R. Jeff Buhr

Hyperspectral Imaging for Detecting Fecal and Ingesta Contamination on Poultry Carcasses

A hyperspectral imaging system including camera with prism-grating-prism spectrograph, fiber optic line lighting, motorized lens control, and hyperspectral image processing software was developed for poultry safety inspection, particularly the identification of fecal and ingesta contamination on poultry carcasses. Both spectral and spatial image data between 400 and 900 nm with 512 spectral bands were acquired from fecal and ingesta contaminated poultry carcasses. Four dominant wavelengths (434, 517, 565, and 628 nm) were selected by principal component analysis from visible/near-infrared spectroscopy to apply for wavelength selection of hyperspectral images. A calibration model for the hyperspectral imaging system was developed from calibration lighting sources (HgAr, Kr, and Lasers) for accurate band selection from hyperspectral images to identify spatial and spectral characterization of fecal and ingesta contaminants. Hyperspectral image processing algorithms, specifically band ratio of dual-wavelength (565/517) images and histogram stretching, were effective on the identification of fecal and ingesta contamination of poultry carcasses. This algorithm can be further applied for real-time identification of fecal contamination on poultry carcasses in the processing line. This paper presents the research results that hyperspectral imaging can be used effectively for detecting feces (from duodenum, ceca, and colon) and ingesta on poultry carcasses and demonstrates potential application for on-line processing of poultry for safety inspection.

A hyperspectral imaging system for identification of faecal and ingesta contamination on poultry carcasses

A method and system for detecting faecal and ingesta contaminants on poultry carcasses were demonstrated. A visible/near infrared monochromator, which measured reflectance and principal component analysis were first used to identify key wavelengths from faecal and uncontaminated skin samples. Measurements at 434, 517, 565 and 628 nm were identified and used for evaluation with a hyperspectral imaging system. The hyperspectral imaging system, which was a line-scan (pushbroom) imaging system, consisted of a hyperspectral camera, fibre-optic line lights, a computer and frame grabber. The hyperspectral imaging camera consisted of a high-resolution charge coupled device (CCD) camera, a prism-grating-prism spectrograph, focusing lens, associated optical hardware and a motorised controller. The imaging system operated from about 400 to 900 nm. The hyperspectral imaging system was calibrated for wavelength, distance and percent reflectance and analysis of calibrated images at the key wavelengths indicated that single-wavelength images were inadequate for detecting contaminants. However, a ratio of images at two of the key wavelengths was able to identify faecal and ingesta contaminants. Specifically, the ratio of the 565-nm image divided by the 517-nm image produced good results. The ratio image was then further processed by masking the background and either enhancing the image contrast with a non-linear histogram stretch, or applying a faecal threshold. The results indicated that, for the limited sample population, more than 96% of the contaminants were detected. Thus, the hyperspectral imaging system was able to detect contaminants and showed feasibility, but was too slow for real-time on-line processing. Therefore, a multivariate system operating at 565 and 517 nm, which should be capable of operating at real-time on-line processing speed, should be used. Further research with such a system needs to be conducted.

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

BackgroundPoultry 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.ResultsCecal 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.ConclusionsOver 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.

Visible/NIR Spectroscopy for Characterizing Fecal Contamination of Chicken Carcasses

Zero tolerance of feces on the surfaces of meat and poultry carcasses during slaughter was established as a standard to minimize the likelihood of microbial pathogens. Microbial pathogens can be transmitted to humans by consumption of contaminated meat and poultry. Compliance with zero tolerance in meat processing establishments is currently verified by visual observation. The objective of this study was to investigate the use of visible, near-infrared reflectance spectroscopy as a method to discriminate between uncontaminated poultry breast skin and feces, and to select key wavelengths for use in a hyperpspectral system. Feces (n = 102), uncontaminated poultry breast skin, and skin contaminated with fecal spots were scanned from 400 to 2498 nm. The spectra were reduced by principal component (PC) analysis. The first four PCs explained 99.8% of the spectral variation. PC 1 was primarily responsible for the separation of uncontaminated skin from feces and for the separation of uncontaminated skin from contaminated skin. . A Classification model was able to classify fecal contaminated skin from the spectral data with a success rate of 95% Key wavelengths were identified by intensity of loading weights at 628 nm for PC 1, 565 nm for PC 2 and 434 and 517 nm for PC 4. Visual assessment of loading weights suggests that discrimination was dependent on the spectral variation related to fecal color and myoglobin and/or hemoglobin content of the uncontaminated breast skin.

Hyperspectral Imaging System for Identification of Fecal and Ingesta Contamination on Poultry Carcasses

A method and system for detecting fecal and ingesta contaminates were demonstrated. A visible / near infrared monochromator, which measured reflectance, and principal component analysis were first used to identify key wavelengths from fecal and uncontaminated skin samples. Measurements at 434, 517, 565, and 628 nm were identified and used for evaluation with a hyperspectral imaging system. The hyperspectral imaging system, which was a line-scan (pushbroom) imaging system, consisted of a hyperspectral camera, fiber-optic line lights, a computer, and frame grabber. The hyperspectral imaging camera consisted of a high resolution CCD camera, a prism-grating-prism spectrograph, focusing lens, associated optical hardware, and a motorized controller. The imaging system operated from about 400 to 900 nm. The hyperspectral imaging system was calibrated for wavelength, distance, and percent reflectance, and analysis of calibrated images at the key wavelengths indicated that single-wavelength images were inadequate for detecting contaminates. However, a ratio of images at two of the key wavelengths was able to identify fecal and ingesta contaminates. Specifically, the ratio of the 565-nm image divided by the 517-nm image produced good results. The ratio image was then further processed by masking the background and enhancing the image contrast with a nonlinear-histogram stretch. The results indicated that, for the limited sample population, 100% of contaminates were detected. Thus, the hyperspectral imaging system was able to detect contaminates and showed feasibility, but was too slow for real-time on-line processing. Therefore, a multivariate system operating at 565 and 517 nm, which should be capable of operating at real-time on-line processing speed, should be used. Further research with such a system needs to be conducted.

Genotype Analyses of Campylobacter Isolated from the Gastrointestinal Tracts and the Reproductive Tracts of Broiler Breeder Roosters

Abstract 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, Camplylobacter 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.

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.

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.

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.