Microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems.

The environmental influence of farm management in concentrated animal feeding operations (CAFO) can yield vast changes to the microbial biota and ecological structure of both the pig and waste manure lagoon wastewater. While some of these changes may not be negative, it is possible that CAFOs can enrich antibiotic resistant bacteria or pathogens based on farm type, thereby influencing the impact imparted by the land application of its respective wastewater. The purpose of this study was to measure the microbial constituents of swine-sow, -nursery, and -finisher farm manure lagoon wastewater and determine the changes induced by farm management. A total of 37 farms were visited in the Mid-South USA and analyzed for the genes 16S rRNA, spaQ (Salmonella spp.), Camp-16S (Campylobacter spp.), tetA, tetB, ermF, ermA, mecA, and intI using quantitative PCR. Additionally, 16S rRNA sequence libraries were created. Overall, it appeared that finisher farms were significantly different from nursery and sow farms in nearly all genes measured and in 16S rRNA clone libraries. Nearly all antibiotic resistance genes were detected in all farms. Interestingly, the mecA resistance gene (e.g. methicillin resistant Staphylococcus aureus) was below detection limits on most farms, and decreased as the pigs aged. Finisher farms generally had fewer antibiotic resistance genes, which corroborated previous phenotypic data; additionally, finisher farms produced a less diverse 16S rRNA sequence library. Comparisons of Camp-16S and spaQ GU (genomic unit) values to previous culture data demonstrated ratios from 10 to 10,000:1 depending on farm type, indicating viable but not cultivatable bacteria were dominant. The current study indicated that swine farm management schemes positively and negatively affect microbial and antibiotic resistant populations in CAFO wastewater which has future "downstream" implications from both an environmental and public health perspective.

Effects of bedding materials in applied poultry litter and immobilizing agents on runoff water, soil properties, and bermudagrass growth.

Poultry producers in the United States have begun using different types of bedding materials in production houses. Release into the environment of nutrients from applied poultry litter (PL) made with different bedding materials has not been investigated, and little information is available on nutrient concentrations in soils that receive broiler litter made with such materials. In this greenhouse study, two bedding materials (rice hulls and pine chips) in PL and two nutrient-immobilizing agents (gypsum and biochar) were applied to bermudagrass, and chemical and microbial contents of runoff water, soil properties, and plant growth were evaluated. Treatments with rice hull bedding material in PL had less runoff nutrient and greater soil soluble N and P compared with pine chip bedding. Gypsum and biochar both significantly reduced C, N, P, Cu, and Zn losses from the first runoff event, which were reduced by 26, 30, 37, 38, and 38% and by 25, 24, 30, 29, and 35%, respectively, but only gypsum obviously reduced these nutrients from later events. Potassium, Ca, Mg, and Mn increased by 2, 36, 11, and 9 times, respectively, and soluble P, Cu, and Fe significantly decreased by 68, 72, and 98%, respectively, in soil amended with gypsum. Rice hull PL in combination with gypsum significantly increased the growth of bermudagrass. Our results indicate that rice hull PL posed less risk for nutrient loss than pine chip PL when applied to fields and that gypsum was better than biochar for reducing runoff C, N, P, and Cu.

Land application of manure and Class B biosolids: an occupational and public quantitative microbial risk assessment.

Land application is a practical use of municipal Class B biosolids and manure that also promotes soil fertility and productivity. To date, no study exists comparing biosolids to manure microbial risks. This study used quantitative microbial risk assessment to estimate pathogen risks from occupational and public exposures during scenarios involving fomite, soil, crop, and aerosol exposures. Greatest one-time risks were from direct consumption of contaminated soil or exposure to fomites, with one-time risks greater than 10. Recent contamination and high exposures doses increased most risks. and enteric viruses provided the greatest single risks for most scenarios, particularly in the short term. All pathogen risks were decreased with time, 1 d to14 mo between land application and exposure; decreases in risk were typically over six orders of magnitude beyond 30 d. Nearly all risks were reduced to below 10 when using a 4-mo harvest delay for crop consumption. Occupational, more direct risks were greater than indirect public risks, which often occur after time and dilution have reduced pathogen loads to tolerable levels. Comparison of risks by pathogen group confirmed greater bacterial risks from manure, whereas viral risks were exclusive to biosolids. A direct comparison of the two residual types showed that biosolids use had greater risk because of the high infectivity of viruses, whereas the presence of environmentally recalcitrant pathogens such as and maintained manure risk. Direct comparisons of shared pathogens resulted in greater manure risks. Overall, it appears that in the short term, risks were high for both types of residuals, but given treatment, attenuation, and dilution, risks can be reduced to near-insignificant levels. That being said, limited data sets, dose exposures, site-specific inactivation rates, pathogen spikes, environmental change, regrowth, and wildlife will increase risk and uncertainty and remain areas poorly understood.

Temporal flux and spatial dynamics of nutrients, fecal indicators, and zoonotic pathogens in anaerobic swine manure lagoon water.

Confined animal feeding operations (CAFOs) often use anaerobic lagoons for manure treatment. In the USA, swine CAFO lagoon water is used for crop irrigation that is regulated by farm-specific nutrient management plans (NMPs). Implementation of stricter US environmental regulations in 2013 will set soil P limits; impacting land applications of manure and requiring revision of NMPs. Precise knowledge of lagoon water quality is needed for formulating NMPs, for understanding losses of N and C in ammonia and greenhouse gas emissions, and for understanding risks of environmental contamination by fecal bacteria, including zoonotic pathogens. In this study we determined year-round levels of nutrients and bacteria from swine CAFO lagoon water. Statistical analysis of data for pH, electrical conductivity (EC), inorganic and organic C, total N, water-soluble and total minerals (Ca, Cu, Fe, K, Mg, Mn, P, and Zn) and bacteria (Escherichia coli, enterococci, Clostridium perfringens, Campylobacter spp., Listeria spp., Salmonella spp., and staphylococci) showed that all differed significantly by dates of collection. During the irrigation season, levels of total N decreased by half and the N:P ratio changed from 9.7 to 2.8. Some seasonal differences were correlated with temperature. Total N and inorganic C increased below 19 °C, and decreased above 19 °C, consistent with summer increases in ammonia and greenhouse gas emissions. Water-soluble Cu, Fe, and Zn increased with higher summer temperatures while enterococci and zoonotic pathogens (Campylobacter, Listeria, and Salmonella) decreased. Although their populations changed seasonally, the zoonotic pathogens were present year-round. Increasing levels of E. coli were statistically correlated with increasing pH. Differences between depths were also found. Organic C, total nutrients (C, Ca, Cu, Fe, Mg, Mn, N, P, and Zn) and C. perfringens were higher in deeper samples, indicating stratification of these parameters. No statistical interactions were found between collection dates and depths.

Nutrients and bacteria in common contiguous Mississippi soils with and without broiler litter fertilization.

In Mississippi, spent poultry litter is used as fertilizer. Nutrient and bacterial levels in litter and nutrient levels in litter-fertilized (L+) soil are known, but less is known of bacterial levels in L+ soil. This study compared contiguous L+ and non-litter-fertilized (L-) soils comprising 15 soil types on five farms in April through May 2009. Levels of pH; NO-N; and Mehlich-3-extractable (M3) and water-extractable (WE) P, Ca, K, and Cu were higher in L+ than in L- soil. Total C; total N; NH-N; and M3 and WE Na, Fe, and Zn did not differ in L+ and L- soil. Bacterial levels were higher in 0- to 5-cm than in 5- to 10-cm cores. Levels were higher in L+ than in L- soil for culturally determined heterotrophic plate counts and staphylococci and were lower for total bacteria estimated by quantitative polymerase chain reaction (qPCR) of 16S rRNA, but cultural levels of thermotolerant coliforms, , , and enterococci were not different. Cultural presence/absence (CPA) tests and qPCR for spp., spp., and spp. detected only spp., which did not differ in L+ (CPA = 77% positive samples; mean qPCR = 0.65 log genomic units [gu] g) and L- (CPA = 70% positive samples; mean qPCR = 0 log gu g) soils. Litter applications were associated with higher levels of pH, P, Cu, heterotrophic plate counts, and staphylococci. Fecal indicator and enteric pathogen levels were not affected. We conclude that, although some litter-derived nutrients and bacteria persisted between growing seasons in L+ soils, enteric pathogens did not.

Comparison of selected nutrients and bacteria from common contiguous soils inside and outside swine lagoon effluent spray fields after long-term use.

Swine (Sus scofa domestica) lagoon effluent is a valuable resource. In the U.S. Mid-South it is applied from April to September to fertilize grass hay in spray-irrigated fields. Lagoon levels of nutrients and bacteria, and soil levels of nutrients have been documented, but little was known of effluent bacterial levels in soil. The present study examined levels of selected effluent bacteria and nutrients in soils inside and outside spray fields after >15 yr of effluent irrigation. Samples were collected February to March 2009 from contiguous soils spanning adjacent irrigated and nonirrigated areas. Separate soil cores for bacterial and nutrient tests were collected in pairs <10 cm apart. Five cores each were collected at 15-m intervals and combined, respectively, to comprise inside and outside samples from each of 20 soils (four each from five farms/spray fields). Analyses of data combined across all soils showed higher pH and Mehlich-3-extracrable (M3-) P, Mg, K, Na, Cu, and Zn inside than outside spray fields, while total N, total C, M3-Ca, and M3-Mn did not differ. Bacterial levels were higher inside than outside spray fields for heterotrophic plate counts, thermotolerant coliforms, Staphylococcus spp., and Clostridium perfringens, but levels of Escherichia coli and Enterococcus spp. were not different. Cultural presence/absence tests for three pathogens (Listeria spp., Campylobacter spp., and Salmonella spp.) detected only Listeria spp., which did not differ inside (23% positive samples) and outside (28% positive). Molecular tests detected all three pathogens at low levels that were not different inside and outside. We found no evidence of cumulative buildup of Campylobacter spp., Listeria spp., or Salmonela s. in spray field soils.

Characterization of selected nutrients and bacteria from anaerobic swine manure lagoons on sow, nursery, and finisher farms in the Mid-South USA.

Swine (Sus scrofa domestica) production in the Mid-South USA comprises sow, nursery, and finisher farms. A 2007 packing plant closure started a regional shift from finisher to sow and nursery farms. Changes in manure stored in lagoons and land-applied as fertilizer were expected but were unknown because nutrient and bacterial levels had not been characterized by farm type. The objectives of this study were to quantify selected nutrients and bacteria, compare levels by farm types, and project impacts of production shifts. Nutrients and bacteria were characterized in 17 sow, 10 nursery, and 10 finisher farm lagoons. Total and thermotolerant coliforms, Escherichia coli (Migula) Castellani and Chalmers, Enterococcus spp., Clostridium perfringens (Veillon and Zuber) Hauduroy et al., Campylobacter spp., Listeria spp., and Salmonella spp. were evaluated. Highest levels were from total coliforms (1.4- 5.7x10(5) cfu 100 mL(-1)), which occurred with E. coli, Campylobacter spp., C. perfringens, and Enterococcus spp., in every lagoon and virtually every sample. Lowest levels were from Listeria spp. and Salmonella spp. (

Rainfall simulation in greenhouse microcosms to assess bacterial-associated runoff from land-applied poultry litter.

Runoff water following a rain event is one possible source of environmental contamination after a manure application. This greenhouse study used a rainfall simulator to determine bacterial-associated runoff from troughs of common bermudagrass [Cynodon dactylon (L.) Pers.] that were treated with P-based, N-based, and N plus lime rates of poultry (Gallus gallus) litter, recommended inorganic fertilizer, and control. Total heterotrophic plate count (HPC) bacteria, total and thermotolerant coliforms, enterococci, staphylococci, Clostridium perfringens, Salmonella, and Campylobacter, as well as antibiotic resistance profiles for the staphylococci and enterococci isolates were all monitored in runoff waters. Analysis following five rainfall events indicated that staphylococci, enterococci, and clostridia levels were related to manure application rate. Runoff release of staphylococci, enterococci, and C. perfringens were approximately 3 to 6 log10 greater in litter vs. control treatment. In addition, traditional indicators such as thermotolerant and total coliforms performed poorly as fecal indicators. Some isolated enterococci demonstrated increased antibiotic resistance to polymixin b and/or select aminoglyocosides, while many staphylococci were susceptible to most antimicrobials tested. Results indicated poultry litter application can lead to microbial runoff following simulated rain events. Future studies should focus on the use of staphylococci, enterococci, and C. perfringens as indicators.

Recovery of Salmonella from bermudagrass exposed to simulated wastewater.

Most confined swine (Sus scrofa) feeding operations in the southeastern United States hold manure in lagoons and apply effluent on bermudagrass [Cynodon dactylon (L.) Pers.] as fertilizer. Salmonella enterica subsp. enterica (ex Kauffman and Edwards) Le Minor and Popoff, has been reported in Mississippi lagoons, but levels and potential for contamination of bermudagrass were unknown. A laboratory method was developed to examine Salmonella contamination of bermudagrass and levels of Salmonella were determined in lagoons. The U.S. Environmental Protection Agency (EPA) worst case water was used to simulate effluent in exposing bermudagrass to Salmonella. Exposed leaves were washed and bacteria enumerated. Contamination of leaves exposed to 10(6) cfu mL(-1) varied from 0 to 10(4) cfu per leaf within and among eight bermudagrass cultivars and five Salmonella isolates. No differences (P < 0.05) occurred between cultivars (n = 20) or isolates (n = 10). Data fitted (R2 = 0.93) to a contamination equation (y = 5 x 10(-6)X6.623) described the relationship between levels (Log10 cfu mL(-1)) of exposure (x) and contamination (y). In fall 2007 Salmonella levels from six lagoons ranged from 1.9 to 2.8 log10 MPN 100 mL(-1) and were below the threshold for contamination predicted by the equation. These preliminary results must be tested with effluents in the field, but considered alongside work of others, which report lagoon Salmonella levels to be highest in fall, suggest that Salmonella levels in effluents from these lagoons may be too low to produce measurable contamination on bermudagrass.

EPA worst case water microcosms for testing phage biocontrol of Salmonella.

A microplate method was developed as a tool to test phages for their ability to control Salmonella in aqueous environments. The method used EPA (U.S. Environmental Protection Agency) worst case water (WCW) in 96-well plates. The WCW provided a consistent and relatively simple defined turbid aqueous matrix, high in total organic carbon (TOC) and total dissolved salts (TDS), to simulate swine lagoon effluent, without the inconvenience of malodor and confounding effects from other biological factors. The WCW was originally defined to simulate high turbidity and organic matter in water for testing point-of-use filtration devices. Use of WCW to simulate lagoon effluent for phage testing is a new and innovative application of this matrix. Control of physical and chemical parameters (TOC, TDS, turbidity, temperature, and pH) allowed precise evaluation of microbiological parameters (Salmonella and phages). In a typical application, wells containing WCW were loaded with Salmonella enterica susp. enterica serovar Typhimurium (ATCC14028) and treated with phages alone and in cocktail combinations. Mean Salmonella inactivation rates (k, where the lower the value, the greater the inactivation) of phage treatments ranged from -0.32 to -1.60 versus -0.004 for Salmonella controls. Mean log(10) reductions (the lower the value, the greater the reduction) of Salmonella phage treatments were -1.60 for phage PR04-1, -2.14 for phage PR37-96, and -2.14 for both phages in a sequential cocktail, versus -0.08 for Salmonella controls. The WCW microcosm system was an effective tool for evaluating the biocontrol potential of Salmonella phages.

Characterization of Salmonella bacteriophages isolated from swine lagoon effluent.

Four Salmonella bacteriophages that had been originally isolated from swine manure lagoons were characterized and compared to each other and to well-known Salmonella phages P22 and Felix 01. Host ranges of the lagoon phages were similar to each other in spot tests on reference strains of Salmonella, but differed slightly from each other on a panel of Salmonella lagoon strains. In single-step growth at 35 degrees C the lagoon phages had latent periods of 15 to 20 min and burst sizes from 100 to 230. The lagoon phages and P22 were purified by cesium chloride (CsCl) gradient centrifugation and used to produce specific antisera and DNA. The lagoon phages were indistinguishable from each other but distinct from P22 and Felix 01 in immunodiffusion and infectivity neutralization tests and in restriction digest analysis.