VTechWorks Repository :: Browsing by Author "Knowlton, Katharine F."

Browsing by Author "Knowlton, Katharine F."

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Abundance of Antibiotic Resistance Genes in Feces Following Prophylactic and Therapeutic Intramammary Antibiotic Infusion in Dairy Cattle
Willing, Brittany Faith (Virginia Tech, 2013-12-04)
Prophylactic and therapeutic antibiotic treatments have the potential to increase excretion of antibiotic resistance genes (ARGs) by dairy cattle through selection pressure on the gut microbiome. The objective of these studies was to evaluate the effect of cephapirin benzathine administered prophylactically at the end of lactation and pirlimycin hydrochloride administered therapeutically during a clinical mastitis infection on the abundance and relative abundance of ARGs in dairy cow feces. For prophylactic treatment using cephapirin benzathine, nineteen end-of-lactation cows were used. Treatment cows (n = 9) received cephapirin benzathine as an intramammary infusion prior to dry-off, and control cows (n =10) received no antibiotics. All cows received an internal non-antibiotic teat sealant. Fecal grab samples were collected for each cow on d -2 (baseline, used as covariate), d 1, 3, 5, 7, and once per week until d 49. Fecal samples were collected in sterile containers, then freeze-dried and subject to DNA extraction. The abundance of ampC, blaCMY-2, ermB, sul1, tetO, tetW, integrase-specific gene int1, and 16S rRNA were quantified using quantitative polymerase chain reaction (qPCR). The genes ampC and blaCMY-2 encode resistance to ß-lactam antibiotics, ermB to macrolides, sul1 to sulfonamides, tetO and tetW to tetracyclines, and int1 a class-1 integrase gene that facilitates horizontal transfer of ARGs across bacteria. The 16S rRNA gene was used as a representation of bacterial population. Absolute abundance was defined as number of ARG copies per gram of freeze-dried feces, while relative abundance was defined as ARG copy numbers per copy of 16S rRNA gene, which is indicative of the proportion of bacteria carrying ARGs. Non-normal data were logarithmically transformed and were statistically analyzed using PROC GLIMMIX in SAS 9.2. Abundance and relative abundance of sul1 and blaCMY-2 were below the limit of quantification in most samples and therefore not suitable for statistical comparisons. The int1 gene was not detectable in any sample. There were significant interactions between treatment and day for the abundance and relative abundance of ampC, tetO, and tetW. The abundance and relative abundance of ampC increased with time in control cows while remaining constant in antibiotic treated cows through the dry period. Antibiotics may act to stabilize the gut microbiome in response to diet and housing changes. There was a significant main effect of treatment for ermB with a significantly greater proportion of bacteria carrying ermB in control cows when compared to antibiotic treated cows. The tetracycline resistance genes tetO and tetW behaved similarly with a significant treatment by day interaction for the abundance and relative abundance of both genes. The relative abundance of both tetO and tetW were greater in control cows when compared to antibiotic treated cows on days 3, 5, 7, and 14. The abundance of both tetO and tetW resistance genes increased in antibiotic treated cows from day 1 to 49. There was also a significant increase in tetW relative abundance when comparing day 1 to 49. Administering long-acting antibiotics as intramammary dry treatment changed fecal bacteria composition during the dry period perhaps by stabilizing GI bacteria through dietary and housing changes. However, the use of prophylactic dry cow treatment does not uniformly or predictably lead to changes in fecal ARGs. In a second study, after clinical mastitis detection and identification, 6 lactating dairy cows received therapeutic mastitis treatment (pirlimycin hydrochloride as an intramammary infusion). Fecal grab samples were collected from each cow on d 0, 3, 9, and 12. Collection and analytical methods were as previously described. Abundance and relative abundance of sul1 and blaCMY-2 were again below the limit of quantification and therefore not suitable for statistical comparison. The int1 gene was not detected in any sample. The abundance of 16S rRNA genes decreased with day and relative abundance ermB, tetO, and tetW increased with day. There was no significant effect of day on the relative abundance of ampC or the abundance of ampC, ermB, tetO, and tetW in feces of cows with clinical mastitis. Administering fast-acting antibiotics as therapeutic intramammary mastitis treatment to dairy cows increased the relative abundance (gene copies per 16S rRNA) of selected ARGs but not the total abundance of ARGs in feces. The use of antibiotics for prevention and treatment of bacterial infections does not uniformly or predictably increase ARGs.
AgroSeek: a system for computational analysis of environmental metagenomic data and associated metadata
Liang, Xiao; Akers, Kyle; Keenum, Ishi M.; Wind, Lauren L.; Gupta, Suraj; Chen, Chaoqi; Aldaihani, Reem; Pruden, Amy; Zhang, Liqing; Knowlton, Katharine F.; Xia, Kang; Heath, Lenwood S. (2021-03-10)
Background Metagenomics is gaining attention as a powerful tool for identifying how agricultural management practices influence human and animal health, especially in terms of potential to contribute to the spread of antibiotic resistance. However, the ability to compare the distribution and prevalence of antibiotic resistance genes (ARGs) across multiple studies and environments is currently impossible without a complete re-analysis of published datasets. This challenge must be addressed for metagenomics to realize its potential for helping guide effective policy and practice measures relevant to agricultural ecosystems, for example, identifying critical control points for mitigating the spread of antibiotic resistance. Results Here we introduce AgroSeek, a centralized web-based system that provides computational tools for analysis and comparison of metagenomic data sets tailored specifically to researchers and other users in the agricultural sector interested in tracking and mitigating the spread of ARGs. AgroSeek draws from rich, user-provided metagenomic data and metadata to facilitate analysis, comparison, and prediction in a user-friendly fashion. Further, AgroSeek draws from publicly-contributed data sets to provide a point of comparison and context for data analysis. To incorporate metadata into our analysis and comparison procedures, we provide flexible metadata templates, including user-customized metadata attributes to facilitate data sharing, while maintaining the metadata in a comparable fashion for the broader user community and to support large-scale comparative and predictive analysis. Conclusion AgroSeek provides an easy-to-use tool for environmental metagenomic analysis and comparison, based on both gene annotations and associated metadata, with this initial demonstration focusing on control of antibiotic resistance in agricultural ecosystems. Agroseek creates a space for metagenomic data sharing and collaboration to assist policy makers, stakeholders, and the public in decision-making. AgroSeek is publicly-available at https://agroseek.cs.vt.edu/ .
Ammonia Emissions and Animal Agriculture
Gay, Susan Wood; Knowlton, Katharine F. (Virginia Cooperative Extension, 2005-09-01)
Provides an overview of ammonia production associated with animal agriculture and explains why it is receiving greater attention from those concerned with environmental quality.
Ammonia Emissions from Dairy Manure Storage Tanks Affected by Diets and Manure Removal Practices
Li, Lifeng (Virginia Tech, 2009-08-07)
The objectives of this study were to determine: 1) ammonia emission rates from stored scraped and flushed manure from dairy cows fed either normal or low N diet; and 2) seasonal effects on ammonia emission rates from stored scraped and flushed dairy manure. Four pilot-scale tanks were used for manure storage with different treatments - scraped manure for normal diet (NS), flushed manure for normal diet (NF), scraped manure for low N diet (LS), and flushed manure for low N diet (LF). The first part of the study lasted for 1 month and four treatments were all investigated; the second part of the study lasted for 12 months and two tanks with treatments NS and NF were investigated. Dynamic flux chambers and a photoacoustic gas analyzer were used to measure ammonia emission rates. There was no significant change of the N content of manure as the dietary N content is reduced (from 17.8% to 15.9% crude protein). However, ammonia emission rates from manure storage tanks were reduced by 33% (from 27.4 ± 38.1 to 18.4 ± 21.9 mg m⁻²h⁻¹; P<0.0001 based on paired t-test). Flushing manure reduced emission rates by 72% compared to scraping manure (from 35.6 ± 39.6 to 10.1 ± 8.2 mg m⁻²h⁻¹; P<0.0001 based on paired t-test). Ammonia emission rates for NS, NF, LS and LF were 43.9 ± 48.0, 10.9 ± 8.7, 27.4 ± 27.3, and 9.3 ± 7.8 mg m-2 h-1, respectively. The chamber headspace temperature for NS, NF, LS and LF were 26.0 ± 6.9, 25.8 ± 6.8, 26.6 ± 6.5, and 27.2 ± 6.7 °C, respectively. The manure pH for NS, NF, LS, and LF were 6.3 ± 0.1, 6.4 ± 0.3, 6.4 ± 0.1, and 6.1 ± 0.1, respectively. Both dietary N reduction and manure flushing are recommended to reduce ammonia emission rates from dairy manure storage tanks. Ammonia emission rates were higher in summer and fall, due to higher air temperature and higher manure pH. The pH of scraped manure was 7.2 ± 0.6, 6.7 ± 0.2, 6.5 ± 0.3 and 7.0 ± 0.3 for fall, winter, spring and summer, respectively. The pH of flushed manure was 6.8 ± 0.4, 6.7 ± 0.4, 6.4 ± 0.3 and 6.8 ± 0.4 for fall, winter, spring and summer, respectively. Ammonia emission rates from scraped manure for fall, winter, spring, and summer were 7.4 ± 8.6, -0.5 ± 1.2, 1.1 ± 1.9, and 5.8 ± 2.7 mg m⁻²h⁻¹, respectively. Ammonia emission rates from flushed manure for fall, winter, spring, and summer were 3.9 ± 4.2, -0.5 ± 0.9, 0.8 ± 1.4, and 4.4 ± 1.2 mg m⁻²h⁻¹, respectively. Seasonal changes of air temperature and manure pH were key factors affecting ammonia emissions from manure storage in this study. Seasonal climate conditions including precipitations (rainstorms and snows) and icing can cause reduction of ammonia emissions from manure storage in open air. More attention should be paid to reduce ammonia emissions in warmer seasons, e.g., by covering the storage facilities.
Antibiotic resistance gene abundance in feces of calves fed pirlimycin-dosed whole milk
Littier, Heather Melissa (Virginia Tech, 2015-08-31)
Exposure to antibiotics has the potential to increase the incidence and proliferation of antibiotic resistance genes (ARG) in the gut and fecal microbiome. Non-saleable, antibiotic-containing milk from cows treated with antibiotics (waste milk) is commonly fed to dairy calves but the effects of ingestion of antibiotics at an early age on the gut microbiome and the development of ARG in the naive gut are not well understood. Pirlimycin, a lincosamide antibiotic acting against Gram positive bacteria through inhibiting protein synthesis by binding to the 50S ribosome, is commonly used as mastitis therapy. Lincosamides are also considered highly important in human medicine, often used against Staphylococcus aureus and Clostridium difficile infections. Emerging microbial resistance to pirlimycin is of concern for both animal and human health. The objective of this study was to determine the effect of early lincosamide antibiotic exposure on the abundance of ARG in feces of milk-fed calves. Eight female Holstein calves were blocked by age, paired by block, and randomly assigned to pasteurized whole milk (control; n = 4) or milk containing 0.2 mg/L of pirlimycin (treatment; n = 4). Calves were enrolled after receiving two colostrum feedings and were fed 5.68 L of pasteurized whole milk, treatment, or control, divided into two daily feedings, from d 1 to d 50 of age. After weaning calves were fed non-medicated starter grain ad libitum. Fecal samples were collected weekly until 85 d of age and freeze-dried. DNA was extracted using QiaAmp® Fast DNA Stool Mini Kit and qPCR was used to quantify the absolute abundance (gene copies/g of wet feces) and relative abundance (gene copies/copies of 16S rRNA genes) of erm(B), tet(O), tet(W) and 16S rRNA genes. Data was analyzed using PROC GLIMMIX in SAS. Abundance of 16S rRNA genes, tet(O) and tet(W) were not different between control and pirlimycin-fed calves nor were the relative abundance of tet(O) (mean = 0.050 tet(O) copies/16S rRNA genes) or tet(W) (0.561 tet(W) copies/16S rRNA genes). While abundance of erm(B) was higher in pirlimycin-fed calves compared to control calves (6.46 and 6.04 log gene copies/g wet feces; P = 0.04) the relative abundance of erm(B) (0.273 gene copies/16S rRNA genes) in feces of calves was not influenced by treatment. There was an effect of day (P < 0.10) for absolute abundance of tet(O), tet(W), and erm(B) indicating that the levels change with time as the fecal microbiome develops. This study suggests that feeding pirlimycin-containing non-saleable milk to growing calves may increase environmental loading of erm(B), which codes for resistance to highly important macrolide and lincosamide antibiotics. Additional research is needed on effects of feeding waste milk to calves on other fecal ARG and on the post-excretion and post-application fate of these genes.
Antibiotic resistance genes in the faeces of dairy cows following short-term therapeutic and prophylactic antibiotic administration
Feng, Xin; Chambers, Lindsey R.; Knowlton, Katharine F. (2019-12-06)
The objective of the research was to quantify three antibiotic resistance genes (tetQ, cfxA and mefA) in the faeces of dairy cows following therapeutic and prophylactic antibiotic treatments. Manure collected from dairy cows treated with either no antibiotic, pirlimycin hydrochloride (PIRL), ceftiofur crystalline free acid (CCFA) or cephapirin benzathine (CEPH) were submitted to quantitative PCR analysis. No treatment effects on the abundance of the tetQ and cfxA were observed. There was a trend for the abundance of the mefA to be increased in cows treated with PIRL (P = 0.07). Overall, the results showed no difference of measured three ARGs from cows receiving different antibiotics. Considering the limited scope of our investigation, further investigation is needed to provide more information on ARGs excretion from cows that received therapeutic and prophylactic antibiotic treatment.
Antimicrobial Resistance Mitigation [ARM] Concept Paper
Vikesland, Peter J.; Alexander, Kathleen A.; Badgley, Brian D.; Krometis, Leigh-Anne H.; Knowlton, Katharine F.; Gohlke, Julia M.; Hall, Ralph P.; Hawley, Dana M.; Heath, Lenwood S.; Hession, W. Cully; Hull, Robert Bruce IV; Moeltner, Klaus; Ponder, Monica A.; Pruden, Amy; Schoenholtz, Stephen H.; Wu, Xiaowei; Xia, Kang; Zhang, Liqing (Virginia Tech, 2017-05-15)
The development of viable solutions to the global threat of antimicrobial resistance requires a transdisciplinary approach that simultaneously considers the clinical, biological, social, economic, and environmental drivers responsible for this emerging threat. The vision of the Antimicrobial Resistance Mitigation (ARM) group is to build upon and leverage the present strengths of Virginia Tech in ARM research and education using a multifaceted systems approach. Such a framework will empower our group to recognize the interconnectedness and interdependent nature of this threat and enable the delineation, development, and testing of resilient approaches for its mitigation. We seek to develop innovative and sustainable approaches that radically advance detection, characterization, and prevention of antimicrobial resistance emergence and dissemination in human-dominated and natural settings...
ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization
Liang, Xiao; Zhang, Jingyi; Kim, Yoonjin; Ho, Josh; Liu, Kevin; Keenum, Ishi M.; Gupta, Suraj; Davis, Benjamin; Hepp, Shannon L.; Zhang, Liqing; Xia, Kang; Knowlton, Katharine F.; Liao, Jingqiu; Vikesland, Peter J.; Pruden, Amy; Heath, Lenwood S. (Frontiers, 2023-09-15)
Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.
Battling Resistance: Judicious Antibiotic Use in Beef and Dairy Cattle
Gleason, Claire B.; Maurer, Ellen; Knowlton, Katharine F.; Vallotton, Amber (Virginia Cooperative Extension, 2017-11-06)
Discusses the use of antibiotics in the dairy and beef cattle industry, and the increasing drug-resistance of many strains of bacteria.
Biochemical Lignin Related Processes in Landfills
Irani, Ayesha (Virginia Tech, 2005-12-16)
The objective of this study was to determine how the key features of bioreactor landfills; increased temperature, moisture and microbial activity, affect the biological stability of the landfill material. In the first part of the study the solubilization and degradation of lignin in paper exposed to these bioreactor landfill conditions are explored. The solubility of the lignin in paper was observed at different temperatures and over 27 weeks at 55°C and the anaerobic bioconversion of office paper, cardboard and Kraft lignin was observed in bench-scale reactors over 8 weeks. As the temperature rose, lignin solubility increased exponentially. With extended thermal treatment, the dissolution of lignin continues at a constant rate. This rate increases 15 times for paper and 1.5 times for cardboard in the presence of rumen inoculum compared to un-inoculated systems. At around 6 weeks the inter-monomeric linkages between the solubilized lignin molecules began breaking down, releasing monomers. In cardboard and Kraft lignin, a significant amount of the monomers mineralize to CO₂ and CH₄ during this time period. The results indicate that small, but significant rates of lignin solubilization and anaerobic lignin degradation are likely to occur in bioreactor landfills due to both higher temperature and microbial activity. In the second part of the study, field data from the Outer Loop Recycling and Disposal Facility in Louisville, Kentucky was evaluated to determine the effectiveness of an anaerobic-aerobic landfill bioreactor (AALB) vs. the control landfill that is managed as a traditional landfill. Moisture, temperature, elevation and the amount of time the MSW has spent in the landfills (age) were measured and compared to determine the factors that affect the biological stability of the landfill. The results showed that the MSW in the AALB is more biologically stable than the MSW in the control landfill, indicating that they are more degraded. Additionally, elevation or location of the MSW was the key factor in determining the extent of MSW stability within the AALB and temperature is the key factor in determining the biological stability of the MSW in the control landfill. Higher temperatures correlated with a more biologically stable waste. The cellulose to lignin ratio (C/L ratio) and biochemical methane potential (BMP) were the main biological stability parameters used.
Calcium and Phosphorus Metabolism in Jersey and Holstein Cows During Early Lactation
Taylor, Megan Sands (Virginia Tech, 2007-09-11)
The objective of this dissertation was to assess the dynamics of calcium (Ca) and phosphorus (P) metabolism in dairy cattle. Hypocalcemia, or a drop in blood Ca, is a common condition near parturition. All cows experience some degree of hypocalcemia. Maintenance of blood Ca within the acceptable range of 8 to 10 mg/dl is a balancing act between the demand for Ca for milk production and the cow's homeostatic mechanisms to maintain blood Ca. These homeostatic mechanisms include bone resorption that is driven by Ca demand however both Ca and P are released when bone is resorbed. These times of bone resorption and bone mineral replenishment have not been accounted for in current mineral recommendations. For the first study, it was postulated that dairy producers could administer 25-hydroxyvitamin D₃ (25-OH) in the prepartum period to prevent hypocalcemia. Twenty-seven multiparous Jersey cows were randomly assigned to receive an oral bolus containing corn starch (control, CON) or corn starch plus 15 mg of 25-hydroxyvitamin D₃ (25-OH) or 15 mg of vitamin D₃ (D₃) at 6 d prior to expected parturition. Jugular blood samples were collected at -14, -13, -5, -4, -3, -2, -1 d prior to expected calving, on the day of calving, and 1, 3, 5, 7, 9, 11, 13, 28, 56, and 84 d with respect to calving. Samples were analyzed for 25-OH, Ca, P, magnesium, osteocalcin (OC), and parathyroid hormone (PTH). Blood Ca, P, and Mg decreased near the time of calving and then increased over time. Serum 25-hydroxyvitamin D₃ was higher for cows dosed with 25-OH (119.0 pg/ml) compared with those dosed with D₃ (77.5 pg/ml) or CON (69.3 pg/ml). Cows dosed with 25-OH tended to have lower serum PTH concentration, but treatments did not affect serum Ca, P, or Mg. Serum OC was higher in second lactation cows compared with cows entering their third or fourth lactation but OC was unaffected by treatment. Although results indicated a 60% increase in serum 25-OH due to a single oral dose of 25-OH prior to calving, the amount administered in this study apparently was not sufficient for initiation of any improvement in Ca homeostasis at parturition. Due to the intimate relationship of Ca and P in bone, it was postulated for the second study that dietary Ca would affect bone mobilization and Ca and P balance in the lactating dairy cow. Eighteen Holstein cows were blocked by parity and calving date and randomly assigned to one of three dietary treatments: high (1.03%, HI), medium (0.78%, MED), or low (0.52%, LOW) dietary Ca. Dietary P was 0.34% in all diets. Total collection of milk, urine, and feces was conducted 2 wk prior to calving and in wk 2, 5, 8, 11, and 20 of lactation. Blood samples were collected at -14 and -10 d prior to calving and 0, 1, 3, 5, 10, 14, 21, 28, 35, 56, 70, 84, 98, and 140 d after calving. Blood samples were analyzed for Ca, P, PTH, OC, and deoxypyridinoline (DPD). Rib bone biopsies were conducted within 10 d of calving and during wk 11 and 20 of lactation. Dietary Ca concentration affected Ca balance, with cows consuming the HI Ca diet in positive Ca balance for all weeks with the exception of wk 11. Interestingly, all cows across all treatments had a negative Ca balance at wk 11, possibly the result of timed estrous synchronization that occurred during wk 11. At wk 20, Ca balances were 61.2, 29.9, and 8.1 g/d for the HI, MED, and LOW diets, respectively. Phosphorus balances across all treatments and weeks were negative. Dietary Ca concentration did not affect P balance in the weeks examined for this study but there was a clear effect of parity on balance, markers of bone metabolism, and bone P. Regardless of dietary treatment, serum OC concentration peaked around d 35 of lactation. Simultaneously, DPD concentration began to decrease, which may indicate a switch from net bone resorption to net bone formation after day 35. This was not reflected in balance measures however, this information may help refine dietary mineral recommendations for lactating dairy cows and ultimately reduce P excretion into the environment. Ultimately from the first study it is clear that oral dosing with 25-OH at 6 d prior to expected calving is not justified. However, we learned that parity has an effect on bone formation with younger animals resorbing and forming more bone and that net formation appears to occur after 30 days in milk. Both of these points were corroborated in the second study. Additionally, the second study demonstrated that dietary Ca content has no effect on P balance from 2 to 20 wk of lactation. Finally, the rib bone does not appear to be a sensitive indicator of bone metabolism or at least not at the time points we measured.
Combined effects of composting and antibiotic administration on cattle manure–borne antibiotic resistance genes
Keenum, Ishi M.; Williams, Robert K.; Ray, Partha P.; Garner, Emily; Knowlton, Katharine F.; Pruden, Amy (2021-04-01)
Background Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days. Results Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition. Conclusions This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.
Compatibility, Yield, and Quality of Matua Prairie Grass, Bromus willdenowii (Kunth), with Legumes
Guay, Jennifer Fincham (Virginia Tech, 2003-12-08)
Matua prairie grass has a potential to extend the grazing season in Virginia due to its higher early spring and fall production. However, little is known about the compatibility of Matua prairie grass with legumes or the effects of legumes on the yield and quality of Matua prairie grass/legume mixtures. An experiment was conducted in 1998 and 1999 to investigate the botanical composition, yield, and chemical composition of Matua prairie grass grown with legumes. Legume treatments consisting of ladino clover (Trifolium repens), red clover (Trifolium pratense), alfalfa (Medicago sativa), and annual lespedeza (Lespedeza stipulacea) were drilled into a Matua prairie grass stand. Nitrogen was applied once each fall at two treatment levels of 0 or 84 kg/ha. The experiment was arranged in a randomized split block design with four replications. Legume treatments had no effect on percentage Matua prairie grass or total dry matter yield in 1998. However, in 1999 the ladino clover and red clover treatments increased (P<0.05) total dry matter yield, but also resulted in a substantial decrease (P<0.05) in percentage Matua prairie grass. Nitrogen application in the fall of 1998 had a residual effect (P<0.05) on the percentage Matua prairie grass and yield in 1999. The highest response to nitrogen fertilization occurred in the harvest immediately after fertilization, in October of 1999, which resulted in the largest increase (P<0.05) in percentage Matua prairie grass and yield, and the greatest decrease (P<0.05) in percentage legumes. The legume and nitrogen treatments similarly influenced the chemical composition of the Matua prairie grass/legume mixed forage. Ladino clover, red clover, and alfalfa treatments generally improved forage quality as indicated by a decrease (P<0.05) in NDF, ADF, hemicellulose, and cellulose, and an increase (P<0.05) in CP and IVDMD. Nitrogen fertilization did not influence the chemical composition of the forages to the same extent as the legume treatments, as a decrease in fiber components and an increase in CP and IVDMD were observed due to nitrogen. Overall, alfalfa appeared to be most compatible with Matua prairie grass, and the incorporation of alfalfa into a Matua prairie grass stand resulted in some improvements in total dry matter yield and nutritive value of the forage, without the detrimental suppression of Matua prairie grass.
Conservation of Nitrogen via Nitrification and Chemical Phosphorus Removal for Liquid Dairy Manure
DeBusk, Jo (Virginia Tech, 2007-11-27)
The objectives of this study were to (1) determine an intermittent aeration strategy that could be used to conserve nitrogen (N) via nitrification in dairy manure, (2) determine the effect of recycled flush water on the bio-availability of N during nitrification, and (3) determine effective and economical dosages of chemicals to remove phosphorus (P) from liquid dairy manure. Intermittent aeration strategies, defined in terms of time the aerator is on and off (ON h:OFF h), could be used to conserve N in dairy manure. Testing of four treatments (continuous aeration [100%], 1h:0.33h [75%], 1h:0.67h [60%], and 1h:1h [50%]) showed that only treatments using air provided for 100% and 75% of the time could support nitrification. The 100% and 75% aeration treatments conserved an average of 38% and 25% of influent total ammonia nitrogen (TAN) as nitrite-N+nitrate-N, respectively. Less than 2% of influent TAN was conserved using 60% and 50% treatments. The effect of manure handling technique on N bioavailability and nitrification was tested using flushed and scraped dairy manure. Nitrification was inhibited in scraped manure. Four aluminum- and iron-based salts and five cationic polyacrylamide polymers were evaluated for P removal using jar tests. Ferric chloride (FeCl3·6H2O), aluminum sulfate (Al2[SO4]3·13H2O, alum), and Superfloc 4512 were selected for further study. Polymer addition enhanced floc size and improved P removal. Treatment of manure (0.89% total solids) from Tank 2 at Virginia Tech's dairy using either FeCl3 or alum in combination with polymer resulted in more than 90% P removal. Chemical treatment and transport of P-rich sludge from a 2,270 cubic meter storage tank would result in an estimated 40% cost savings over transport of the entire manure volume offsite for land application elsewhere. The manure treatment strategies tested provide some solutions to dairy farmers regarding adjustment of N:P ratios so that manure can be applied to meet nutrient needs of crops while adhering to regulations set forth by nutrient management plans.
Development and Comparison of 17beta-Estradiol Sorption Isotherms for Three Agriculturally Productive Soils From Different Physiographic Regions in Virginia
Kozarek, Jessica Lindberg (Virginia Tech, 2005-08-10)
Natural steroid estrogens such as 17beta-estradiol in low nanogram per liter concentrations can adversely affect the reproductive health of aquatic organisms. The overall goal of this research was to quantify the sorption of 17beta-estradiol to three soils considered to be agriculturally productive from different physiographic regions in Virginia to aid in modeling the concentration of estrogens available for transport in runoff from agricultural fields. Batch equilibrium experiments were conducted with various concentrations of 17beta-estradiol (E2) in a background solution of 5 mM calcium chloride and 100 mg/L sodium azide added to four separate soil samples representative of productive agricultural soils from three different physiographic regions of Virginia. Groseclose loam, Myatt sandy loam and Cecil loam were supplied by the Crop and Soil Environmental Sciences Department at Virginia Tech. All soils were collected from the plow layer (0 to 15 cm) except for an additional Cecil soil sample from the Bt horizon. The concentration of E2 in the liquid phase was measured by gas chromatography/mass spectrometry (GC/MS) and was used to find the time to reach equilibrium and to develop sorption isotherms for each soil. The time required to reach equilibrium for all soils was less than 24 hours. A linear isotherm provided the best fit to model the sorption of E2 to Cecil and Myatt soils (R2 = 0.94 and 0.96, respectively). For Groseclose soil, the general form of the Freundlich isotherm fit best (R2 = 0.98), although the linear isotherm also provided a good fit (R2 = 0.93). The sorption of E2 to agricultural soil appears to be related to the organic carbon content of each soil (Pearson coefficient, 0.82). Attempts to analyze and create isotherms for conjugated E2 by deconjugating with metholysis were unsuccessful.
Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion
Ray, Partha; Knowlton, Katharine F.; Shang, Chao; Xia, Kang (PLOS, 2014-11-06)
Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg−1 and 0.96 µg L−1 for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg−1) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L−1). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.
Dietary phosphorus and water quality
Myers, Kelly; Knowlton, Katharine F.; Jones, Gerald M. (Virginia Cooperative Extension, 2000)
There is a direct relationship between phosphorus levels in dairy cattle rations and the amount of phosphorus excreted in manure. Phosphorus accumulates in surface water and can pollute ponds, streams and lakes.
Dietary phosphorus effects on characteristics of mechanically separated dairy manure
Knowlton, Katharine F.; Love, Nancy G.; Parsons, C. A. (American Society of Agricultural and Biological Engineers, 2005)
One approach to reduce nutrient losses from livestock farms is to apply biological waste treatment systems such as biological nitrogen (N) removal or enhanced biological phosphorus (P) removal (EBPR) to reduce the nutrient content of land-applied waste. The EBPR process takes advantage of the ability of P-accumulating organisms (PAOs) to sequester excess P as polyphosphate granules in their cytoplasms, yielding a P-depleted liquid effluent and a P-enriched biomass. Biological N removal systems result in the conversion of organic or ammonia-N to innocuous N-2 gas. Understanding the variation in parameters such as chemical oxygen demand (COD), total and volatile suspended solids (TSS and VSS), and ammonia-N (NH3-N) is necessary to design these systems. Our objectives were to evaluate the effects of diet and manure separation on parameters important to reactor design. Waste was collected from nine cows fed a high P diet (0.47% P), a low P diet (0.32 % P), or low P with exogenous phytase plus cellulase (0.32 % P), in a replicated Latin square design (three 3 X 3 squares). Total collection of milk, urine, and feces was conducted on days 19 to 21 of each period, a mixed slurry (urine, feces, and water) was created, and slurry was separated mechanically to generate liquid effluent. Slurry contained more COD, solids, N, and P than liquid effluent, but the COD:P ratio was similar in the two wastes. The ratio of COD:N was higher in slurry than in separator effluent, but the ratio in both wastes was sufficient to support biological N removal. The P content of slurry, liquid effluent, and manure solids from cows fed low P was lower than from cows fed high P, and the COD content of effluent was higher with the low P diet. The COD:P ratio of all wastes was sufficient to support EBPR and biological N removal, but variation was observed with diet. Waste from cows fed low P had a higher COD:P ratio than that of cows fed high P, and waste from cows fed the enzyme-supplemented diet had a lower COD:N ration than that of cows fed the control diet. Dairy manure slurry and effluent will support EBPR and biological N removal. Dietary effects on parameters important to the design of advanced waste treatment systems were observed, but were not of a magnitude that would affect reactor design.
Digestion of inositol phosphates by dairy cows: Method development and application
Ray, Partha Pratim (Virginia Tech, 2012-05-02)
Successful implementation of dietary P management strategies demand improved understanding of P digestion dynamics in ruminants and this is not possible without a reliable and accurate phytate (Pp) quantification method. The objective of the first study was to develop a robust, accurate, and sensitive method to extract and quantify phytate in feeds, ruminant digesta and feces. Clean-up procedures were developed for acid and alkaline extracts of feed, ruminant digesta and feces and clarified extracts were analyzed for Pp using high performance ion chromatography (HPIC). The quantified Pp in acid and alkaline extracts was comparable for feed but alkaline extraction yielded greater estimates of Pp content for digesta and feces than did acid extraction. Extract clean-up procedures successfully removed sample matrix interferences making alkaline extraction compatible with HPIC. The developed method was applied to investigate the disappearance of Pp from the large intestine of dairy heifers. Eight ruminally- and ileally-cannulated crossbred dairy heifers were used and each heifer was infused ileally with 0, 5, 15, or 25 g/d Pp and total fecal collection was conducted. On average 15% of total Pp entering the large intestine was degraded but the amount of infused Pp did not influence the degradability of Pp. Net absorption of P from the large intestine was observed. A feeding trial was conducted to investigate the effect of dietary Pp supply on ruminal and post-ruminal Pp digestion. Six ruminally-and ileally-cannulated crossbred lactating cows were used and dietary treatments were low (0.10% Pp), medium (0.18% Pp), and high (0.29% Pp) Pp, and a high inorganic P (Pi; 0.11% Pp; same total P content as high Pp). Ruminal Pp digestibility increased linearly with dietary Pp. As in the infusion study, net disappearance of Pp from the large intestine was only 16% of total Pp entering the large intestine and not influenced by dietary Pp. Fecal P excretion increased linearly with increasing dietary Pp but was not affected by form of dietary P. In lactating cows Pp digestibility was not affected by dietary Pp and fecal P excretion was regulated by total dietary P rather than by form of dietary P.
Effect of composting and soil type on dissipation of veterinary antibiotics in land-applied manures
Chen, Chaoqi; Ray, Partha P.; Knowlton, Katharine F.; Pruden, Amy; Xia, Kang (2018)
The objective of this study was to determine the fate of commonly used veterinary antibiotics in their naturally excreted form when manure-based amendments are applied to soil. Beef cattle were administered sulfamethazine, tylosin, and chlortetracycline and dairy cows were treated with pirlimycin. The resulting manure was composted for 42 d under static or turned conditions and applied at agronomic N rates to sandy, silt, and silty clay loam soils and compared with amendment with corresponding raw manures in sacrificial microcosms over a 120-day period. Antibiotic dissipation in the raw manure-amended soils followed bi-phasic first order kinetics. The first phase half-lives for sulfamethazine, tylosin, chlortetracycline, and pirlimycin ranged from 6.0 to 18, 2.7 to 3.7, 23 to 25, and 5.5–8.2 d, respectively. During the second phase, dissipation of sulfamethazine was negligible, while the half-lives for tylosin, chlortetracycline, and pirlimycin ranged from 41 to 44, 75 to 144, and 87–142 d, respectively. By contrast, antibiotic dissipation in the compost-amended soils followed single-phase first order kinetics with negligible dissipation of sulfamethazine and half-lives of tylosin and chlortetracycline ranging from 15 to 16 and 49–104 d, respectively. Pirlimycin was below the detection limit in the compost-amended soils. After incubating 120 d, antibiotics in compost-amended soils (up to 3.1 μg kg−1) were significantly lower than in manure-amended soils (up to 19 μg kg−1, p < .0001), with no major effect of soil type on the dissipation. Risk assessment suggested that composting can reduce antibiotic resistance selection potential in manure-amended soils.

Browsing by Author "Knowlton, Katharine F."

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