Genetic Characteristics of Salmonella Isolates Recovered From Reused Broiler Litter Over Three Successive Flocks.

Salmonella infections are a leading cause of bacterial food-borne illness worldwide. Infections are highly associated with the consumption of contaminated food, and in particular, chicken meat. The severity of Salmonella infections depends on the presence of antimicrobial resistance genes and virulence factors. While there are many studies which have investigated Salmonella strains isolated from postharvest chicken samples, there is a gap in our understanding of the genetic properties that influence the persistence of Salmonella in preharvest and in particular their makeup of antimicrobial resistance genes and virulence factors. We used whole genome sequencing and hierarchical clustering to characterize and classify the genetic diversity of Salmonella enterica isolates (n = 55) recovered from the litter of commercial broiler chicken raised in four colocated broiler houses of one integrated farm over three consecutive flocks. The chicken were raised under a newly adopted "No Antibiotics Ever" program, and copper sulfate was administered via drinking water. In-silico serovar prediction identified three S. enterica serovars: Enteritidis (n = 12), Kentucky (n = 40), and Senftenberg (n = 3). Antimicrobial susceptibility testing revealed that only one S. Kentucky isolate was resistant to streptomycin, while the remaining isolates were susceptible to all antibiotics tested. Metal resistance operons, including copper and silver, were identified chromosomally and on plasmids in serovar Senftenberg and Kentucky isolates, respectively, while serovar Enteritidis carried several virulence factors on plasmids. Serovar Kentucky isolates harboring metal resistance operons were the only Salmonella isolates recovered from the litter of third flock cohort. These results suggest that there might be environmental selection for Salmonella strains carrying plasmid-associated metal resistance and virulence genes, which could play a role in their persistence in litter.

Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter.

IMPORTANCE: Campylobacter is a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat. Campylobacter is common in the microbiota of avian and mammalian gut; however, acquisition of antimicrobial resistance genes (ARGs) and virulence factors (VFs) may result in strains that pose significant threat to public health. Although there are studies investigating the genetic diversity of Campylobacter strains isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. Here, we show that Campylobacter jejuni and Campylobacter coli differ in their carriage of antimicrobial resistance and virulence factors may also differ in their ability to persist in litter during consecutive grow-out of broiler flocks. We found that presence/absence of virulence factors needed for evasion of host defense mechanisms and gut colonization played an integral role in differentiating Campylobacter strains.

Broiler house environment and litter management practices impose selective pressures on antimicrobial resistance genes and virulence factors of Campylobacter.

Campylobacter infections are a leading cause of bacterial diarrhea in humans globally. Infections are due to consumption of contaminated food products and are highly associated with chicken meat, with chickens being an important reservoir for Campylobacter. Here, we characterized the genetic diversity of Campylobacter species detected in broiler chicken litter over three consecutive flocks and determined their antimicrobial resistance and virulence factor profiles. Antimicrobial susceptibility testing and whole genome sequencing were performed on Campylobacter jejuni (n = 39) and Campylobacter coli (n = 5) isolates. All C. jejuni isolates were susceptible to all antibiotics tested while C. coli (n =4) were resistant to only tetracycline and harbored the tetracycline-resistant ribosomal protection protein (TetO). Virulence factors differed within and across grow houses but were explained by the isolates' flock cohort, species and multilocus sequence type. Virulence factors involved in the ability to invade and colonize host tissues and evade host defenses were absent from flock cohort 3 C. jejuni isolates as compared to flock 1 and 2 isolates. Our results show that virulence factors and antimicrobial resistance genes differed by the isolates' multilocus sequence type and by the flock cohort they were present in. These data suggest that the house environment and litter management practices performed imposed selective pressures on antimicrobial resistance genes and virulence factors. In particular, the absence of key virulence factors within the final flock cohort 3 isolates suggests litter reuse selected for Campylobacter strains that are less likely to colonize the chicken host.

Management and environmental factors influence the prevalence and abundance of food-borne pathogens and commensal bacteria in peanut hull-based broiler litter.

In this study, we conducted a longitudinal sampling of peanut hull-based litter from a farm under a "no antibiotics ever" program. Our objective was to determine broiler management practices and environmental factors that are associated with the occurrence of food-borne pathogens (Salmonella and Campylobacter) and the abundance of commensal bacteria (Escherichia coli, Enterococcus spp., and Staphylococcus spp.). Litter (n = 288) was collected from 4 broiler houses over three consecutive flocks, starting with a complete house cleanout and fresh peanut hull. Litter was sampled at the beginning of each grow-out cycle and at the end of the cycle. Logistic and linear regression models were used to model the relationships between pathogen prevalence, commensal abundance and management practices, and environmental factors. The number of flocks raised on litter, grow-out period, broiler house, litter pH, litter moisture, and house temperature were associated with the prevalence of pathogens and the abundance of commensal bacteria in litter. The final logistic model for pathogens showed that a higher probability of detecting Salmonella in litter was associated with the number of flocks raised on litter and the grow-out period. A higher probability of detecting Campylobacter in litter was associated with the number of flocks raised on litter, broiler house and the sections of the house, and the pH of litter. Our results suggest that management practices and environmental factors affect Salmonella and Campylobacter differently and suggest that each pathogen will require its own tailored intervention to stop their persistence in broiler litter.

Poultry manureshed management: Opportunities and challenges for a vertically integrated industry.

Manureshed management seeks to address systemic imbalances in nutrient distributions at scales beyond the farmgate and potentially across county and state boundaries. The U.S. poultry industry, which includes broilers, layers, pullets, and turkeys, has many characteristics that are compatible with achieving a vision of manureshed management, including a history of engaging in local and regional programs to better distribute manure resources. Despite widespread vertical integration that supports large-scale strategic decision making and dry manures that favor off-farm transport, there are still many challenges to poultry manureshed management that require engaging stakeholders other than just the poultry industry. Analysis of county-level nutrient budgets highlights the industry's "mega-manureshed," extending from the Mid-Atlantic, across the southeast, and into northwest Arkansas, Oklahoma, and Texas. The analysis also identifies areas with legacy nutrient build-up that are still present today. Implementing manureshed management in the U.S. poultry industry requires comprehensive consideration of manure treatment technologies, alternative uses such as bioenergy production, market development for treated manure products, transport of manure nutrients from source to sink areas, and manure brokering programs that promote manure nutrient distribution. Fortunately, past and present evolution and innovation within the industry places it as a likely leader of the manureshed vision.

Riparian land cover and hydrology influence stream dissolved organic matter composition in an agricultural watershed.

Dissolved organic matter (DOM) represents an essential component of the carbon cycle and controls biogeochemical and ecological processes in aquatic systems. The composition and reactivity of DOM are determined by the spatial distribution of its sources and its residence time in a watershed. While the effects of agricultural land cover on DOM quality have been reported across spatial and temporal scales, the influence of riparian land cover on stream DOM composition has received little attention. Furthermore, the combined effects of riparian land cover and streamflow rates on DOM composition require investigation. To this end, a multi-year (2016-2018) DOM characterization study was conducted using bi-weekly water samples collected from seven sub-watersheds nested within the Little River Experimental Watershed (LREW) near Tifton, Georgia, USA. DOM optical properties were determined to assess compositional variations using UV-Vis and excitation-emission matrix (EEM) fluorescence spectroscopy coupled with parallel factor (PARAFAC) analysis. PARAFAC analysis indicated that DOM in the LREW was dominated by three humic-like fluorescing components of terrestrial, microbial, and anthropogenic origin and a protein-like component. DOM composition was influenced by riparian land cover and hydrology, and shifted towards recently produced, low molecular weight DOM with low aromaticity as the percentage of agricultural land within riparian wetlands increased. During periods of high discharge and high baseflow, the DOM pool was dominated by recalcitrant and terrestrial-derived material but shifted towards protein-like and microbial-derived with increasing cropland in the riparian area. The results of this two-year study indicate that the replacement of forested riparian buffers with agricultural land can result in altered DOM composition which may affect carbon cycling and downstream water quality in agricultural watersheds.

Metals in Soil and Runoff from a Piedmont Hay Field Amended with Broiler Litter and Flue Gas Desulfurization Gypsum.

Flue gas desulfurization gypsum (FGDG) from coal-fired power plants is readily available for agricultural use in many US regions. Broiler litter (BL) provides plant available N, P, and K but can be a source of unwanted As, Cu, and Zn. As a source of Ca and S, FGDG can reduce losses of P and other elements in runoff from BL-amended areas. Rainfall simulation plots (2.0 m) were established on a Piedmont Cecil soil growing 'Coastal' bermudagrass ( L.) for hay. Accumulation and transport of As, Cu, Cd, Cr, Hg, Pb, and Zn were evaluated after annual BL applications (13.5 Mg ha) with four FGDG rates (0, 2.2, 4.5, 9.0 Mg ha) and two FGDG treatments (0 and 9 Mg ha) without BL. Runoff As concentrations were sixfold greater with BL than without ( ≤ 0.01) and were similar to BL with FGDG at 2.2, 4.5 or 9.0 Mg ha ( ≤ 0.10). Runoff concentrations of target elements did not increase where FGDG was applied alone. After three annual applications of FGDG and BL, soil concentrations of As, Cr, Pb, Hg, and Cu were well below levels of environmental concern. Our findings indicate that runoff losses of As from BL application are not reduced with FGDG but support other research indicating no identifiable environmental risks from FGDG beneficial use in agricultural systems.

Flue gas desulfurization gypsum: implication for runoff and nutrient losses associated with broiler litter use on pastures on ultisols.

Gypsum has been shown to reduce runoff on soils prone to crust formation in the southeastern United States. Increased infiltration from gypsum applications could therefore help reduce runoff P and other nutrient losses from application of broiler litter (BL), a nutrient-rich fertilizer. In rainfall simulation experiments in June 2009 and May 2011, runoff and nutrient (N, P, Ca, Mg) losses in runoff were compared among treatments consisting of 0, 2.2, 4.5, and 9.0 Mg ha flue gas desulfurization gypsum (FGDG) combined with 13.5 Mg ha of BL and two control treatments of (0-0) and (9.0-0.0) Mg ha (FGDG-BL). A randomized block design with three replications was set up on a Cecil (Typic Kanhapludult) soil growing Coastal bermudagrass ( L.) for hay near Watkinsville, Georgia. Amendments were applied each April from 2009 to 2011. A reduction in runoff of 30% each year from the (9.0-13.5) compared with the (0-0) treatment was not statistically significant. Gypsum was effective in reducing concentration and load in one of the two study years: P and NH-N in 2009 (up to 83%) and NO-N in 2011 (up to 73%). A combination of factors related to weather extremes, timing of FGDG and BL applications, and their implication on soil and vegetation responses at different landscape positions might have caused the different responses between years. Additional studies focused on isolating the impacts of such factors would be helpful to ascertain the effectiveness of multiyear applications of FGDG as a best management practice to reduce P and other nutrient losses in soils of the southeastern United States.

Survival dynamics of fecal bacteria in ponds in agricultural watersheds of the Piedmont and Coastal Plain of Georgia.

Animal agriculture in watersheds produces manure bacteria that may contaminate surface waters and put public health at risk. We measured fecal indicator bacteria (commensal Escherichia coli and fecal enterococci) and manure pathogens (Salmonella and E. coli 0157:H7), and physical-chemical parameters in pond inflow, within pond, pond outflow, and pond sediments in three ponds in agricultural watersheds. Bishop Pond with perennial inflow and outflow is located in the Piedmont, and Ponds A and C with ephemeral inflow and outflow in the Coastal Plain of Georgia. Bromide and chloride tracer experiments at Bishop Pond reflected a residence time much greater than that estimated by two models, and indicated that complete mixing within Bishop Pond was never obtained. The long residence time meant that fecal bacteria were exposed to solar UV-radiation and microbial predation. At Bishop Pond outflow concentrations of fecal indicator bacteria were significantly less than inflow concentrations; such was not observed at Ponds A and C. Both Salmonella and E. coli 0157:H7 were measured when concomitant concentrations of commensal E. coli were below the criterion for surface water impairment indicating problems with the effectiveness of indicator organisms. Bishop Pond improved down stream water quality; whereas, Ponds A and C with ephemeral inflow and outflow and possibly greater nutrient concentrations within the two ponds appeared to be less effective in improving down stream water quality.

Runoff water quality during drought in a zero-order georgia piedmont pasture: nitrogen and total organic carbon.

Approximately 11% of the Southern Piedmont (1.8 million ha) is used for pasture and hay production, mostly under low-input management. Few studies have investigated in the region long-term nitrogen and carbon losses in surface runoff, which can be significant. We present 1999 to 2009 hydrologic and water quality data from a rotationally grazed, 7.8-ha, zero-order pasture (W1) near Watkinsville in the Georgia Piedmont. Annual rainfall was 176 to 463 mm below the long-term average (1240 mm) in 7 of the 11 yr. There were 20 runoff events during 86 mo of below-average rainfall (deficit period), compared with 54 events during 46 mo of nondeficit period. Mean event flow-weighted concentration (in mg L) was 0.96 for nitrate-nitrogen (NO-N), 0.97 for ammonium-nitrogen (NH-N), 3.70 for total nitrogen (TN), and 9.12 for total organic carbon (TOC) ( = 43-47; limited due to instrument problem). Nutrient loads (in kg ha per event) averaged 0.04 for NO-N, 0.03 for NH-N, 0.19 for TN, and 0.54 for TOC. Total loads for N and TOC were 6 to 11 times greater from nondeficit than from deficit periods. The observed N concentrations, while well below maximum drinking water standard limits, could pose risk for eutrophication, which can be stimulated at lower concentrations. However, the ability of headwater streams, such as the one downstream of W1, to reduce nutrient concentrations might partially alleviate this concern. The results of this study point to the need to use a long-term dataset that includes measurements made in drought and wet years when evaluating the efficacy of water quality standards.

Comparative die-off of Escherichia coli O157:H7 and fecal indicator bacteria in pond water.

In situ and in vitro experiments were performed to assess the effects of solar radiation and predation by indigenous microflora on the relative die-off rates of a toxigenic strain of Escherichia coli O157:H7, commensal E. coli, and fecal enterococci in surface waters from ponds in agricultural watersheds. The objective of these experiments was to discern a mechanism of persistence of E. coli O157:H7 in surface waters compared to fecal indicator bacteria. Results of these experiments indicated that E. coli and fecal enterococci were affected by both insolation and apparent predation; whereas E. coli O157:H7 appeared to be resistant to both of these environmental stressors. The number of days to reach 99% die-off (T(99)-values) for E. coli O157:H7 was significantly greater than that for the indicator bacteria. The capacity to prolong die-off may be connected to the apparent persistence of E. coli O157:H7 in surface waters.

17beta-Estradiol and testosterone in drainage and runoff from poultry litter applications to tilled and no-till crop land under irrigation.

Thirteen million [corrected] metric tons of poultry litter are produced annually by poultry producers in the U.S. Poultry litter contains the sex hormones estradiol and testosterone, endocrine disruptors that have been detected in surface waters. The objective of this study was to evaluate the potential impact of poultry litter applications on estradiol and testosterone concentrations in subsurface drainage and surface runoff in irrigated crop land under no-till and conventional-till management. We conducted an irrigation study in fall of 2001 and spring of 2002. Four treatments, no-till plus poultry litter, conventional-till plus poultry litter, no-till plus conventional fertilizer, and conventional-till plus conventional fertilizer, were evaluated. Flow-weighted concentration and load ha(-1) of the two hormones were measured in drainage and runoff. Soil concentrations of estradiol and testosterone were measured. Based on comparisons to the conventional fertilizer (and control) treatments, poultry litter did not add to the flow-weighted concentration or load ha(-1) of either estradiol or testosterone in subsurface drainage or surface runoff. Significant differences were, however, observed between tillage treatments: flow-weighted concentrations of estradiol were greater for no-till than conventional-till plots of the June irrigation; and runoff loads of both estradiol and testosterone were less from no-till than conventional-till plots for the November irrigation. Although the differences between no-till and conventional-tillage appeared to affect the hydrologic transport of both hormones, the differences appeared to have inconsequential environmental impact.

Temporal assessment of the impact of exposure to cow feces in two watersheds by multiple host-specific PCR assays.

Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

Rainfall and tillage effects on transport of fecal bacteria and sex hormones 17beta-estradiol and testosterone from broiler litter applications to a Georgia Piedmont Ultisol.

Poultry litter provides nutrients for crop and pasture production; however, it also contains fecal bacteria, sex hormones (17beta-estradiol and testosterone) and antibiotic residues that may contaminate surface waters. Our objective was to quantify transport of fecal bacteria, estradiol, testosterone and antibiotic residues from a Cecil sandy loam managed since 1991 under no-till (NT) and conventional tillage (CT) to which either poultry litter (PL) or conventional fertilizer (CF) was applied based on the nitrogen needs of corn (Zea mays L) in the Southern Piedmont of NE Georgia. Simulated rainfall was applied for 60 min to 2 by 3-m field plots at a constant rate in 2004 and variable rate in 2005. Runoff was continuously measured and subsamples taken for determining flow-weighted concentrations of fecal bacteria, hormones, and antibiotic residues. Neither Salmonella, nor Campylobacter, nor antimicrobial residues were detected in litter, soil, or runoff. Differences in soil concentrations of fecal bacteria before and after rainfall simulations were observed only for Escherichia coli in the constant rainfall intensity experiment. Differences in flow-weighted concentrations were observed only for testosterone in both constant and variable intensity rainfall experiments, and were greatest for treatments that received poultry litter. Total loads of E. coli and fecal enterococci, were largest for both tillage treatments receiving poultry litter for the variable rainfall intensity. Load of testosterone was greatest for no-till plots receiving poultry litter under variable rainfall intensity. Poultry litter application rates commensurate for corn appeared to enhance only soil concentrations of E. coli, and runoff concentrations of testosterone above background levels.

Fecal bacteria and sex hormones in soil and runoff from cropped watersheds amended with poultry litter.

The application of poultry litter to agricultural fields can provide plant nutrients for crops and forage production, but fecal bacteria and the sex hormones estradiol and testosterone are components of litter that can be detrimental to the environment. Our objective was to determine if applications of poultry litter to small watersheds would contribute to the load of fecal bacteria and sex hormones to soil and runoff. We, therefore, investigated the fate and transport of fecal bacteria, estradiol and testosterone from surface applied poultry litter to four small watersheds. Poultry litter was applied to meet the nitrogen requirements of pearl millet (Pennisetum glaucum L.) in 2000 and grain sorghum [Sorgham bicolor (L.) Moench] in 2001. Neither Salmonella nor Campylobacter were detected in the litter but the fecal indicator bacteria were. The average load of total coliforms,Escherichia coli, and fecal enterococci applied with the litter was 12.2, 11.9, and 12.7 log10 cells ha(−1), respectively. The average load of estradiol and testosterone was 3.1 and 0.09 mg ha(-1), respectively.Runoff events first occurred seven months after the first litter application in 2000, and three weeks after the second application in 2001.Only for the 25 July 2001 runoff event three weeks after the second litter application, were the concentrations of total coliforms, E. coli,and fecal enterococci in runoff greater than background concentrations which were on average 5.2, 1.1, and 2.9 log10 MPN 100 ml(−1),respectively [corrected]. Average background levels of total coliforms, fecal enterococci,and E. coli in surface soil were 8.2, 7.9, and 3.5 log (10) cells kg(−1) soil. At the rate of litter application the concentrations of estradiol and testosterone in the litter did not appear to impact the background levels in the soil and runoff. Because concentrations of sex hormones in litter from other broiler operations are known to be greater than in the litter we applied, further study on the connection between concentrations of sex hormones in poultry litter and operational practices is recommended.