Proof of concept for developing novel feeds for cattle from wasted food and crop biomass to enhance agri-food system efficiency.

Modern agri-food systems generate large amounts of crop-based biomass that are unfit for direct human consumption but potentially suitable for livestock feeding in production of meats, milk, and eggs. This study aims to develop novel feeds for cattle from some of those biomass materials through the natural microbial-driven processes of ensiling. Fruit and vegetables resembling supermarket discards were ensiled alone or co-ensiled with corn crop residues, mushroom wastes, etc. via laboratory experiments. Longitudinal sample analyses showed that (co-)ensiling was successful, with pH and fermentation acids changing rapidly into desirable ranges (pH < 4.5, the acids 5-13% DM with lactic acid dominating). The (co-)ensiled products had key nutritional parameters comparable to those of good quality forages commonly used on dairy farms. Additionally, in vitro incubation experiments indicated that the ensiled products could substitute certain conventional feeds while maintaining diet digestibility. Findings from this pilot study provide a proof of principle that quality novel feeds for cattle can be generated by co-ensiling food discards and low-value crop residues. Future research and animal feeding trials to demonstrate the utility of this approach can help societies more effectively utilize untapped biomass resources, strengthening the regenerative capacity of agri-food systems towards a more sustainable food future.

Educational interventions to address misconceptions about antibiotic residues in milk can alter consumer perceptions and may affect purchasing habits.

The industrialization of the agri-food industry and resultant decrease in the number of people employed on farms has contributed to a knowledge gap among consumers about food production processes. A commonly reported concern of dairy consumers is the use of antibiotics in dairy animals, even though these drugs are an important tool for promoting animal health and welfare and food safety. The extent to which consumers are aware of antibiotic residue avoidance practices in dairy production is unknown, and it is unclear whether acquisition of such knowledge could affect purchasing behavior and perceptions of dairy farming. The objectives of this study were to assess consumers' perceptions about the quality and production of dairy products in the United States and determine whether educational materials on processes that limit the occurrence of antibiotic residues in milk can change consumers' perceptions of dairy products and purchasing behaviors. We surveyed 804 consumers and assigned them to 1 of 3 interventions: (1) a control arm (reading the content of the Dairy page of the USDA's myplate.gov website); (2) an educational brochure on the processes that prevent antibiotic residues in milk; and (3) a video on the same processes. We found that a majority (86.1%) of participants believe that the quality of dairy products in the United States is high, although many had concerns about the treatment of dairy animals and chemicals (pesticides, antibiotics, hormones) in dairy products. Compared with the control intervention, the brochure was associated with a significant decrease in the level of concern consumers had about chemicals in their milk [-0.20 points on a Likert scale, 95% confidence interval (CI), -0.32 to -0.08] and a significantly increased comfort in purchasing conventional dairy products (odds ratio 2.43, 95% CI, 1.62 to 3.66). The video was associated with even stronger effects: a 0.29-unit decrease in the level of concern about chemicals in milk (95% CI, -0.42 to -0.016) and 2.94 times greater odds of purchasing conventional dairy products (95%, CI 1.92 to 4.49). Although consumer food decision making is complex and driven by multiple factors, it appears that education about the processes that promote food safety can reassure consumers about their concerns and potentially affect purchasing habits.

Using Structural Equation Modeling to Understand Interactions Between Bacterial and Archaeal Populations and Volatile Fatty Acid Proportions in the Rumen.

Microbial syntrophy (obligate metabolic mutualism) is the hallmark of energy-constrained anaerobic microbial ecosystems. For example, methanogenic archaea and fermenting bacteria coexist by interspecies hydrogen transfer in the complex microbial ecosystem in the foregut of ruminants; however, these synergistic interactions between different microbes in the rumen are seldom investigated. We hypothesized that certain bacteria and archaea interact and form specific microbial cohorts in the rumen. To this end, we examined the total (DNA-based) and potentially metabolically active (cDNA-based) bacterial and archaeal communities in rumen samples of dairy cows collected at different times in a 24 h period. Notably, we found the presence of distinct bacterial and archaeal networks showing potential metabolic interactions that were correlated with molar proportions of specific volatile fatty acids (VFAs). We employed hypothesis-driven structural equation modeling to test the significance of and to quantify the extent of these relationships between bacteria-archaea-VFAs in the rumen. Furthermore, we demonstrated that these distinct microbial networks were host-specific and differed between cows indicating a natural variation in specific microbial networks in the rumen of dairy cows. This study provides new insights on potential microbial metabolic interactions in anoxic environments that have broader applications in methane mitigation, energy conservation, and agricultural production.

Short communication: Comparison of the fecal bacterial communities in diarrheic and nondiarrheic dairy calves from multiple farms in southeastern Pennsylvania.

Diarrhea is a major cause of illness and death in preweaned calves and causes significant economic losses to producers. A better understanding of the fecal microbiota in diarrheic and nondiarrheic calves could lead to improved treatment and prevention strategies. The purpose of this study was to compare the fecal microbiota of diarrheic and nondiarrheic calves to improve our understanding of what constitutes a healthy fecal microbiota in preweaned calves. At each of 7 farms, fecal samples were obtained from 1 to 3 diarrheic Holstein dairy calves (2 to 17 d old at sampling time) and age-matched (within 5 d) nondiarrheic controls for a total of 20 samples. Calves were fed either acidified bulk milk, pasteurized or unpasteurized waste milk, or milk replacer depending on farm. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq (Illumina Inc., San Diego, CA) platform, and analyzed using QIIME2. Firmicutes and Bacteroidetes were the most abundant phyla in both groups; Fusobacteria was numerically more abundant in the diarrheic group, whereas Proteobacteria and Actinobacteria were numerically more abundant in the nondiarrheic group. At the genus level, Bacteroides was the most abundant genus in both groups and was numerically more abundant in the nondiarrheic group. Results from the mixed-effects regression model showed that Faecalibacterium and Butyricimonas were more abundant in the nondiarrheic calves, whereas Clostridium and Peptostreptococcus were more abundant in the diarrheic calves. Our results indicate that commensal bacteria acquired in the neonatal period may have been replaced with potential pathogens in diarrheic calves, which may have contributed to the incidence of diarrhea either directly or indirectly.

Clostridioides difficile on dairy farms and potential risk to dairy farm workers.

Clostridioides difficile causes severe colitis in people and is a significant enteric pathogen in many species of animals, including swine, horses, and potentially cattle. C. difficile is shed in feces, and transmission occurs horizontally via the fecal-oral route. Livestock has been suggested as a potential reservoir for C. difficile, and while studies have shown that swine and farm workers can be colonized with identical clones of C. difficile, the zoonotic transmission of C. difficile from livestock to people has not been definitively demonstrated. The goal of this study was to determine whether dairy calves and dairy farm workers harbored genetically similar isolates of C. difficile. First, we validated a glove juice protocol for detecting C. difficile on farm workers' hands. We then visited 23 farms and collected 1) fecal samples from 92 dairy calves, 2) hand rinsates from 38 dairy farm workers, and 3) fecal samples from five of the dairy farm workers who were willing to submit them. All samples underwent anaerobic culture and qPCR to detect C. difficile. C. difficile was detected on 15 of the farms (65.2%, 95% confidence interval (CI) 42.7%-83.6%) and in 28 calves (30.4%, 95% CI 21.2-40.9%) but in none of the hand rinsates or human fecal samples. Thus, the zoonotic transmission of C. difficile on dairy farms could not be demonstrated, and dairy farmers did not appear to be at increased risk of acquiring C. difficile via the fecal-oral route.

Temporal changes in the fecal bacterial community in Holstein dairy calves from birth through the transition to a solid diet.

The development of a robust microbiome is critical to the health of dairy calves, but relatively little is known about the progression of the microbiome through the weaning transition. In this study, fecal samples were obtained from ten female Holstein calves at 6 timepoints between 2-13 weeks of age. Calves were fed acidified milk until weaning at 8 weeks old and had access to starter grain throughout the study. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq platform, and analyzed using the QIIME2 pipeline. Bacterial richness, estimated by number of observed species, and bacterial diversity, estimated by Shannon diversity index, both differed significantly between timepoints and both increased over time (P <0.05), with the largest increases occurring during weaning. Weighted and unweighted UniFrac analysis showed significant differences (P <0.05) between bacterial communities across timepoints; betadisper analysis revealed that the microbiomes of individual calves became more similar with time. Throughout the study, Firmicutes was the dominant phylum, followed by Bacteroidetes. Thirteen bacterial genera were found to be significantly influenced by time, including Faecalibacterium, Clostridium, unclassified S24-7, Collinsella, Sharpea, and Treponema. Unclassified Ruminococcaceae was the most prevalent genus at timepoints 1, 3, 5, and 6, but different amplicon sequence variants were detected at each timepoint suggesting the presence of different species of Ruminococcaceae at different times. Bacteroides was the most prevalent genus at timepoint 2, and Prevotella was most prevalent at timepoint 4. Our results indicate that there is considerable variation in the calf microbiome pre-weaning, but the microbial community stabilizes and becomes similar to the adult microbiome at weaning. Further studies to describe the phylogeny and functionality of core microbiota through the weaning transition are needed to improve health and reduce diarrhea in the neonatal period.

A step towards personalizing next line therapy for resected pancreatic and related cancer patients: A single institution's experience.

BACKGROUND: There is a lack of precision medicine in pancreatic ductal adenocarcinoma (PDA) and related cancers, and outcomes for patients with this diagnosis remain poor despite decades of research investigating this disease. Therefore, it is necessary to explore novel therapeutic options for these patients who may benefit from personalized therapies. OBJECTIVE: Molecular profiling of hepatopancreaticobiliary malignancies at our institution, including but not limited to PDA, was initiated to assess the feasibility of incorporating molecular profiling results into patient oncological therapy planning. METHODS: All eligible patients from Thomas Jefferson University (TJU) with hepatopancreaticobiliary tumors including PDA, who agreed to molecular testing profiling, were prospectively enrolled in a registry study from December 2014 to September 2017 and their tumor samples were tested to identify molecular markers that can be used to guide therapy options in the future. Next generation sequencing (NGS) and protein expression in tumor samples were tested at CLIA-certified laboratories. Prospective clinicopathologic data were extracted from medical records and compiled in a de-identified fashion. RESULTS: Seventy eight (78) patients were enrolled in the study, which included 65/78 patients with PDA (local and metastatic) and out of that subset, 52/65 patients had surgically resected PDA. Therapy recommendations were generated based on molecular and clinicopathologic data for all enrolled patients. NGS uncovered actionable alterations in 25/52 surgically resected PDAs (48%) which could be used to guide therapy options in the future. High expression of three proteins, TS (p = 0.005), ERCC1 (p = 0.001), and PD-1 (p = 0.04), was associated with reduced recurrence-free survival (RFS), while TP53 mutations were correlated with longer RFS (p = 0.01). CONCLUSIONS: The goal of this study was to implement a stepwise strategy to identify and profile resected PDAs at our institution. Consistent with previous studies, approximately half of patients with resected PDA harbor actionable mutations with possible targeted therapeutic implications. Ongoing studies will determine the clinical value of identifying these mutations in patients with resected PDA.

The distribution of microbiomes and resistomes across farm environments in conventional and organic dairy herds in Pennsylvania.

BACKGROUND: Antimicrobial resistance is a serious concern. Although the widespread use of antimicrobials in livestock has exacerbated the emergence and dissemination of antimicrobial resistance genes (ARG) in farm environments, little is known about whether antimicrobial use affects distribution of ARG in livestock systems. This study compared the distribution of microbiomes and resistomes (collections of ARG) across different farm sectors in dairy herds that differed in their use of antimicrobials. Feces from heifers, non-lactating, and lactating cows, manure storage, and soil from three conventional (antimicrobials used to treat cows) and three organic (no antimicrobials used for at least four years) farms in Pennsylvania were sampled. Samples were extracted for genomic DNA, processed, sequenced on the Illumina NextSeq platform, and analyzed for microbial community and resistome profiles using established procedures. RESULTS: Microbial communities and resistome profiles clustered by sample type across all farms. Overall, abundance and diversity of ARG in feces was significantly higher in conventional herds compared to organic herds. The ARG conferring resistance to betalactams, macrolide-lincosamide-streptogramin (MLS), and tetracyclines were significantly higher in fecal samples of dairy cows from conventional herds compared to organic herds. Regardless of farm type, all manure storage samples had greater diversity (albeit low abundance) of ARG conferring resistance to aminoglycosides, tetracyclines, MLS, multidrug resistance, and phenicol. All soil samples had lower abundance of ARG compared to feces, manure, and lagoon samples and were comprised of ARG conferring resistance to aminoglycosides, glycopeptides, and multi-drug resistance. The distribution of ARG is likely driven by the composition of microbiota in the respective sample types. CONCLUSIONS: Antimicrobial use on farms significantly influenced specific groups of ARG in feces but not in manure storage or soil samples.

Comparison of Two Sampling Techniques for Evaluating Ruminal Fermentation and Microbiota in the Planktonic Phase of Rumen Digesta in Dairy Cows.

The objective of this experiment was to compare ruminal fluid samples collected through rumen cannula (RC) or using an oral stomach tube (ST) for measurement of ruminal fermentation and microbiota variables. Six ruminally cannulated lactating Holstein cows fed a standard diet were used in the study. Rumen samples were collected at 0, 2, 4, 6, 8, and 12 h after the morning feeding on two consecutive days using both RC and ST techniques. Samples were filtered through two layers of cheesecloth and the filtered ruminal fluid was used for further analysis. Compared with RC, ST samples had 7% greater pH; however, the pattern in pH change after feeding was similar between sampling methods. Total volatile fatty acids (VFA), acetate and propionate concentrations in ruminal fluid were on average 23% lower for ST compared with RC. There were no differences between RC and ST in VFA molar proportions (except for isobutyrate), ammonia and dissolved hydrogen (dH2) concentrations, or total protozoa counts, and there were no interactions between sampling technique and time of sampling. Bacterial ASV richness was higher in ST compared with RC samples; however, no differences were observed for Shannon diversity. Based on Permanova analysis, bacterial community composition was influenced by sampling method and there was an interaction between sampling method and time of sampling. A core microbiota comprised of Prevotella, S24-7, unclassified Bacteroidales and unclassified Clostridiales, Butyrivibrio, unclassified Lachnospiraceae, unclassified Ruminococcaceae, Ruminococcus, and Sharpea was present in both ST and RC samples, although their relative abundance varied and was influenced by an interaction between sampling time and sampling method. Overall, our results suggest that ruminal fluid samples collected using ST (at 180 to 200 cm depth) are not representative of rumen pH, absolute values of VFA concentrations, or bacterial communities >2 h post-feeding when compared to samples of ruminal fluid collected using RC. However, ST can be a feasible sampling technique if the purpose is to study molar proportions of VFA, protozoa counts, dH2, and ammonia concentrations.

Multiplatform profiling of pancreatic neuroendocrine tumors: Correlative analyses of clinicopathologic factors and identification of co-occurring pathogenic alterations.

BACKGROUND: Multi-omic profiling of pancreatic neuroendocrine tumors (PanNETs) was performed to correlate genomic, proteomic, and molecular pathway alterations with clinicopathologic factors and identify novel co-occurring pathogenic alterations of potential clinical relevance to PanNET management. METHODS: PanNETs referred to Perthera, Inc. having undergone molecular profiling for precision matched therapeutic purposes were screened. Correlative analyses were performed using Fisher's exact test across individual pathogenic alterations or altered molecular pathways and clinicopathologic variables. Associations were visualized by hierarchical clustering. Prognostic associations with overall survival (OS) were identified using Cox regression for pathogenic alterations and pathway-level alterations. Hazard ratios (HR) and odds ratios (OR) were reported with 95% confidence intervals (CI). RESULTS: From 12/2014-1/2019, 46 patients with predominantly locally advanced and metastatic PanNETs were included. MEN1 alterations by next-generation sequencing (NGS) were less associated with having high-grade PanNETs and metastatic disease at diagnosis (p ≤ 0.05). Genomic alterations associated with increased replicative stress (primarily driven by RB1 and TP53) correlated with higher grade (OR 6.87 [95% CI: 1.57-35.18], p = 0.0043) and worse OS (HR 13.62 [95% CI: 1.51-122.5], p = 0.0198). Other significant associations included: ERCC1 protein expression with DAXX or MEN1 alterations (NGS), PTEN (NGS) with ARID1A or TP53 alterations (NGS), and history of diabetes coincided with cell cycle pathway alterations but was mutually exclusive with replicative stress pathway alterations. CONCLUSIONS: We identified several molecular signatures of potential clinical significance for therapeutic targeting and prognostication in PanNETs warranting prospective validation. Our findings are hypothesis generating and can inform larger molecular profiling efforts in PanNETs.

Quantification of antibiotic use on dairy farms in Pennsylvania.

Antibiotic use data are critical for drawing conclusions about the epidemiological connections between antibiotic use in farms animals, antibiotic resistance, animal health, and human health. The goal of this study was to quantitatively and qualitatively characterize antibiotic use on dairy farms in Pennsylvania, the state with second largest number of dairy farms nationally. A survey was sent to 10% of the 6,580 dairy farms registered in Pennsylvania and completed by 235 producers (response rate of 36%). Data on antibiotic use in the previous month and in the previous 6 mo were collected based on farmer self-report, using either recall or treatment records. Two metrics were used to quantify antibiotic consumption: animal-defined daily doses (ADD) and days of therapy (DOT), a metric used in human medicine for purposes of antimicrobial stewardship. Across all farms, 24,444 ADD and 19,029 DOT were reported, representing treatment incidences of 4.2 ADD/1,000 animal-days and 3.3 DOT/1,000 animal-days. These rates were generally lower than those found in other states and countries. The main indication for antibiotic use was mastitis, and first-generation cephalosporins were the most commonly used class of antibiotic for all indications, followed by penicillins and third-generation cephalosporins. Trends in use were similar for ADD and DOT, but the numbers of recorded DOT and associated treatment incidences were generally lower than the number of ADD and associated treatment incidences. Rates of treatment were significantly associated with herd size. This study is the first to quantify antibiotic use on dairy farms in Pennsylvania and the first to use the DOT metric in a dairy setting.

Identification of a novel metabolic-related mutation (IDH1) in metastatic pancreatic cancer.

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme implicated in cancer cell metabolic reprogramming. This is underscored by the detection of functional, somatic IDH1 mutations frequently found in secondary glioblastoma. To our knowledge, there has never been a reported, validated case of an IDH1 mutation in a pancreatic ductal adenocarcinoma (PDA). Herein, we present a case of a patient with metastatic PDA that harbored a potentially actionable, albeit rare, IDH1 mutation. As part of the Know Your Tumor project (Pancreatic Cancer Action Network), a 48-year-old female was diagnosed with metastatic PDA and subsequently started on standard of care chemotherapy, during which her hepatic lesions progressed. Detailed molecular profiling was performed on a biopsy from a liver lesion that demonstrated an IDH1 mutation, R132H. This mutation was confirmed by an independent sequencing reaction from the tumor sample, and by immunohistochemistry using an antibody specific for the IDH1 R132H mutation. The patient subsequently received a mutant IDH1 inhibitor (AG-120, Agios Pharmaceuticals, Cambridge, MA), but with no response. IDH1 mutations are common in certain cancer types, but have not been reported in PDA. We report the first case of an IDH1 mutation in this tumor type, perhaps providing a rare opportunity for a targeted therapy as a treatment option for PDA.

Erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations.

The objective of the current study was to investigate characteristics of Erysipelothrix spp. from slaughter condemnations. Specimens from 70 carcasses with lesions suspect for swine erysipelas were collected at an abattoir in Iowa from October 2007 to February 2009. Erysipelothrix spp. were isolated from 59 of 70 carcasses (84.3%). Abattoir inspectors classified lesions as acute, subacute, or chronic; 8 of 8 (100%) were acute cases, 31 of 32 (96.9%) were subacute cases, and 20 of 30 (66.6%) were chronic cases that were isolation positive. The following serotypes were identified: 1a (40.7%; 24/59), 2 (49.2%; 29/59), 7 (1/59), 10 (1/59), 11 (1/59), and untypeable (5.1%; 3/59). Serotypes 1a and 2 were identified in pigs with acute, subacute, or chronic clinical manifestations, whereas serotypes 7, 10, and 11 were only present in chronic cases. Fifty-seven of the 59 isolates were determined to belong to E. rhusiopathiae, and 2 of 59 of the isolates were determined to be E. tonsillarum by multiplex real-time polymerase chain reaction. Surface protective antigen (spa) A was detected in all E. rhusiopathiae isolates but not in E. tonsillarum serotypes 7 and 10. The results of the present study indicate that E. rhusiopathiae serotypes 1a and 2 continue to be commonly isolated from condemned pig carcasses and that spaA is the exclusive spa type in U.S. abattoir isolates. Interestingly, E. tonsillarum, thought to be avirulent for swine, was isolated from systemic sites from 3.4% of the carcasses that were negative for E. rhusiopathiae, indicating the potential importance of this genotype in erysipelas pathogenesis.

Development and validation of an immunohistochemical method for rapid diagnosis of swine erysipelas in formalin-fixed, paraffin-embedded tissue samples.

The objective of the study was to develop an immunohistochemical (IHC) assay for rapid detection of Erysipelothrix rhusiopathiae. Serotypes 1a, 1b, and 2 are most frequently associated with clinical disease in pigs. Antiserum against serotypes 1a, 1b, and 2 was produced in rabbits, pooled, and applied to formalin-fixed, paraffin-embedded tissue sections of pigs (lungs, heart, spleen, and skin). The results obtained with the IHC assay were compared with direct culture on tissue samples from experimentally inoculated pigs either treated (n = 6) with antibiotics or untreated (n = 8) as well as on samples from field cases (n = 170) submitted to the Veterinary Diagnostic Laboratory at Iowa State University. The agreement between direct culture and IHC staining was found to be substantial. The results of the present study indicate that the IHC assay is highly sensitive and specific in detecting E. rhusiopathiae antigen in formalin-fixed, paraffin-embedded tissues. Results indicated that the IHC is particularly useful in cases in which pigs had been treated with antibiotics prior to submission and in which direct cultures of organs were negative. In addition, the IHC was found to be useful for detection of E. rhusiopathiae antigen in skin lesions, which are often culture negative.

Comparison of conventional direct and enrichment culture methods for Erysipelothrix spp. from experimentally and naturally infected swine.

The objective of the current study was to compare the diagnostic performance of a direct isolation method for Erysipelothrix spp. with a broth-based enrichment technique. Samples were obtained from three sources: 1) experimentally inoculated pigs, 2) porcine tissue samples submitted to the Iowa State University Veterinary Diagnostic Laboratory (Ames, IA), and 3) tissues from condemned carcasses at an abattoir. Culture plates from direct isolation and broth-based technique were evaluated for growth at 24 and 48 hr. Results indicated that the broth enrichment method was markedly more sensitive for the isolation of Erysipelothrix spp. To the authors' knowledge, this is the first comparison of direct culture and broth-based enrichment methods for the isolation of Erysipelothrix spp. Interestingly, in several samples, a Gram-positive bacterium with almost identical growth characteristics to Erysipelothrix spp. was detected and identified as a Vagococcus sp. through 16S ribosomal RNA gene sequencing. The results of this study indicate that the broth-based enrichment method should be used for the isolation of Erysipelothrix spp. from clinical samples with a history suggestive of erysipelas and that Vagococcus spp. is potentially an important differential diagnosis.