late fall for 2 years for a total of 126 samples. Free-catch water and sediment grab samples were taken at each site; Moore's swabs were placed for up to 3 days at most sites. A total of 56 isolates of thermophilic Campylobacter were recovered. Thirteen samplings were positive by two or three methods, and 26 samplings were positive by only one method; once by Moore's swab only and 25 times by free-catch water only. Campylobacter was detected at 58% of cattle pasture sites, 30% of forested sites and 81% of WPC sites. Twenty-one of the isolates carried antimicrobial resistance genes, mostly blaOXA-61. Free-catch water samples were more efficient than Moore's swabs or sediment samples for recovery of Campylobacter, which was more likely to be detected in streams near cattle pastures and human communities than in forested land. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of environmental water in transmitting Campylobacter was investigated, and methods for recovery of the organism were compared. The sequence types of recovered Campylobacter correlated with adjacent land use without regard to the method used to isolate the organisms. Sequence types and antimicrobial resistance genes associated with cattle were most prevalent near pastures. Even though types were recurrent at a given site, types appeared to be lost or replaced as the water flowed downstream.

Synbiotic supplementation to decrease Salmonella colonization in the intestine and carcass contamination in broiler birds.

In vitro and in vivo experiments were conducted to study the effects of synbiotic supplementation on Salmonella enterica ser. Enteritidis (SE) proliferation, cecal content load, and broiler carcass contamination. Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici culture supernatants decreased (P < 0.05) the in vitro proliferation of SE at 1:1 supernatant: pathogen dilution. A total of 240 Cobb-500 broiler chicks were randomly allotted to three treatment groups (8 replicates/group with 10 birds/replicate): control (basal diet), antibiotic (Virginiamycin at 20 mg/kg feed), synbiotic (PoultryStar® ME at 0.5 g/kg feed containing L. reuteri, E. faecium, B. animalis, P. acidilactici and a Fructooligosaccharide) from day of hatch. At 21 d of age, all birds in experimental groups were orally inoculated with 250 µl of 1 X 109 CFU SE. Antibiotic supplementation increased (P < 0.05) body weight and feed consumption, compared to the control group. Birds in the synbiotic supplementation had intermediate body weight and feed consumption that were not significantly different from both the control and antibiotic group at 42 d of age in SE infected birds. No significant effects were observed in feed efficiency at 42 d of age among the groups. Antibiotic and synbiotic supplementation decreased (P < 0.05) SE load in cecal contents by 0.90 and 0.85 log units/ g and carcass SE load by 1.4 and 1.5 log units/mL of rinsate compared to the control group at 42 d of age (21 dpi). The relative abundance of IL-10, IL-1, TLR-4, and IFNγ mRNA was decreased (P < 0.05) in the antibiotic and synbiotic supplementation groups compared to the control birds at 42 d of age (21 dpi). It can be concluded that synbiotic supplementation decreased SE proliferation in vitro and decreased SE load in the cecal contents and broiler carcass.

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.

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.

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.

Sampling naturally contaminated broiler carcasses for Salmonella by three different methods.

Postchill neck skin maceration (NSM) and whole-carcass rinsing (WCR) are frequently used methods to detect salmonellae from processed broilers. These are practical, nondestructive methods, but they are insensitive and may result in false negatives (20 to 40%). Neck skin samples comprise only 4% of the skin from the broiler carcass by weight, while WCR will not detect firmly attached Salmonella organisms and only 7.5% of the rinsate is utilized. Whole-carcass enrichment (WCE) involves incubation of the whole carcass overnight in a preenrichment broth and can recover as few as 8 inoculated Salmonella cells per carcass. The objective of this study was to use NSM, WCR, and WCE sampling to detect naturally occurring Salmonella from the same commercially processed broiler either prechill or postchill. Ten carcasses were obtained prechill and another 10 postchill on each of two replicate days from each of two commercial processing plants. From each carcass, 8.3 g of neck skin was sampled, and then the carcass was rinsed with 400 ml of 1% buffered peptone water. Thirty milliliters was removed and incubated (WCR), and the remaining 370 ml of broth and the carcass were incubated at 37°C for 24 h (WCE). Overall, Salmonella organisms were detected on 21, 24, and 32 of 40 prechill carcasses by NSM, WCR, and WCE, respectively, while 2, 2, and 19 of 40 postchill carcasses were positive by the respective methods. Prechill carcasses were 64% (77 of 120) positive for Salmonella, while postchill carcasses were 19% (23 of 120) positive. Commercial processing reduced the positive-sample prevalence by 45%. Salmonella organisms were detected on 20% (24 of 120) of the samples from plant 1 and 63% (76 of 120) of the carcasses from plant 2. This study demonstrates significant differences in the results for Salmonella prevalence among sampling methods both before and after immersion chilling, as well as between processing plants on days that samples were taken.

Apparent attachment of Campylobacter and Salmonella to broiler breeder rooster spermatozoa.

It has been demonstrated that horizontal and vertical transmission of Salmonella and Campylobacter can occur in broiler breeder flocks. The mechanism of this transmission is still unclear. Previously negative broiler breeder flocks have been reported to become positive with Salmonella, Campylobacter, or both after the introduction of "spike" roosters at 45 wk of age. To determine whether the rooster semen is a possible source of transmission to hens for colonization, we evaluated the association of both Salmonella and Campylobacter spp. to segments (head, midpiece, and tail) of individual spermatozoa after artificial inoculation. Salmonella typhimurium, Salmonella heidelberg, and Salmonella montevideo, or Campylobacter jejuni (in 0.85% saline) was added to a freshly collected (by abdominal massage) aliquot of pooled semen from roosters housed in individual cages. The semen and bacteria solutions were incubated 1 h at room temperature. Samples were fixed using Karnosvsky and Zamboni fixatives for 24 h prior to centrifuging and rinsing in 0.1 M cacodylate x HCl buffer. Individual aliquot samples were then subjected to both scanning (JSM-5800) and transmission (JEM-1210) electron microscopy. The scanning electron microscopy showed that Salmonella was associated with all 3 segments (head, midpiece, and tail) of the spermatozoa and apparently equally distributed. Campylobacter was mainly associated with the midpiece and tail segments; few isolates were located on the head segment. The transmission electron microscopy showed apparent attachment of Salmonella and Campylobacter to the spermatozoa.

Movement and persistence of Salmonella in broiler chickens following oral or intracloacal inoculation.

The dissemination of Salmonella into various lymphoid-like organs in young broiler chicks after oral and intracloacal inoculation was studied. A three-strain cocktail of Salmonella Typhimurium, Salmonella Montevideo, and Salmonella Enteritidis was administered either orally or intracloacally to day-old chicks. After 1 h, 1 day, or 1 week, the ceca, thymus, liver and gallbladder, spleen, and bursa were sampled for the presence of Salmonella. There was a marked difference in the recovery of Salmonella 1 h postinoculation. Only 6 of 50 samples from orally inoculated chicks were positive compared with 33 of 50 samples from cloacally inoculated samples. In comparison, 24 h and 1 week after inoculation, there was no difference in the number of positive samples between oral or cloacal inoculation. The rapidity of the translocation of the Salmonella from the cloacal inoculum compared to the oral inoculum is likely due to the transient time required for Salmonella to move through the alimentary tract. The method of inoculation did not affect the distribution of serogroups. Of the three serotypes in the composite inoculum, the Salmonella Enteritidis (group D) was recovered only twice in replication 1 and not at all in replication 2. Both the Salmonella Typhimurium (serogroup B) and the Salmonella Montevideo (serogroup C1) were recovered extensively throughout the study.

Salmonella prevalence in free-range and certified organic chickens.

Many consumers assume that broiler chickens grownunder traditional commercial conditions will have more Salmonella than free-range or organic chickens, which usually are less crowded, have access to outside spaces during grow out, and are fed special diets. Despite these perceptions, there is a lack of published information about the microbiological status of free-range and organic chickens. A total of 135 processed free-range chickens from four different commercial free-range chicken producers were sampled in 14 different lots for the presence of Salmonella. Overall, 9 (64%) of 14 lots and 42 (31%) of 135 of the carcasses were positive for Salmonella. No Salmonella were detected in 5 of the 14 lots, and in one lot 100% of the chickens were positive for Salmonella. An additional 53 all-natural (no meat or poultry meal or antibiotics in the feed) processed chickens from eight lots were tested; 25% ofthe individual chickens from 37% of these lots tested positive for Salmonella. Three lots of chickens from a single organicfree-range producer were tested, and all three of the lots and 60% of the individual chickens were positive for Salmonella.The U.S. Department of Agriculture Food Safety and Inspection Service reported that commercial chickens processed from 2000 to 2003 had a Salmonella prevalence rate of 9.1 to 12.8%. Consumers should not assume that free-range or organicconditions will have anything to do with the Salmonella status of the chicken.

Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-life broiler breeder hens.

Campylobacter and Salmonella are known to cause acute bacterial gastroenteritis in humans. Raw poultry products have been implicated as a significant source of these infections. Five trials were conducted to determine whether Campylobacter and Salmonella spp. exist naturally in the mature and immature ovarian follicles of late-life broiler breeder hens. Broiler breeder hens ranging from 60 to 66 wk of age were obtained from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. Overall, Campylobacter was found in 7 of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca. Campylobacter was found in at least one of each sample of mature follicles and in ceca in each of the five trials. Salmonella was found in 0 of 55 immature follicles, 1 of 47 mature follicles, and 8 of 55 ceca. In this study, the recovery rate of Salmonella from late-life broiler breeder hen ovarian follicles was relatively low. However, the recovery rate of Campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs. Determining how Campylobacter contaminated these ovarian follicles and how many chicks could be colonized from this source are the next steps in helping to elucidate a better understanding of this ecology and the control of Campylobacter in poultry production.

Presence of Campylobacter jejuni in various organs one hour, one day, and one week following oral or intracloacal inoculations of broiler chicks.

Day-old broiler chicks (n=30) were obtained from a commercial hatchery and inoculated, either orally or intracloacally, with a characterized strain of Campylobacter jejuni. At 1 hr, 1 day, and 1 wk after inoculation, broilers (n = 5) from the orally and intracloacally inoculated groups along with control birds (n=4) were humanely killed by cervical dislocation. The broilers from the control and treatment groups were aseptically opened, and the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca were aseptically removed and individually analyzed for C. jejuni. Overall, C. jejuni was isolated after oral inoculation from 13% (10/ 75), 17% (13/75), and 28% (14/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 10% (4/ 40), 8% (3/40), 10% (4/40), 25% (10/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively. Following the intracloacal route of inoculation, C. jejuni was recovered from 32% (24/75), 8% (6/75), and 16% (8/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 5% (2/40), 5% (2/40), 5% (2/40), 45% (18/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively, for all sampling periods. Campylobacter spp. were not recovered from sample sites examined from the control broilers from trial one, trial two, or trial three samples examined after 1 hr and 1 day. However, one control sample was positive from the 1-wk sampling from repetition three; therefore, those data were omitted. The rapid movement of Campylobacter to internal organs following both oral and intracloacal inoculation may be significant, particularly if it persists in these organs as reservoirs throughout the 65-wk life cycle of breeding birds.

Recovery of Campylobacter jejuni in feces and semen of caged broiler breeder roosters following three routes of inoculation.

We previously reported the recovery of Campylobacter (naturally colonized) from the ductus deferens of 5 of 101 broiler breeder roosters, and four of those five positive roosters had previously produced Campylobacter-positive semen samples. Those results prompted further evaluation to determine if inoculation route influenced the prevalence or level of Campylobacter contamination of semen, the digestive tract, or reproductive organs. Individually caged roosters, confirmed to be feces and semen negative for Campylobacter, were challenged with a marker strain of Campylobacter jejuni either orally using 1.0 ml of a diluted cell suspension (log(10)4.3 to 6.0 cells), by dropping 0.1 ml of suspension (log(10)5.3 to 7.0 cells) on the everted phallus immediately after semen collection or by dip coating an ultrasound probe in the diluted cell suspension (log(10)4.3 to 6.0 cells) and then inserting the probe through the vent into the colon. Six days postinoculation, individual feces and semen samples were again collected and cultured for Campylobacter. Seven days postinoculation, roosters were killed, the abdomen aseptically opened to expose the viscera, and one cecum, one testis, and both ductus deferens were collected. The samples were then suspended 1:3 (weight/volume) in Bolton enrichment broth for the culture of Campylobacter. Samples were also directly plated onto Cefex agar to enumerate Campylobacter. Campylobacter was recovered 6 days after challenge from feces in 82% of samples (log(10)4.1 colony-forming units [CFU]/g sample), 85% of semen samples (log(10)2.9 CFU/ml), and on the seventh day postchallenge from 88% of cecal samples (log(10)5.8 CFU/g sample). Campylobacter was not directly isolated from any testis sample but was detected following enrichment from 9% (3/33) of ductus deferens samples. Roosters challenged with Campylobacter orally, on the phallus, or by insertion of a Campylobacter dip-coated ultrasound probe were all readily colonized in the ceca and produced Campylobacter-positive semen and feces on day 6 after challenge. The low prevalence of recovery of Campylobacter from the ductus deferens samples and failure to recover from any testis sample suggests that semen may become Campylobacter positive while traversing the cloaca upon the everted phallus. The production of Campylobacter-positive semen could provide a route in addition to fecal-oral for the horizontal transmission of Campylobacter from the rooster to the reproductive tract of the hen.

Detection of Salmonella from chicken rinses and chicken hot dogs with the automated BAX PCR system.

The BAX system with automated PCR detection was compared with standard cultural procedures for the detection of naturally occurring and spiked Salmonella in 183 chicken carcass rinses and 90 chicken hot dogs. The automated assay procedure consists of overnight growth (16 to 18 h) of the sample in buffered peptone broth at 35 degrees C, transfer of the sample to lysis tubes, incubation and lysis of the cells, transfer of the sample to PCR tubes, and placement of tubes into the cycler-detector, which runs automatically. The automated PCR detection assay takes about 4 h after 16 to 24 h of overnight preenrichment. The culture procedure consists of preerichment, enrichment, plating, and serological confirmation and takes about 72 h. Three trials involving 10 to 31 samples were carried out for each product. Some samples were spiked with Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Montevideo, and Salmonella Enteritidis at 1 to 250 cells per ml of rinse or 1 to 250 cells per g of meat. For unspiked chicken rinses, Salmonella was detected in 2 of 61 samples with the automated system and in 1 of 61 samples with the culture method. Salmonella was recovered from 111 of 122 spiked samples with the automated PCR system and from 113 of 122 spiked samples with the culture method. For chicken hot dogs, Salmonella was detected in all 60 of the spiked samples with both the automated PCR and the culture procedures. For the 30 unspiked samples, Salmonella was recovered from 19 samples with the automated PCR system and from 10 samples with the culture method. The automated PCR system provided reliable Salmonella screening of chicken product samples within 24 h.

Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation.

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm. These results suggest that C. perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out. In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing. In the first trial, C. perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively. In the second trial, the incidence of C. perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and fromprocessed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively. The genetic relatedness of the isolated strains as determined by ribotyping suggests that C. perfringens may be transmitted between facilities within the integrated broiler chicken operation.

Serotyping and ribotyping of Salmonella using restriction enzyme PvuII.

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point-based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.