Research Note: Role of darkling beetles (Alphitobius diaperinus) and litter in spreading and maintaining Salmonella Enteritidis and Campylobacter jejuni in chicken flocks.

Salmonella and Campylobacter are common foodborne pathogens in chickens, but their persistence mechanisms within flocks are not fully understood. In this study, 4 groups of SPF Leghorn chickens (n = 50) were orally inoculated with 108Salmonella Enteritidis and 108Campylobacter jejuni, housed in BSL-2 rooms inside containers with autoclaved bedding and beetles (n = 200). Phase I (wk 1-3): the infected chickens remained in the containers and were then euthanized while beetles and litter remained in the container (group A), beetles were removed and litter remained in the container (group B), beetles remained and litter was removed (group C), and beetles and litter were removed (group D). Phase II (wk 5-7): autoclaved bedding was added to containers in groups C and D, and new SPF chickens (n = 50) were introduced and kept. Phase III (wk 8-20): all chickens were euthanized, and the litter and/or beetles remained in the containers for 17 wk. The prevalence of Salmonella Enteritidis and Campylobacter was significantly higher when detected by PCR compared to culture. In phase II, when infected chickens were removed and new chickens were introduced, 1 fecal sample in group B and 3 litter samples in groups B and C were found positive for Salmonella Enteritidis, and Campylobacter was still detected in groups A, B, and C litter samples, but not in beetles. In phase III, when all chickens were removed, Salmonella Enteritidis was identified in beetle samples from group A and the litter samples of all tested groups A, B, and C, and C. jejuni was positive in litter samples from groups A and B but not in the beetle. Sixty-nine days after removing infected chickens, culturable Salmonella was still found in beetles. Salmonella and Campylobacter were detectable in litter up to 127 d after removing infected chickens. This study highlights the transmission of Salmonella and Campylobacter via beetles and litter to new flocks in successive rearing cycles. Intensive control programs should target insect exclusion and implement strict poultry litter management or litter changes between flocks.

16S rRNA gene-based assessment of common broiler chicken sampling methods: Evaluating intra-flock sample size, cecal pair similarity, and cloacal swab similarity to other alimentary tract locations.

16S rRNA gene sequencing for characterization of microbiomes has become more common in poultry research and can be used to both answer specific research questions and help inform experimental design choices. The objective of this study was to use 16S rRNA gene sequencing to examine common sampling practices in broiler chicken studies such as: the required number of birds selected from a flock to adequately capture microbiome diversity, the differences between cecal pairs within the same bird, and whether cloacal swabs are representative of other alimentary tract (AT) locations. To do this, nine market age broilers were euthanized and immediately sampled in ten AT locations: crop, gizzard, proventriculus, duodenum, jejunum, ileum, cecal samples from each pouch, colon, and cloacal swab. DNA was extracted and subjected to 16S rRNA gene amplification and sequencing. Each location within the broiler AT hosts distinct microbial communities. When each sampling location was considered, it was found that sampling after 2.8 birds (range 2-4) resulted in less than 10% new amplicon sequencing variants (ASV) being added while sampling after 7.6 birds (range 6-10) increases new observed ASVs by less than 1%. Additionally, when cecal pairs from the same bird were evaluated, it was found that cecal pair mates are an adequate replication if interested in the total cecal microbiome but may be less useful if a rare lineage is of interest. Furthermore, when compared to other AT locations, the cecal microbiome was enriched in Firmicutes and Bacteroides while several lineages, most notably Lactobacillus, were under-represented. Finally, when cloacal swabs were compared to other AT locations, community similarity exhibited a direct distance relationship, i.e., the more aborad samples were the more similar they were to the swab. These findings indicate that while cloacal swabs can approximate overall changes in microbiome composition, they are not adequate for inferring changes to specific taxa in other parts of the AT tract-even those that are highly abundant within the microbial community. These data provide new insights guiding appropriate sample size selection within flocks and add to the consensus data regarding cecal pair similarity and destructive versus non-destructive sampling methods.

Animal welfare assessment of on-farm euthanasia methods for individual, heavy turkeys.

On-farm euthanasia of poultry, including turkeys, may not be possible for most people as birds gain weight; thus alternative mechanical methods have been developed. Our objective was to compare mechanical cervical dislocation with the Koechner Euthanizing Device (KED), captive bolt euthanasia with the Turkey Euthanasia Device (TED), head-only CO2 euthanasia (CO2), and electric euthanasia as potential humane methods for euthanizing individual, heavy turkeys. We assessed their impact on loss of brain stem reflexes, acute distress (corticosterone, CORT), kill success, torn skin, and blood loss. Turkeys (n = 174) were euthanized on 3 sampling days, while birds were restrained using a mobile bird euthanasia apparatus. Brain stem reflexes recorded were the cessation and return of induced nictitating membrane reflex (loss of consciousness and brain stem dysfunction), mouth gaping reflex (brain stem dysfunction), and musculoskeletal movements (spinal cord dysfunction). Overall, KED resulted in more frequent (at 4 min: KED 7 of 14; electric 0 of 13; TED 0 of 11; CO2 2 of 14 birds on day 1) and longer durations of the induced nictitating reflex compared to the other methods (means of day 2 and 3: KED 233; electric 15; TED 15; CO2 15 s). The mouth gaping reflex endured the longest after KED euthanasia (means of day 2 and 3: KED 197; electric 15; TED 51; CO2 15 s). Musculoskeletal movements endured longest after KED euthanasia (means of day 2 and 3: KED 235; electric 15; TED 219; CO2 15 s). Returning reflexes were more frequent after KED and TED compared to CO2 and electric euthanasia, where it was absent. CO2, electric, and TED euthanasia showed comparable kill success (success: CO2 42 out of 43; electric 44 of 45; TED 42 of 44), with KED resulting in most unsuccessful kills (unsuccessful: 8 out of 42). CORT responses were inconsistent. Torn skin and blood loss occurred more frequently after KED and TED compared to CO2 and electric applications. Therefore, we conclude that, based on a comparison of these 4 methods, the most discernibly humane was electric euthanasia, which consistently resulted in quick loss of consciousness within 15 s, no returning reflexes, and no torn skin or blood loss.

Euthanasia: Manual versus Mechanical Cervical Dislocation for Broilers.

The aim was to assess the onset of brain stem death for two euthanasia methods-manual cervical dislocation (CD) versus the Koechner Euthanizing Device (KED). Over three days broilers of 36 (n = 60), 42 (n = 80), or 43 days old (n = 60) were euthanized. On days 2 and 3, a treatment was added in which the bird's head was extended at a ~90° angle after application of the KED (KED+). On those days, gap size was recorded between the skull and atlas vertebra by 1-cm increments. The onset of brain death was assessed by recording the nictitating membrane reflex, gasping reflex and musculoskeletal movements (sec). Additionally, skin damage and blood loss were recorded (y/n). On all days, CD resulted in quicker loss of reflexes and movements compared to KED or KED+. Reflexes returned in 0⁻15% of CD birds, 50⁻55% of KED birds, and 40⁻60% of KED+ birds, possibly regaining consciousness. Skin damage occurred in 0% of CD birds, 68⁻95% of KED birds, and 85⁻95% of KED+ birds. On day 2 (p = 0.065) and 3 (p = 0.008), KED birds had or tended to have a narrower skull-to-atlas gap compared to CD and KED+ birds. Based on our results, CD would be the recommended method for broilers.

Neutralization of Bactericidal Activity Related to Antimicrobial Carryover in Broiler Carcass Rinse Samples.

Studies were conducted to examine the ability of three chemicals to neutralize residual antibacterial activity of commercial antimicrobial chemicals used in poultry processing. Chemical antimicrobial interventions used in poultry processing may have potential for carryover into whole poultry carcass buffered peptone water (BPW) rinses collected for monitoring Salmonella contamination. Such carryover may lead to false-negative results due to continuing bactericidal action of the antimicrobial chemicals in the rinse. To simulate testing procedures used to detect Salmonella contamination, studies were conducted by separately adding test neutralizers (highly refined soy lecithin, sodium thiosulfate, or sodium bicarbonate) to BPW and using these solutions as carcass rinses. Control samples consisted of BPW containing no additional neutralizing agents. One of four antimicrobial solutions (cetylpyridinium chloride, peroxyacetic acid, acidified sodium chlorite, and a pH 1 hydrochloric:citric acid mix) was then added to the rinses. The four antimicrobial solutions were prepared at maximum allowable concentrations and diluted with modified BPW rinses to volumes simulating maximum carryover. These solutions were then inoculated with a mixed culture of five nalidixic acid-resistant Salmonella serovars at 106 CFU/mL. The inoculated rinse was stored at 4°C for 24 h, and Salmonella was enumerated by direct plating on brilliant green sulfa agar supplemented with nalidixic acid. Results indicate that incorporation of optimal concentrations of three neutralizing agents into BPW neutralized the demonstrated carryover effects of each of the four antimicrobial solutions tested, allowing recovery of viable Salmonella at 106 CFU/mL (P > 0.05), equivalent to recovery from carcass rinses with no antimicrobial carryover. Incorporation of these neutralizers in BPW for Salmonella monitoring may reduce false-negative results and aid regulatory agencies in accurate reporting of Salmonella contamination of poultry.

Effect of Simulated Sanitizer Carryover on Recovery of Salmonella from Broiler Carcass Rinsates.

Numerous antimicrobial chemicals are currently utilized as processing aids with the aim of reducing pathogenic bacteria on processed poultry carcasses. Carryover of active sanitizer to a carcass rinse solution intended for recovery of viable pathogenic bacteria by regulatory agencies may cause false-negative results. This study was conducted to document the potential carryover effect of five sanitizing chemicals commonly used as poultry processing aids for broilers in a postchill dip. The effect of postdip drip time on the volume of sanitizer solution carryover was first determined by regression of data obtained from 10 carcasses. The five sanitizer solutions were diluted with buffered peptone water at 0-, 1-, and 5-min drip time equivalent volumes as determined by the regression analysis. These solutions were then spiked to 10(5) CFU/ml with a mixture of five nalidixic acid-resistant Salmonella enterica serovars, stored at 4°C for 24 h, and finally enumerated by plate count on brilliant green sulfa agar containing nalidixic acid. At the 0- and 1-min drip time equivalents, no Salmonella recovery was observed in three of the five sanitizers studied. At the 5-min drip time equivalent, one of these sanitizers still exhibited significant (P ≤ 0.05) bactericidal activity. These findings potentially indicate that the currently utilized protocol for the recovery of Salmonella bacteria from postchill sanitizer interventions may lead to false-negative results due to sanitizer carryover into the carcass rinsate.

Successional changes in the chicken cecal microbiome during 42 days of growth are independent of organic acid feed additives.

BACKGROUND: Poultry remains a major source of foodborne bacterial infections. A variety of additives with presumed anti-microbial and/or growth-promoting effects are commonly added to poultry feed during commercial grow-out, yet the effects of these additives on the gastrointestinal microbial community (the GI microbiome) as the bird matures remain largely unknown. Here we compared temporal changes in the cecal microbiome to the effects of formic acid, propionic acid, and medium-chain fatty acids (MCFA) added to feed and/or drinking water. RESULTS: Cecal bacterial communities at day of hatch (n = 5 birds), 7d (n = 32), 21d (n = 27), and 42d (n = 36) post-hatch were surveyed using direct 454 sequencing of 16S rRNA gene amplicons from each bird in combination with cultivation-based recovery of a Salmonella Typhimurium marker strain and quantitative-PCR targeting Clostridium perfringens. Treatment effects on specific pathogens were generally non-significant. S. Typhimurium introduced by oral gavage at day of hatch was recovered by cultivation from nearly all birds sampled across treatments at 7d and 21d, but by 42d, S. Typhimurium was only recovered from ca. 25% of birds, regardless of treatment. Sequencing data also revealed non-significant treatment effects on genera containing known pathogens and on the cecal microbiome as a whole. In contrast, temporal changes in the cecal microbiome were dramatic, highly significant, and consistent across treatments. At 7d, the cecal community was dominated by three genera (Flavonifractor, Pseudoflavonifractor, and a Lachnospiracea sequence type) that accounted for more than half of sequences. By 21d post-hatch, a single genus (Faecalibacterium) accounted for 23-55% of sequences, and the number of Clostridium 16S rRNA gene copies detected by quantitative-PCR reached a maximum. CONCLUSIONS: Over the 42 d experiment, the cecal bacterial community changed significantly as measured by a variety of ecological metrics and increases in the complexity of co-occurrence networks. Management of poultry to improve animal health, nutrition, or food safety may need to consider the interactive effects of any treatments with the dramatic temporal shifts in the taxonomic composition of the cecal microbiome as described here.

Effects of broiler carcass scalding and chilling methods on quality of early-deboned breast fillets.

The impact of scalding and chilling methods on quality of broiler breast fillets (pectoralis major) was evaluated. In 4 replications, 6- to 7-wk-old male and female broilers were slaughtered and scalded either at 60°C for 1.5 min (hard scalding) or 52.8°C for 3 min (soft scalding). Following evisceration, the carcasses were either air-chilled (0.5°C, 120 min) or immersion-chilled in water and ice (79 L/carcass, 0.5°C, 40 min, air agitated). Breast fillets were removed from the carcass within 4 h postmortem. Quality attributes including fillet color (both dorsal-bone and ventral-skin sides), pH, total moisture content, water-holding capacity (drip loss and cook loss), and Warner-Bratzler shear force were determined. Significant interactions between replication and scalding were found for pH, ventral side redness (a*) value, and cook loss and between replication and chilling for pH and ventral side a* and yellowness (b*) values. There were no interactions (P > 0.05) between chilling and scalding methods for any of the measurements. Immersion chilling resulted in higher (P < 0.05) ventral side lightness (L*) values, dorsal side b* values, drip loss, cook loss, and shear force compared with air chilling. No significant differences (P > 0.05) between the 2 scalding methods were observed for any of the quality attributes. These results indicate that broiler carcass chilling method has a much greater impact on quality of breast meat than scalding method and that the influence of chilling on breast meat quality is independent of scalding treatment.

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters.

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)-polymerase chain reaction (PCR). In 9 of 12 roosters, Campylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.

Genotype analyses of Campylobacter isolated from distinct segments of the reproductive tracts of broiler breeder hens.

Campylobacter isolated from feces and from the oviduct of six broiler breeder hens were genotyped by using flaA SVR DNA sequence analyses. A diversity of genotypes was observed among fecal and oviduct isolates. Comparison of isolates from the oviducts of individual hens revealed variable results. In three cases (hen 2, hen 3, and hen 6), analyses indicated that isolates from all regions of the individual hen's reproductive tract were closely related; isolates from hen 1 and hen 4 were diverse. Comparison of the Campylobacter isolates between hens revealed that in two cases, hens 1 and 3 and hens 4 and 6, certain isolates possessed identical flaA SVR sequence types. Comparisons of Campylobacter isolates recovered from a distinct region of the oviduct were found to have increased diversity as sampling progressed down the oviduct. This study further demonstrates that Campylobacter is present within the reproductive tract of breeder hens and that this presence may enable vertical transmission of Campylobacter from the breeder hen to the broiler offspring.