Ty B. Schmidt

Proteomic analysis of the response of Listeria monocytogenes to bile salts under anaerobic conditions

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. The infectious process depends on survival in the high bile-salt conditions encountered throughout the gastrointestinal tract, including the gallbladder. However, it is not clear how bile-salt resistance mechanisms are induced, especially under physiologically relevant conditions. This study sought to determine how the L. monocytogenes strains EGDe (serovar 1/2a), F2365 (serovar 4a) and HCC23 (serovar 4b) respond to bile salts under anaerobic conditions. Changes in the expressed proteome were analysed using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. In general, the response to bile salts among the strains tested involved significant alterations in the presence of cell-wall-associated proteins, DNA repair proteins, protein folding chaperones and oxidative stress-response proteins. Strain viability correlated with an initial osmotic stress response, yet continued survival for EGDe and F2365 involved different mechanisms. Specifically, proteins associated with biofilm formation in EGDe and transmembrane efflux pumps in F2365 were expressed, suggesting that variations exist in how virulent strains respond and adapt to high bile-salt environments. These results indicate that the bile-salt response varies among these serovars and that further research is needed to elucidate how the response to bile salts correlates with colonization potential in vivo.

Survival of Escherichia coli O157:H7 Transformed with Either the pAK1-lux or pXEN-13 Plasmids in In Vitro Bovine Ruminal and Fecal Microbial Fermentations

The use of luminescent plasmids in bacteria may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding patterns of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth characteristics of E. coli O157:H7 in mixed ruminal and fecal microbial fluid cultures would be altered when transformed with one of the two luminescent plasmids: pAK1-lux (PAK) or pXEN-13 (XEN). Transformants harboring the luminescent plasmids were compared to the non-transformed parental strain (wild type [WT]) after incubating in mixed ruminal or fecal microbial fluid media for 6 h in triplicate (n=3). The transformants and WT exhibited similar growth rates. Within mixed ruminal microbial fluid fermentations and mixed fecal microbial fluid, all transformants grew similarly (p=0.28) through the 6-h study. The reflective light unit (RLU; photons/pixel per second) photonic emissions of each plasmid within ruminal fluid differed at 0 h (p=0.002) and 2 h (p=0.02) and within fecal fluid at 0 h (p=0.009) and 2 h (p=0.04). The RLU remained the same within rumen fluid at 4 h (p=0.22) and 6 h (p=0.80) and within fecal fluid at 4 h (p=0.06) and 6 h (p=0.29). Growth of E. coli O157:H7 transformed with the bioluminescent plasmids was not altered in comparison to the WT, suggesting that both plasmids may serve as useful models for in vivo studies.

Survival of O157:H7 and Non-O157 Serogroups of Escherichia coli in Bovine Rumen Fluid and Bile Salts

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

Use of Bioluminescent Escherichia coli to Determine Retention During the Life Cycle of the Housefly, Musca domestica (Diptera: Muscidae, L)

Researchers have documented that the housefly (Musca domestica) can serve as a vector for the spread of foodborne pathogens to livestock, food, and humans. Most studies have investigated Musca domestica as a vector only after the fly comes into contact or consumes the pathogen as an adult. The objective of this study was to determine whether the larvae of Musca domestica could ingest Escherichia coli from bovine manure and whether the E. coli could survive the metamorphosis process and be transmitted. Larvae (n=960) were incubated in sterilized bovine manure inoculated with 0, 3, 5, and 8 log10 colony-forming units (CFU)/mL of bioluminescent E. coli for 24 (larvae stage), 48 (larvae stage), 120 (pupae stage), and 192 h (adult stage). Larvae incubated for 24 h in bovine manure possessed 0.0, 2.7, 2.9, and 3.5 log(10) CFU/mL of E. coli, from inoculated with 0, 3, 5, and 8 log(10) CFU/mL of E. coli, respectively. Concentrations of E. coli within the pupae were 0.0, 1.7, 1.9, and 2.2 log(10) CFU/mL for each inoculation concentration, respectively. Flies that emerged from the pupae stage contained 0.0, 1.3, 2.2, and 1.7 log(10) CFU/mL of E. coli from larvae incubated in manure inoculated with concentrations of E. coli, respectively. These results suggest the housefly can emerge with quantities of E. coli. While this was an enteropathogenic E. coli (EPEC), these data may suggest that if the fly is capable of retaining similar concentrations of an enterohemorrhagic E. coli (EHEC), these concentrations may be capable of initiating illness in humans. Furthermore, the E. coli concentration within and on adult flies is related to environmental exposure. It must be noted that larvae were incubated in sterilized bovine manure, and there was no other bacterial competition for the E. coli. Thus, the rate of positive flies and concentrations present when flies emerged may vary under more realistic conditions.

National Beef Quality Audit–2016: In-plant survey of carcass characteristics related to quality, quantity, and value of fed steers and heifers

The National Beef Quality Audit (NBQA)-2016 used in-plant cooler assessments to benchmark the current status of the fed steer and heifer beef industry in the United States. In-plant cooler assessments ( = 9,106 carcasses) were conducted at 30 facilities, where approximately 10% of a single days production were evaluated for USDA quality grade (QG) and yield grade (YG) factors. Frequencies of evaluated traits were 66.5% steer and 33.4% heifer sex classes and 82.9% native, 15.9% dairy-type, and 1.2% estimated breed types. Mean USDA YG factors were 1.42 cm for adjusted fat thickness, 89.5 cm for LM area, 390.3 kg for HCW, and 1.9% for KPH. Mean USDA YG was 3.1, with a frequency distribution of 9.6% YG 1, 36.7% YG 2, 39.2% YG 3, 12.0% YG 4, and 2.5% YG 5. Mean USDA QG traits were Small for marbling score, A for overall maturity, A55 for lean maturity, and A for skeletal maturity. Mean USDA QG was Select with a frequency distribution of QG of 3.8% Prime, 67.3% Choice, 23.2% Select, and 5.6% lower score. Lower score included dark cutter (1.9%), blood splash (0.1%), and hard bone, which are USDA overall maturity scores of C or older (1.8%). Marbling score distributions were 0.85% Slightly Abundant or greater, 7.63% Moderate, 23.54% Modest, 39.63% Small, 23.62% Slight, and 0.83% Traces or less. Carcasses that were Choice or Select and USDA YG 2 or 3 accounted for 70.7% of the carcasses evaluated. Compared with the previous NBQA, we found a numerical increase in mean USDA YG, USDA QG, adjusted fat thickness, HCW, LM area, and marbling score with an increase in dairy-type carcasses and percentage of carcasses grading USDA Prime and Choice as well as frequency of USDA YG 4 and 5. The findings from this study will be used by all segments of the industry to understand and improve the quality of fed steer and heifer beef that is being produced.

The metabolic, stress axis, and hematology response of zilpaterol hydrochloride supplemented beef heifers when exposed to a dual corticotropin-releasing hormone and vasopressin challenge,,

The objective of this study was to determine the metabolic, stress, and hematology response of beef heifers supplemented with zilpaterol hydrochloride (ZH) when exposed to an endocrine stress challenge. Heifers ( = 20; 556 ± 7 kg BW) were randomized into 2 treatment groups: 1) control (CON), no ZH supplementation, and 2) zilpaterol (ZIL), supplemented with ZH at 8.33 mg/kg (DM basis). The ZIL group was supplemented ZH for 20 d, with a 3-d withdrawal period. On d 24, heifers received an intravenous bolus of corticotropin-releasing hormone (CRH; 0.3 µg/kg BW) and arginine vasopressin (VP; 1.0 µg/kg BW) to activate the stress axis. Blood samples were collected at 30-min intervals for serum and 60-min intervals for plasma and whole blood, from -2 to 8 h relative to the challenge at 0 h (1000 h). Samples were analyzed for glucose, insulin, NEFA, blood urea nitrogen (BUN), cortisol, epinephrine, norepinephrine, and complete blood cell counts. Following the challenge, cattle were harvested over a 3-d period. Liver, LM, and biceps femoris (BF) samples were collected and analyzed for glucose, lactate, and glycolytic potential (GP). There was a treatment ( ≤ 0.001) effect for vaginal temperature (VT), with ZIL having a 0.1°C decrease in VT when compared with CON. A treatment × time effect ( = 0.002) was observed for NEFA. A treatment effect was observed for BUN; ZIL had decreased BUN concentrations compared with CON ( < 0.001) prior to the challenge; however, no treatment × time effect was observed. There was also a treatment effect for cortisol ( ≤ 0.01) and epinephrine ( = 0.003); ZIL had decreased cortisol and epinephrine during the CRH/VP challenge when compared with CON. There was a time effect for total white blood cells, lymphocytes, and monocytes; each variable increased ( ≤ 0.01) 2 h postchallenge. Additionally, neutrophil counts decreased ( ≤ 0.01) in response to CRH/VP challenge in both treatment groups. Glucose concentrations within the LM were greater ( = 0.03) in CON when compared with ZIL. Lactate concentrations and GP within the BF were greater in CON ( = 0.05) when compared with ZIL. These data suggest there are some variations observed between treatments in terms of response to the CRH/VP challenge; however, in the environmental conditions of this trial, none of the variations observed suggest that the supplementation of ZH detrimentally alters the ability of cattle to effectively respond to stressful stimuli.

Effect of Citrus Byproducts on Survival of O157:H7 and Non-O157 Escherichia coli Serogroups within In Vitro Bovine Ruminal Microbial Fermentations.

Citrus byproducts (CBPs) are utilized as a low cost nutritional supplement to the diets of cattle and have been suggested to inhibit the growth of both Escherichia coli O157:H7 and Salmonella. The objective of this study was to examine the effects in vitro that varying concentrations of CBP in the powdered or pelleted variety have on the survival of Shiga-toxin Escherichia coli (STEC) serotypes O26:H11, O103:H8, O111:H8, O145:H28, and O157:H7 in bovine ruminal microorganism media. The O26:H11, O111:H8, O145:H28, and O157:H7 serotypes did not exhibit a change in populations in media supplemented with CBP with either variety. The O103:H8 serotype displayed a general trend for an approximate 1log10 reduction in 5% powdered CBP and 20% pelleted CBP over 6 h. There was a trend for reductions in populations of a variant form of O157:H7 mutated in the stx1 and stx2 genes in higher concentrations of CBP. These results suggest that variations exist in the survival of these serotypes of STEC within mixed ruminal microorganism fluid media when supplemented with CBP. Further research is needed to determine why CBPs affect STEC serotypes differently.

National Beef Quality Audit–2016: Transportation, mobility, live cattle, and carcass assessments of targeted producer-related characteristics that affect value of market cows and bulls, their carcasses, and associated by-products

Abstract The National Beef Quality Audit–2016 marks the fourth iteration in a series assessing the quality of live beef and dairy cows and bulls and their carcass counterparts. The objective was to determine the incidence of producer-related defects, and report cattle and carcass traits associated with producer management. Conducted from March through December of 2016, trailers (n = 154), live animals (n = 5,470), hide-on carcasses (n = 5,278), and hide-off hot carcasses (n = 5,510) were surveyed in 18 commercial packing facilities throughout the United States. Cattle were allowed 2.3 m2 of trailer space on average during transit indicating some haulers are adhering to industry handling guidelines for trailer space requirements. Of the mixed gender loads arriving at processing facilities, cows and bulls were not segregated on 64.4% of the trailers surveyed. When assessed for mobility, the greatest majority of cattle surveyed were sound. Since the inception of the quality audit series, beef cows have shown substantial improvements in muscle. Today over 90.0% of dairy cows are too light muscled. The mean body condition score for beef animals was 4.7 and for dairy cows and bulls was 2.6 and 3.3, respectively. Dairy cattle were lighter muscled, yet fatter than the dairy cattle surveyed in 2007. Of cattle surveyed, most did not have horns, nor any visible live animal defects. Unbranded hides were observed on 77.3% of cattle. Carcass bruising was seen on 64.1% of cow carcasses and 42.9% of bull carcasses. However, over half of all bruises were identified to only be minor in severity. Nearly all cattle (98.4%) were free of visible injection-site lesions. Current results suggest improvements have been made in cattle and meat quality in the cow and bull sector. Furthermore, the results provide guidance for continued educational and research efforts for improving market cow and bull beef quality.

National Beef Quality Audit-2016: Transportation, mobility, and harvest-floor assessments of targeted characteristics that affect quality and value of cattle, carcasses, and by-products

Abstract The National Beef Quality Audit-2016 (NBQA-2016) was conducted to assess current transportation, mobility, and quality characteristics of U.S. fed steers and heifers. Data were collected at 17 beef processing facilities between March and November 2016. About 8,000 live cattle were evaluated for transportation and mobility, and about 25,000 carcasses were evaluated on the slaughter floor. Cattle were in transit to the slaughter facility for a mean duration of 2.7 h from a mean distance of 218.5 km using trailers with dimensions ranging from 17.84 m2 to 59.09 m2. Area allotted per animal averaged 1.13 m2 and ranged from 0.85 m2 to 2.28 m2. A total of 96.8% of cattle received a mobility score of 1 (walks easily, no apparent lameness). Identification types (35.1% had multiple) were lot visual tags (61.5%), individual tags (55.0%), electronic tags (16.9%), metal-clip tags (9.2%), bar-coded tags (0.05%), wattles (0.01%), and other (2.6%). Cattle were black-hided (57.8%), Holstein (20.4%), red-hided (10.5%), yellow-hided (4.8%), gray-hided (2.9%), brown-hided (1.3%), and white-hided (1.1%). Unbranded hides were observed on 74.3% of cattle; 18.6% had brands located on the butt, 6.3% on the side, and 1.3% on the shoulder (values exceed 100% due to multiple brands). For hide-on carcasses, 37.7% displayed no mud or manure; specific locations for mud or manure were legs (40.8%), belly (33.0%), tail region (15.5%), side (6.8%), and top-line (3.9%). Cattle without horns represented 83.3% of the sample, and cattle that did have horns measured: < 2.54 cm (5.5%), 2.54 to 12.7 cm (8.3%), and > 12.7 cm (2.9%). Carcasses without bruises represented 61.1% of those sampled, whereas 28.2% had 1, 8.2% had 2, 2.1% had 3, and 0.3% had 4 bruises. Of those carcasses with a bruise, the bruise was located on the loin (29.7%), round (27.8%), chuck (16.4%), rib (14.4%), and brisket/plate/flank (11.6%). Frequencies of offal condemnations were livers (30.8%), lungs (18.2%), viscera (16.3%), hearts (11.1%), heads (2.7%), and tongues (2.0%). Compared to NBQA-2011, fewer cattle were identified for traceability, fewer were black-hided, a greater number were Holstein cattle, more with no brand and no horns, fewer without bruises, more liver, lung, and viscera condemnations, and fewer heads and tongues were condemned. The NBQA remains an influential survey for the U.S. beef industry to provide benchmarks and strategic plans for continued improvement of beef quality and consistency.

National Beef Quality Audit – 2016: Survey of carcass characteristics through instrument grading assessments

The instrument grading assessment portion of the National Beef Quality Audit (NBQA) - 2016 allows the unique opportunity to evaluate beef carcass traits over the course of a year. One week of instrument grading data was collected each month from 5 beef processing corporations encompassing 18 facilities from January 2016 through December 2016 ( = 4,544,635 carcasses). Mean USDA yield grade (YG) was 3.1 with 1.37 cm fat thickness (FT), 88.9 cm LM area, 393.6 kg HCW, and 2.1% KPH. Frequency distribution of USDA YG was 9.5% YG 1, 34.6% YG 2, 38.8% YG 3, 14.6% YG 4, and 2.5% YG 5. Increases in HCW and FT since the NBQA-2011 were major contributors to differences in mean YG and the (numerically) increased frequency of YG 3, 4, and 5 carcasses found in the current audit. Mean marbling score was Small, and the distribution of USDA quality grades was 4.2% Prime, 71.4% Choice, 21.7% Select, and 2.7% other. Frequency of carcasses grading Prime on Monday (6.43%) was numerically higher than the average frequency of carcasses grading Prime overall (4.2%). Monthly HCW means were 397.6 kg in January, 397.2 kg in February, 396.5 kg in March, 389.3 kg in April, 384.8 kg in May, 385.0 kg in June, 386.1 kg in July, 394.1 kg in August, 399.1 kg in September, 403.9 kg in October, 406.5 kg in November, and 401.9 kg in December. Monthly mean marbling scores were Small in January, Small in February, Small in March, Small in April, Small in May, Small in June, Small in July, Small in August, Small in September, Small in October, Small in November, and Small in December. Both mean HCW and mean marbling score declined in the months of May and June. The month with the greatest numerical frequency of dark cutters was October (0.74%). Comparison of overall data from in-plant carcass and instrument grading assessments revealed close alignment of information, especially for YG (3.1 for in-plant assessment versus 3.1 for instrument grading) and marbling (Small for in-plant assessment versus Small for instrument grading). These findings allow the beef industry access to the greatest volume of beef value-determining characteristics for the U.S. fed steer and heifer population than ever reported, resulting in potentially more precise targeting of future quality and consistency efforts.