Combination of a urease inhibitor and a plant essential oil to control coliform bacteria, odour production and ammonia loss from cattle waste

AIM: To evaluate urea hydrolysis, volatile fatty acid (VFA) production (odour) and coliforms in cattle waste slurries after a urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and a plant oil component (thymol) were added. METHODS AND RESULTS: Faeces from cattle fed a diet of 70% corn silage and 30% alfalfa haylage, urine and distilled water in the ratio 50 : 35 : 15 were blended at high speed for 1 min. Triplicate aliquots of 750 ml were amended with NBPT plus or minus thymol and reblended for 1 min, and were poured into 1.6 l wide-mouth jars covered 90% with a lid. After 56 days, thymol (2000 mg kg(-1) waste) in combination with NBPT (80 mg kg(-1) waste) retained 5.2 g of an initial 9.2 g of urea in cattle waste slurries, compared with less than 1 g of urea retained when NBPT was the only additive (P < 0.05). Another experiment using excreta from cattle fed 76.25% high moisture corn, 19.25% corn silage and a 4.5% supplement, blended at a low speed, gave a similar response with urea hydrolysis; and the two treatments, thymol alone and thymol in combination with NBPT, reduced VFA production (P < 0.01) and eliminated all coliform bacteria by day 1. A third experiment indicated coliforms disappeared in the no addition treatment after 8 days; however, they were viable at 6.6 x 10(4) CFU g(-1) waste beyond 35 days in the NBPT treatment. CONCLUSIONS: Thymol supplements the effect of NBPT by increasing the inhibitory period for hydrolysis of urea in cattle waste slurries and nitrogen retention in the waste. SIGNIFICANCE AND IMPACT OF THE STUDY: Thymol and NBPT offer the potential to reduce odour and pathogens in cattle manure, and increase the fertilizer value.     

Salmonella enterica Serovar Typhimurium and Escherichia coli Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P ≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20°C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of S. enterica serovar Typhimurium and E. coli in manure-fertilized soil. The number of indigenous E. coli in soil was never significantly lower (P < 0.05) than that of S. enterica serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne S. enterica serovar Typhimurium.     

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium,Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10⁶ colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10⁶ CFU/ml for 96–120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms. Received: 2 October 2001 / Accepted: 31 December 2001     

Effects of Cattle Feeding Regimen and Soil Management Type on the Fate of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Manure, Manure-Amended Soil, and Lettuce

Survival of the green fluorescent protein-transformed human pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was studied in a laboratory-simulated lettuce production chain. Dairy cows were fed three different roughage types: high-digestible grass silage plus maize silage (6:4), low-digestible grass silage, and straw. Each was adjusted with supplemental concentrates to high and low crude protein levels. The pathogens were added to manure, which was subsequently mixed (after 56 and 28 days for E. coli O157:H7 and Salmonella serovar Typhimurium, respectively) with two pairs of organically and conventionally managed loamy and sandy soil. After another 14 days, iceberg lettuce seedlings were planted and then checked for pathogens after 21 days of growth. Survival data were fitted to a logistic decline function (exponential for E. coli O157:H7 in soil). Roughage type significantly influenced the rate of decline of E. coli O157:H7 in manure, with the fastest decline in manure from the pure straw diet and the slowest in manure from the diet of grass silage plus maize silage. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although decline was significantly faster in the manure derived from straw than in the manure from the diet of grass silage plus maize silage. The pH and fiber content of the manure were significant explanatory factors and were positively correlated with the rate of decline. With E. coli O157:H7 there was a trend of faster decline in organic than in conventional soils. No pathogens were detected in the edible lettuce parts. The results indicate that cattle diet and soil management are important factors with respect to the survival of human pathogens in the environment.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa. Methods and Results: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g^?1 in manure and manure‐amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro‐Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high‐density inocula and enhanced the decline of low‐density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure‐amended soil than in manure. The 7 log CFU g^?1E. coli O157:H7 and Salm. Typhimurium survived for 49–84 and 63–98 days, while at 4 log CFU g^?1, persistence was 21–28 and 35–42 days, respectively. Conclusions: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. Significance and Impact of the Study: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.     

Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10⁻⁸ ml/min and 1.91 × 10⁻⁸ ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10⁻² CFU/ml for S. Typhimurium and 4.58 × 10⁻⁵ CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.     

Effects of the Dicarboxylic Acids Malate and Fumarate on E. coli O157:H7 and Salmonellaenterica Typhimurium Populations in Pure Culture and in Mixed Ruminal Microorganism Fermentations

The dicarboxylic acids malate and fumarate increase ruminal pH, reduce methane production, increase propionate and total volatile fatty acid (VFA) production, and reduce lactic acid accumulation in a manner similar to ionophores. These acids stimulate the ruminal bacterium Selenomonas ruminantium to ferment lactate to produce propionate. Thus, dicarboxylic acids have been suggested as nonantibiotic modifiers of the ruminal fermentation, but their impact on ruminal microbial ecology remains unknown. This study was designed to examine what effects these modifiers may have on intestinal pathogen populations such as Escherichia coli O157:H7 and S. enterica Typhimurium prior to their widespread incorporation into cattle rations. Pure cultures of E. coli O157:H7 strain 933 and S. enterica Typhimurium were grown with malate and fumarate added at 0, 1, 5, 10, and 20 mM (v/v; n = 3). Neither dicarboxylic acid inhibited (p > 0.1) the growth rate or final populations of E. coli O157:H7 or S. enterica Typhimurium. Ruminal fluid was collected from cattle (n = 2) and E. coli O157:H7 and S. enterica Typhimurium were added to separate ruminal fermentations incubated for 24 h at 39°C. Fumarate and malate were added at concentrations of 0, 5, 10, and 20 mM (v/v; n = 2) and incubated for 24 h at 39°C. Malate or fumarate addition did not affect (p > 0.1) populations of E. coli O157:H7 or S. enterica Typhimurium. However, the final pH was increased (p < 0.05), the acetate:propionate ratio was decreased (p < 0.05), and the total VFA production was increased (p < 0.05) by ≥10 mM dicarboxylic acid addition. These results confirm that dicarboxylic acids can modify ruminal fermentation, but they do not affect populations of critical foodborne pathogens. Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Kinetic model‐based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions

Aim: Present a kinetic model‐based approach for using isothermal data to predict the survival of manure‐borne enteric bacteria under dynamic conditions in an agricultural environment. Methods and Results: A model to predict the survival of Salmonella enterica serovar Typhimurium under dynamic temperature conditions in soil in the field was developed. The working hypothesis was that the inactivation phenomena associated with the survival kinetics of an organism in an agricultural matrix under dynamic temperature conditions is for a large part due to the cumulative effect of inactivation at various temperatures within the continuum registered in the matrix in the field. The modelling approach followed included (i) the recording of the temperature profile that the organism experiences in the field matrix, (ii) modelling the survival kinetics under isothermal conditions at a range of temperatures that were registered in the matrix in the field; and (iii) using the isothermal‐based kinetic models to develop models for predicting survival under dynamic conditions. The time needed for 7 log CFU g^?1Salmonella Typhimurium in manure and manure‐amended soil to reach the detection limit of the enumeration method (2 log CFU g^?1) under tropical conditions in the Central Agro‐Ecological Zone of Uganda was predicted to be 61–68 days and corresponded with observed CFU of about 2·2–3·0 log CFU g^?1, respectively. The Bias and Accuracy factor of the prediction was 0·71–0·84 and 1·2–1·4, respectively. Conclusions: Survival of Salm. Typhimurium under dynamic field conditions could be for 71–84% determined by the developed modelling approach, hence substantiating the working hypothesis. Significance and Impact of the Study: Survival kinetic models obtained under isothermal conditions can be used to develop models for predicting the persistence of manure‐borne enteric bacteria under dynamic field conditions in an agricultural environment.     

Effect of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">n</Emphasis>-butyl) thiophosphoric triamide on urea metabolism and the assimilation of ammonium by <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study was to perform an in-depth analysis of the effect that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting 4 weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A non-fertilized control was also cultivated. Several parameters related with N metabolism were analysed at the end of growth period. NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.     

Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 10⁹ CFU g⁻¹ on A. thaliana roots and to 2 × 10⁷ CFU g⁻¹ on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.     

Simultaneous Near-Infrared Radiant Heating and UV Radiation for Inactivating Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Powdered Red Pepper (Capsicum annuum L.)

This study was conducted to investigate the efficacy of the simultaneous application of near-infrared (NIR) heating and UV irradiation for reducing populations of food-borne pathogens, including Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 in red pepper powder and to clarify the mechanisms of the lethal effect of the NIR-UV combined treatment. Also, the effect of the combination treatment on quality was determined by measuring changes in color and pungency constituents. Simultaneous NIR-UV combined treatment for 5 min achieved 3.34- and 2.78-log CFU reductions in S. Typhimurium and E. coli O157:H7, respectively, which involved 1.86- and 1.31-log CFU reductions, respectively, which were attributed to the synergistic effect. Through qualitative and quantitative analyses, damage to the cell envelope was identified as the main factor contributing to the synergistic lethal effect of NIR-UV combined treatment. Color values and capsaicin and dihydrocapsaicin content of NIR-UV simultaneously treated red pepper powder were not significantly (P > 0.05) different from those of untreated samples. These results suggest that simultaneous application of NIR and UV treatment can be effectively used to control food-borne pathogens in powdered red pepper without affecting quality.     

Use of urease inhibitors to reduce ammonia loss following application of urea to flooded rice fields

Ammonia (NH₃) volatilization is an important mechanism for nitrogen (N) loss from flooded rice fields following the application of urea into the floodwater. One method of reducing losses is to use a urease inhibitor that retards the hydrolysis of urea by soil urease and allows the urea to diffuse deeper into the soil. The two chemicals that have shown most promise are phenylphosphorodiamidate [PPD] and N(n-butyl)thiophosphorictriamide [NBPT], but they seldom work effectively. PPD decomposes rapidly when the pH departs from neutrality, and NBPT must be converted to the oxygen analogue for it to be effective. Our field studies in Thailand show that the activity of PPD can be prolonged, and NH₃ loss markedly reduced, by controlling the floodwater pH with the algicide terbutryn. A mixture of NBPT and PPD in the presence of terbutryn was even more effective than PPD alone. It appears that during the time when the PPD was effective, NBPT was being converted to the oxygen analogue. The combined urease inhibitor-algicide treatment reduced NH₃ loss from 10 to 0.4 kg N ha^-1.     

Phage cocktail strategies for the suppression of a pathogen in a cross-feeding coculture

Cocktail combinations of bacteria-infecting viruses (bacteriophages) can suppress pathogenic bacterial growth. However, predicting how phage cocktails influence microbial communities with complex ecological interactions, specifically cross-feeding interactions in which bacteria exchange nutrients, remains challenging. Here, we used experiments and mathematical simulations to determine how to best suppress a model pathogen, E. coli, when obligately cross-feeding with S. enterica. We tested whether the duration of pathogen suppression caused by a two-lytic phage cocktail was maximized when both phages targeted E. coli, or when one phage targeted E. coli and the other its cross-feeding partner, S. enterica. Experimentally, we observed that cocktails targeting both cross-feeders suppressed E. coli growth longer than cocktails targeting only E. coli. Two non-mutually exclusive mechanisms could explain these results: (i) we found that treatment with two E. coli phage led to the evolution of a mucoid phenotype that provided cross-resistance against both phages, and (ii) S. enterica set the growth rate of the coculture, and therefore, targeting S. enterica had a stronger effect on pathogen suppression. Simulations suggested that cross-resistance and the relative growth rates of cross-feeders modulated the duration of E. coli suppression. More broadly, we describe a novel bacteriophage cocktail strategy for pathogens that cross-feed.     

Analysis of the ArcA regulon in anaerobically grown <Emphasis Type="Italic">Salmonella enterica sv</Emphasis>. Typhimurium

Background  

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that must successfully adapt to the broad fluctuations in the concentration of dissolved dioxygen encountered in the host. In Escherichia coli, ArcA (Aerobic Respiratory Control) helps the cells to sense and respond to the presence of dioxygen. The global role of ArcA in E. coli is well characterized; however, little is known about its role in anaerobically grown S. Typhimurium.     

Waterborne Pathogen Detection by Use of Oligonucleotide-Based Microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10⁴ S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture

A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genes sipB and sipC of Salmonella and an intragenic segment of gene eae of E. coli O157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.     

A Determination and Comparison of Urease Activity in Feces and Fresh Manure from Pig and Cattle in Relation to Ammonia Production and pH Changes

Ammonia emission from animal production is a major environmental problem and has impacts on the animal health and working environment inside production houses. Ammonia is formed in manure by the enzymatic degradation of urinary urea and catalyzed by urease that is present in feces. We have determined and compared the urease activity in feces and manure (a urine and feces mixture) from pigs and cattle at 25°C by using Michaelis-Menten kinetics. To obtain accurate estimates of kinetic parameters V_max and K''_m, we used a 5 min reaction time to determine the initial reaction velocities based on total ammoniacal nitrogen (TAN) concentrations. The resulting V_max value (mmol urea hydrolyzed per kg wet feces per min) was 2.06±0.08 mmol urea/kg/min and 0.80±0.04 mmol urea/kg/min for pig feces and cattle feces, respectively. The K''_m values were 32.59±5.65 mmol urea/l and 15.43±2.94 mmol urea/l for pig feces and cattle feces, respectively. Thus, our results reveal that both the V_max and K''_m values of the urease activity for pig feces are more than 2-fold higher than those for cattle feces. The difference in urea hydrolysis rates between animal species is even more significant in fresh manure. The initial velocities of TAN formation are 1.53 mM/min and 0.33 mM/min for pig and cattle manure, respectively. Furthermore, our investigation shows that the maximum urease activity for pig feces occurs at approximately pH 7, and in cattle feces it is closer to pH 8, indicating that the predominant fecal ureolytic bacteria species differ between animal species. We believe that our study contributes to a better understanding of the urea hydrolysis process in manure and provides a basis for more accurate and animal-specific prediction models for urea hydrolysis rates and ammonia concentration in manures and thus can be used to predict ammonia volatilization rates from animal production.     

Leaching of bioluminescent Escherichia coli O157:H7 from sheep and cattle faeces during simulated rainstorm events

Aims: Development of a novel inoculation technique to improve the current methods of determining the leaching of Escherichia coli O157:H7 from faeces. Methods and Results: Ruminant faeces were inoculated with a high [c. 10⁷ colony forming units (CFU) g⁻¹] or low (c. 10⁴ CFU g⁻¹) load of a lux-marked strain of E. coli O157:H7 via injection, and subjected to four simulated heavy rainfall events. The population density and metabolic activity of E. coli O157:H7 recovered within the leachate was determined following each simulated rain event and compared with the indigenous E. coli population. The concentration of E. coli O157:H7 in the leachates followed a similar trend to that of nonpathogenic E. coli. Significantly greater densities of generic and pathogenic E. coli were recovered in the leachates generated from sheep faeces compared with cattle faeces. Pathogen metabolic activity was also significantly greater in sheep faeces. Conclusions: Our findings show that E. coli O157:H7 may readily leach from ruminant faeces during rain events. The bacterium leaches more freely from sheep faeces than from cattle faeces and displays greater metabolic activity within sheep leachate. Significance and Impact of the Study: A novel inoculation technique was developed that allowed the determination of both population density and cellular activity of E. coli O157:H7 in leachate derived from faeces.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.