Salmonella Biofilm Formation under Fluidic Shear Stress on Different Surface Materials.

This study characterized biofilm formation of various Salmonella strains on common processing plant surface materials (stainless steel, concrete, rubber, polyethylene) under static and fluidic shear stress conditions. Surface-coupons were immersed in well-plates containing 1 mL of Salmonella (6 log CFU/mL) and incubated aerobically for 48 h at 37 °C in static or shear stress conditions. Biofilm density was determined using crystal violet assay, and biofilm cells were enumerated by plating on tryptic soy agar plates. Biofilms were visualized using scanning electron microscopy. Data were analyzed by SAS 9.4 at a significance level of 0.05. A surface-incubation condition interaction was observed for biofilm density (p < 0.001). On stainless steel, the OD600 was higher under shear stress than static incubation; whereas, on polyethylene, the OD600 was higher under static condition. Enumeration revealed surface-incubation condition (p = 0.024) and surface-strain (p < 0.001) interactions. Among all surface-incubation condition combinations, the biofilm cells were highest on polyethylene under fluidic shear stress (6.4 log/coupon; p < 0.001). Biofilms of S. Kentucky on polyethylene had the highest number of cells (7.80 log/coupon) compared to all other strain-surface combinations (p < 0.001). Electron microscopy revealed morphological and extracellular matrix differences between surfaces. Results indicate that Salmonella biofilm formation is influenced by serotype, surface, and fluidic shear stress.

Roles of Aerotolerance, Biofilm Formation, and Viable but Non-Culturable State in the Survival of Campylobacter jejuni in Poultry Processing Environments.

Campylobacter jejuni is one of the most common causes of foodborne human gastroenteritis in the developed world. This bacterium colonizes in the ceca of chickens, spreads throughout the poultry production chain, and contaminates poultry products. Despite numerous on farm intervention strategies and developments in post-harvest antimicrobial treatments, C. jejuni is frequently detected on broiler meat products. This indicates that C. jejuni is evolving over time to overcome the stresses/interventions that are present throughout poultry production and processing. The development of aerotolerance has been reported to be a major survival strategy used by C. jejuni in high oxygen environments. Recent studies have indicated that C. jejuni can enter a viable but non-culturable (VBNC) state or develop biofilm in response to environmental stressors such as refrigeration and freezing stress and aerobic stress. This review provides an overview of different stressors that C. jejuni are exposed to throughout the poultry production chain and the genotypic and phenotypic survival mechanisms, with special attention to aerotolerance, biofilm formation, and development of the VBNC state.

The Prevalence of Salmonella and Campylobacter on Broiler Meat at Different Stages of Commercial Poultry Processing.

In poultry processing, Salmonella and Campylobacter contaminations are major food safety concerns. Peracetic acid (PAA) is an antimicrobial commonly used in commercial poultry processing to reduce pathogen prevalence so as to meet the USDA-FSIS performance standards. The objective of this study was to determine the prevalence of Salmonella and Campylobacter on broiler meat in various steps of commercial poultry processing in plants that use PAA. Post-pick, pre-chill, post-chill, and drumstick chicken samples were collected from three processing plants and mechanically deboned meat (MDM) was collected from two of the three plants. Each plant was sampled thrice, and 10 samples were collected from each processing step during each visit. Among the 420 samples, 79 were contaminated with Salmonella and 155 were contaminated with Campylobacter. Salmonella and Campylobacter contamination on the post-pick samples averaged 32.2%. Significant reductions in Salmonella and Campylobacter were observed in pre-chill to post-chill samples, where the prevalence was reduced from 34% and 64.4% to nondetectable limits and 1.1%, respectively (p < 0.001). Salmonella and Campylobacter remained undetectable on the drumstick samples in all three processing plants. However, the prevalence of Salmonella and Campylobacter on MDM was similar to the post-pick prevalence, which suggests substantial cross-contamination from post-chill to MDM.

Spoilage Bacteria Counts on Broiler Meat at Different Stages of Commercial Poultry Processing Plants That Use Peracetic Acid.

In poultry processing, spoilage microbes are persistent microorganisms, which affect the quality of broiler meat. Peracetic acid (PAA) is the most common antimicrobial used by commercial processing plants, which can reduce the prevalence of these microbes. The goal of this study was to determine the concentrations of aerobic bacteria, coliforms, lactic acid bacteria, and Pseudomonas on broiler meat in processing plants that use peracetic acid in various concentrations as the primary antimicrobial. Samples were collected from three processing plants at five processing steps: post-pick (defeathering), pre-chill, post-chill, mechanically deboned meat (MDM), and drumsticks. Samples were rinsed in buffered peptone water for bacteria isolation. Over six log CFU/sample of aerobic plate counts (APC), lactic acid bacteria, and coliforms were detected on post-pick samples. All spoilage bacteria were reduced to nondetectable levels on post-chill samples (p < 0.001). However, the presence of all bacteria on mechanically deboned meat (MDM) samples indicated varying degrees of cross contamination from post-chill and MDM samples. These results suggest PAA effectively reduces spoilage microbes in chilling applications irrespective of differences in PAA concentrations. However, due to the levels of spoilage microbes detected in MDM, it may be worth investigating the potential interventions for this stage of processing.

Oxidative stress biomarkers and free amino acid concentrations in the blood plasma of moderately exercised horses indicate adaptive response to prolonged exercise training.

Oxidative stress caused by routine physical stressors may negatively impact the performance of equine athletes; thus, the present study identifies oxidative biomarkers in the blood plasma of exercising horses. Stock-type horses were subject to a standardized moderate-intensity exercise protocol 3 times per week for 8 wk. Exercise protocol followed NRC guidelines consisting of 30% walk, 55% trot, and 15% canter, with a target heart rate (HR) of 90 BPM. Blood plasma was collected in wk 1, 2, 7, and 8 immediately before and 0, 30, 60, and 90 min after exercise and analyzed for total antioxidant capacity (TAC), thiobarbituric acid reactive substance (TBARS), glutathione peroxidase activity (GPx), and superoxide dismutase activity (SOD). Data were analyzed as repeated measures with wk, d, time, and their interactions as fixed effects. The TAC on day 2 (0.40 mM Trolox) was 7.5% greater than on day 3 (P = 0.013). There were wk × d × time interactions for SOD, TBARS, and GPx (P < 0.001). The TBARS remained at pre-exercise baseline (d-1 wk-1; 2.7 µM malondialdehyde) for most collection times within weeks 1, 7, and 8 (P ≥ 0.058); however, TBARS increased by 0.24 to 0.41 µM on day 2 of week 2 post-exercise (P < 0.001) and remained similarly elevated on day 3 pre- and immediately post-exercise (P < 0.001). The GPx similarly remained at baseline (172.6 µM/min; P ≥ 0.621) but increased by 48.18 to 83.4 µM/min at most collection times on days 1 and 2 of week 2 (P ≤ 0.023). The SOD remained at baseline (167.2 U/ mL; P ≥ 0.055) until increasing by 11.28 to 15.61 U/mL at 30 min post-exercise on day 1, week 1 and at most collection times on day 3, week 8 (P ≤ 0.043). Amino acids with antioxidant properties such as Met, Tyr, and Trp drastically decreased from weeks 2 to 8 (P < 0.001). Met and Tyr also decreased from -60 to 90 min (P < 0.047), whereas there was no time effect on Trp concentration (P = 0.841). The current study indicates the time-dependent nature of oxidative stress concerning persistent stressors such as exercise.

Performance horses are subjected to numerous stressors. These stressors may subsequently impact their overall performance. The present study measured oxidative stress biomarkers in the blood of exercising horses. Horses were moderately exercised over an 8-wk period and blood plasma was collected to measure total antioxidant capacity (TAC), thiobarbituric acid reactive substance (TBARS), glutathione peroxidase activity (GPx), and superoxide dismutase activity (SOD). Amino acid concentration was also evaluated. The TAC was greater on day 2 vs. day 3. The TBARS remained at pre-exercise (baseline) at most times except for increasing on day 2 of week 2 post-exercise. The GPx also remained at baseline for most times but increased on days 1 and 2 of week 2. The SOD remained at baseline until increasing at 30 min post-exercise on day 1, week 1 and at most collection times on day 3, week 8. Amino acids with antioxidant properties drastically decreased from weeks 2 to 8. Horses are exposed to a variety of physical stressors on a regular basis that may produce similar effects in the equine stress response. Understanding the response in the equine athlete when exposed to new stressors is crucial in determining how to prevent oxidative damage in future athletes.

Prevalence and Molecular Characteristics of Avian Pathogenic Escherichia coli in "No Antibiotics Ever" Broiler Farms.

Avian pathogenic Escherichia coli (APEC) causes significant economic and welfare concerns to the broiler industry. For several decades, prophylactic supplementation of antimicrobial growth promoters was the primary method to control APEC; however, the recent shift to no antibiotics ever (NAE) production has increased colibacillosis incidence. The objectives of this study were to determine the influence of season, flock age, and sample type on the prevalence and virulence of E. coli and to identify the serogroups and antimicrobial susceptibility of virulent and nonvirulent E. coli in NAE broiler farms. Litter, feces, cloacal swabs, and tracheal swabs were collected from 4 NAE farms during spring and summer seasons, and E. coli was isolated and confirmed by PCR. Confirmed E. coli isolates were tested for 5 APEC-virulence-associated genes (VAGs) using quantitative PCR (qPCR). Further, E. coli isolates with all five VAGs (100 isolates) and E. coli isolates without any VAGs (87 isolates) were screened against 11 antimicrobials through Kirby-Bauer disk diffusion assay, and their serogroups were tested using PCR. Data were analyzed using the GLIMMIX procedure of SAS 9.4, and statistical significance was determined at a P value of ≤0.05. Overall, the prevalence of E. coli was not affected by season, flock age, or sample type. However, the prevalence of all tested VAGs decreased from spring to summer (P ≤ 0.002). The frequency of resistance was highest for tetracycline, and serogroups O8 (31%) and O78 (11%) were most frequent in virulent E. coli. In conclusion, there is a high prevalence of virulent E. coli in NAE farms, especially in the spring season. IMPORTANCE Avian pathogenic Escherichia coli causes one of the most detrimental bacterial diseases to the United States poultry industry, colibacillosis. Colibacillosis leads to decreased performance, early mortality, and subsequent production loss. Previously, colibacillosis was largely mitigated by the use of antimicrobial growth promoters. Due to concerns about antimicrobial resistance, the use of these promoters has been largely removed from the broiler industry. With recent shifts in the poultry industry to NAE broiler production, there is an increase in bacterial disease and mortality. We do not know how this shift to NAE affects APEC prevalence within broiler farms. Therefore, in the current study, we attempted to assess the prevalence and virulence of E. coli within an antibiotic-free broiler environment, assessed antimicrobial susceptibility, and identified the serogroups of virulent and nonvirulent E. coli.

Research Note: Prevalence and molecular characteristics of Clostridium perfringens in "no antibiotics ever" broiler farms.

Clostridium perfringens (C. perfringens) is the etiological agent of necrotic enteritis and gangrenous dermatitis; 2 diseases that cause significant economic and welfare concerns to the broiler industry. Previously, Clostridium-related diseases were managed with the use of antimicrobial growth promoters fed to broilers that improved gut health and performance. The recent shift to no antibiotics ever (NAE) production has increased the incidence of Clostridium-related diseases. The objective of this study was to identify C. perfringens prevalence and toxinotypes in NAE farms. Samples of litter, feces, and cloacal swabs were collected from 4 NAE broiler farms in the summer of 2019, on d 28 and d 56 of one flock cycle. A total of 734 presumptive isolates were obtained from 192 samples collected in the study. Irrespective of the age of flock and sample type, all 192 samples contained at least one colony presumptively identified as C. perfringens on Perfringens agar plate with morphology as a single, round colony with opaque ring and black center. All isolates were further screened using PCR for confirmation, toxinotyping, and identification of virulence-associated genes. Only 9 isolates among the 734 presumptive isolates were confirmed as C. perfringens and all confirmed isolates were toxinotype A with variation in presence of netB, cpb2, and tpeL. More extensive studies are required to assess the prevalence and virulence of C. perfringens in NAE farms.

Advancing Semen Evaluation Using Lipidomics.

Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species.

Lipidomic markers of sperm cryotolerance in cattle.

The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor; n = 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform-methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography-mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P < 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg; P < 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.

Avian Pathogenic Escherichia coli and Clostridium perfringens: Challenges in No Antibiotics Ever Broiler Production and Potential Solutions.

United States is the largest producer and the second largest exporter of broiler meat in the world. In the US, broiler production is largely converting to antibiotic-free programs which has caused an increase in morbidity and mortality within broiler farms. Escherichia coli and Clostridium perfringens are two important pathogenic bacteria readily found in the broiler environment and result in annual billion-dollar losses from colibacillosis, gangrenous dermatitis, and necrotic enteritis. The broiler industry is in search of non-antibiotic alternatives including novel vaccines, prebiotics, probiotics, and housing management strategies to mitigate production losses due to these diseases. This review provides an overview of the broiler industry and antibiotic free production, current challenges, and emerging research on antibiotic alternatives to reduce pathogenic microbial presence and improve bird health.

Application of natural antioxidant extract from guava leaves (Psidium guajava L.) in fresh pork sausage.

The current study was conducted to determine the antioxidant efficacy of guava leaf extract (3000 to 6000 ppm on fat basis) in fresh pork sausage, compared with negative control (CON) and 200 ppm butylated hydroxytoluene (BHT), for 0, 1, 4, 7, 10, and 14 d at 4 °C. The extract provided a total antioxidant capacity of 1505 µmol trolox equivalence/g. From d 4, the extract at 5000 and 6000 ppm provided greater (P < .05) antioxidant capacity than the CON and was either similar (P > .05) or greater (P < .05) than BHT. From d 4, the sausage formulated with 4000 to 6000 ppm of guava leaf extract had less conjugated dienes, lower peroxide and acidic values, less thiobarbituric reactive substances value, and better color (P < .05) than the CON and did not differ from BHT (P > .05). Guava leaf extract at 4000 ppm or greater is effective in preventing oxidation in fresh pork sausage.

Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy.

The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.

Effects of feeding endophyte-infected tall fescue seeds to stocker Angus steers on retail quality attributes of beef strip steaks.

The objective of this study was to determine the effects of feeding endophyte-infected tall fescue seeds to Angus steers during the stocker phase on the quality attributes of beef strip steaks during retail display. Endophyte-infected tall fescue seeds had no effect on steak surface lean color, myoglobin forms, proximate composition, thiobarbituric acid reactive substances, aerobic plate count, pH, activity of superoxide dismutase and metmyoglobin reductase, shear force, and sensory attributes (P ≥ 0.087). However, lightness, redness, oxymyoglobin percentage, and MRA decreased from 45.01, 32.60, 67.61%, and 9.54 µM/min/g, respectively, on d 0 to 40.11, 21.83, 48.95%, and 2.30 µM/min/g, respectively, on d 7 (P ≤ 0.001). Thiobarbituric acid reactive substances were increased by 30% by d 5 (P = 0.015) and APC was increased by 0.5 log CFU/g by d 7 (P ≤ 0.012).

Technological characteristics of pre- and post-rigor deboned beef mixtures from Holstein steers and quality attributes of cooked beef sausage.

The objective of this study was to determine the effects of deboning time (pre- and post-rigor), processing steps (grinding - GB; salting - SB; batter formulation - BB), and storage time on the quality of raw beef mixtures and vacuum-packaged cooked sausage, produced using a commercial formulation with 0.25% phosphate. The pH was greater in pre-rigor GB and SB than in post-rigor GB and SB (P < .001). However, deboning time had no effect on metmyoglobin reducing activity, cooking loss, and color of raw beef mixtures. Protein solubility of pre-rigor beef mixtures (124.26 mg/kg) was greater than that of post-rigor beef (113.93 mg/kg; P = .071). TBARS were increased in BB but decreased during vacuum storage of cooked sausage (P ≤ .018). Except for chewiness and saltiness being 52.9 N-mm and 0.3 points greater in post-rigor sausage (P = .040 and 0.054, respectively), texture profile analysis and trained panelists detected no difference in texture between pre- and post-rigor sausage.

Effect of deboning time on the growth of Salmonella, E. coli, aerobic, and lactic acid bacteria during beef sausage processing and storage.

The objective of the current study was to determine the effects of deboning time, three steps of sausage processing (grinding, salting, and batter formulation), and storage time (of raw materials and cooked sausage) on the growth (log CFU/g) of aerobic bacteria, lactic acid bacteria, and inoculated Salmonella and E. coli. Beef deboning time did not influence bacterial counts (P≥0.138). However, salting of raw ground beef resulted in a 0.4-log reduction in both aerobic plate count (APC) and Salmonella (P≤0.001). Lactic acid bacteria were increased from non-detectable concentration (0.54 log) on d 0 to 3.8 log on d 120 of vacuum storage (P≤0.019). Salmonella counts were increased (P<0.001) over storage time (3.2 to 3.3 log CFU/g from d 0 to 10). Results indicated that salting and batter formulation had a greater impact on bacterial counts than rigor state of raw beef.

Determination of total cholesterol in meat and poultry by gas chromatography: single-laboratory validation.

The primary objective of this study was to determine the intralaboratory performance of a cholesterol determination method that combines direct saponification of a 1 g meat or poultry sample and GC quantification of liberated cholesterol without derivatization. Cholesterol was detected at 11.96 min using a GC-flame ionization detector (FID) system. With a 0.005 mg/mL 5alpha-cholestane internal standard and 0.008 to 0.020 mg/mL cholesterol standard series, the FID response was linearly correlated to standard concentrations with a coefficient of determination of 0.995 and a response factor of 0.66. The LOD and LOQ were 1.24 and 4.00 mg/100 g, respectively. Cholesterol could be analyzed within 6 days of preparation with high precision (CV of 0.92 to 2.69%) and accuracy (recovery of 93.24 to 100.56%). This simplified procedure allows for decreased errors and increased productivity, and the method proved to be reliable and able to withstand practical variation in procedural application. The method has been applied routinely with excellent precision to update data on the cholesterol content of beef, pork, and chicken in the U.S. Department of Agriculture National Nutrient Database for Standard Reference.