This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.
Agricola
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2005, p. 6165-6174, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6165-6174.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Eelco Franz,* Anne D. van Diepeningen, Oscar J. de Vos, and Ariena H. C. van Bruggen

Biological Farming Systems Group, Department of Plant Sciences, Wageningen University and Research Center, Marijkeweg 22, 6709 PG Wageningen, The Netherlands

Received 25 February 2005/ Accepted 24 May 2005


arrow
ABSTRACT
 
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.


arrow
INTRODUCTION
 
Agricultural animals are widely recognized as reservoirs of human enteric pathogens (31, 44). These pathogens are shed in their feces, which in turn could serve as the primary source for contamination of various food products. Most cases of human infection by these pathogens have been linked primarily to the consumption of animal food products. However, various pathogens have been recovered from vegetables (3), and the number of documented disease cases associated with the consumption of raw vegetables has increased in recent years (40, 44). Outbreaks of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium have been associated with the consumption of lettuce (17, 19). Both human enteric pathogens have a principal reservoir in cattle (9, 53).

One possible mechanism of vegetable contamination with these pathogens is the land application of manure as fertilizer (33). The conditions for survival of enteric human pathogens are generally considered to be unfavorable once they are excreted from the animal (46). Possible contamination of vegetables grown in soil enriched with manure will largely depend on the survival capabilities of the pathogen in manure, in soil, and in or on plants. Differences in animal feeding regimens and the absence of synthetic fertilizers, pesticides, and routine use of antibiotics may lead to differences in pathogen prevalence and survival between organic and conventional farming systems. Because animal manure is the major source of fertilization in organic crop production, microbial safety is at the center of attention for organic vegetable production (1). However, it has not been demonstrated that the risk of contamination of fresh vegetables is higher with organic than with conventional production (29).

Diet composition, abrupt changes in diet, or fasting may influence the shedding of E. coli O157:H7 (43). There has been considerable debate concerning the effect of hay feeding versus grain feeding on the shedding and acid resistance of E. coli O157:H7. Grain feeding can create a more acidic environment in the guts of cattle, which leads to the selection for acid-resistant generic E. coli, which may include the considerably acid-resistant E. coli O157:H7 (11, 38). This dietary effect on shedding of E. coli O157:H7 is supported by some epidemiological data (16, 37), but other results point in another direction (39). The hypothesis has also been supported (45) or challenged (15, 20, 25, 48) by experiments conducted with ruminants inoculated with E. coli O157:H7. Besides affecting the shedding of pathogens, the cattle feeding regimen can be expected to affect manure composition and might thereby also affect pathogen survival capabilities in manure.

In bovine manure, E. coli O157:H7 is documented to survive for extended periods of time (5, 26, 28, 55). Salmonella serovar Typhimurium also is capable of survival for considerable periods of time in manure (18) and slurries (18, 24). Survival of excreted pathogens in freshly produced manure will be affected by the manure management system used on the farm: manure is handled as a slurry or as solid manure, applied to fields after a range of storage times, and applied by surface spreading or injection into the soil (31). So far, the potential influence of cattle diet on pathogen survival in manure has not been the subject of research. In manure-amended soil, reported survival times of E. coli O157:H7 vary considerably, from several weeks (32) to several months (5, 21, 23, 28). Long-term survival has also been demonstrated for Salmonella serovar Typhimurium (22, 30).

E. coli O157:H7 and Salmonella may be transferred from manure-amended soil or manure compost-amended soil to leaf and root vegetables and can persist for long periods of time on these vegetables (21, 22, 30). Recently it has been shown that E. coli O157:H7 can become internalized in lettuce by entering the plant through the root system from a planting mixture of manure and soil and can migrate throughout the edible part of the plant (41). Because of the lack of chemical treatments for controlling pathogen invasion in lettuce production, suppression of pathogens must rely solely on the antagonistic capacity of the resident microflora in the different ecological niches. Functional and taxonomic diversity and biomass of soil microbial and faunal communities are frequently higher in organic than in conventional fields and have been correlated with a higher suppression of soilborne plant pathogens (50, 51).

At present there is insufficient information about the influence of cattle diet and manure characteristics on the survival of human pathogens in manure. It is also not known whether organically and conventionally managed soils differ in the capability to suppress human pathogens. Moreover, the possible internalization of human pathogens in the edible parts of leafy vegetables grown in manure-amended soil is scarcely documented. Previous studies on pathogen survival in the agricultural environment focused primarily on single parts of the lettuce production chain, such as manure, soil, or manure-amended soil with crops. In the present study, we simulated the lettuce production chain in the laboratory and monitored the fate of E. coli O157:H7 and Salmonella serovar Typhimurium in three subsequent niches: manure, manure-amended soil, and plant. The objectives of the present study were to determine pathogen survival as a function of cattle diet, soil type, and soil management (organic or conventional). Furthermore, the possibility of (internal) contamination of lettuce after a period of pathogen survival in manure and manure-amended soil was investigated.


arrow
MATERIALS AND METHODS
 
Bacteria.
Escherichia coli O157:H7 strain B6-914 gfp-91 was kindly provided by Pina Fratamico (13). This strain does not produce Shiga-like toxins I and II (Stx1– Stx2–) but contains the pGFP cDNA vector (Clontech Laboratories, Inc., Palo Alto, CA) expressing green fluorescent protein (GFP) and ampicillin resistance. The survival characteristics of the GFP-labeled strain were indistinguishable from those of the wild-type strain (13). In addition, Kudva et al. (26) reported no differences in survival in bovine manure and manure slurry between toxin-positive (Stx1+ Stx2+) and toxin-negative (Stx1– Stx2–) E. coli O157:H7. Two phenotypes of Salmonella enterica serovar Typhimurium, MAE 110 (PagfD1 rdar; aggregate phenotype) and MAE 119 ({Delta}agfD101 saw; wild-type morphology), were kindly provided by Ute Römling (35, 36). These strains were derived from strains MAE 51 and MAE 52, respectively, and both carry kanamycin and gentamicin resistance and the GFP gene on the chromosome after transformation with the PAG408 minitransposon (42). The two strains can be distinguished by their appearance under UV light. The colony appearance of MAE 110 is larger, flatter, more ragged, and less bright than that of MAE 119. Bacteria were stored at –80°C and were checked for viability prior to use.

Cattle feeding and manure collection.
Manure was obtained from an ongoing experiment on the effect of diet on manure quality by the Department of Animal Science of the Wageningen University and Research Center, The Netherlands (J. W. Reijs, personal communication). Dairy cows (Holstein Frisian, 3 to 7 years of age) were housed in one stable under identical conditions. Six pairs (n = 2) of animals were fed six different diets for nearly 9 weeks (from 20 January 2003 until 21 March 2003): high-digestible grass silage (60%) plus maize silage (40%) (GM), low-digestible grass silage (GO), and straw (S), each adjusted with supplemental concentrates to high (H) and low (L) crude protein (CP) levels (Table 1). Fresh manure (without urine) was collected directly from the pairs of cows (with equal amounts of manure from each individual well mixed in a bucket) at the end of the feeding trial (after 9 weeks) and stored at 5°C in 20-liter containers.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Description of the six types of diet fed to dairy cows in an experimental, controlled setup (J. W. Reijs, personal communication)

Soils.
An organically managed sandy soil and a conventionally managed sandy soil, cropped to potatoes, were collected from two neighboring farms in Marknesse (Flevoland, The Netherlands). Organic and conventional loamy soils, cropped to onions, were collected from two neighboring farms in Ens (Flevoland, The Netherlands). Both organic farms were accredited by Skal (the inspection body for organic production in The Netherlands) and thus refrained from the use of artificial fertilizers or pesticides. However, both the organic and conventional farmers used animal manures as fertilizer. Throughout each field, 15 soil samples (1 to 20 cm deep) were collected between the plants with an augur and mixed. Samples were transported in plastic bags to the lab, stored at 5°C, and sieved (4 mm) before use.

Inoculation of manure.
A simulation of the transitions in the lettuce production chain from manure to soil and plants was done separately for E. coli O157:H7 and for a mixture of both Salmonella serovar Typhimurium phenotypes. The inoculum was prepared in Luria-Bertani broth with 50 µg/ml ampicillin for E. coli O157:H7 B6-914 gfp-91 and 50 µg/ml kanamycin for the Salmonella serovar Typhimurium phenotypes. Both phenotypes were grown separately and mixed to equal amounts before inoculation of the manure. Cells were harvested by centrifugation at 3,000 x g (Hermle 2384 K) and washed with and resuspended in 0.1% peptone buffer (Oxoid) to a density of 1 x 109 CFU per milliliter. This cell density was determined spectrophotometrically, taking into account that an optical density at 630 nm of 1 would equal approximately 0.7 x 109 CFU ml–1. The dry weight of the manure was determined by drying overnight at 105°C. Cells were added to a final density of 1 x 107 CFU per gram manure dry weight (gdw–1). For Salmonella serovar Typhimurium a mixture of 0.5 x 107 CFU MAE 110 gdw–1 and 0.5 x 107 CFU MAE 119 gdw–1 was added to manure. After mixing by thoroughly kneading the manure in a plastic bag from the outside by hand, 500 g of the inoculated manure was transferred to a preweighed plastic pot (1 liter), which was closed but had the ability of gas exchange. There were three replicate pots per manure type and the same number of noninoculated pots, which functioned as blanks, with 0.1% peptone buffer added instead of bacterial suspension. The pots were weighed and incubated at 10°C in darkness. At each sampling time, pots were weighed before and after sampling to check for evaporation. The moisture content remained constant (on average around 85%) during the experiment. In addition, at each sampling time, manure samples from the blanks were dried overnight at 105°C to determine their dry weight.

Plate counts of E. coli O157:H7 and Salmonella serovar Typhimurium.
The inoculated pots were sampled over time to determine the survival of the pathogens in manure (at time zero and after 3, 8, 16, 22, 28, 43, 56, 84, and 133 days). At each sampling time, two samples of approximately 1 g of each replica were removed from the middle of each mixture with a sterile spoon and put in separate preweighed dilution tubes with 4.5 ml of 0.1% peptone. Sampling holes were closed. Sample-containing tubes were weighed to determine the exact size of the sample. Samples were vortexed and put in a ultrasonic bath for 30 s (Branson 5200; 120-W output power, 47 kHz). The samples were vortexed again, and 10-fold serial dilutions were made. From the two highest dilutions, 50 µl was plated in duplicate on petri dishes with sorbitol-MacConkey (Oxoid) agar with ampicillin (50 µg/ml) for the enumeration of E. coli O157:H7 or on Luria-Bertani medium with kanamycin (50 µg/ml) for the enumeration of Salmonella serovar Typhimurium. The number of necessary dilutions was estimated based on preliminary counts. This resulted in two plates per dilution, four plates per sample, and thus eight plates per replica. When low cell numbers were expected, 16 or 32 plates per sample were used to increase the detection limit. Cell suspensions were spread on the surface by shaking with 2-mm sterile glass beads. The inoculated plates were sealed with Parafilm and incubated at 37°C for 24 h. Numbers of E. coli O157:H7 and of Salmonella serovar Typhimurium were determined by counting green fluorescent CFU with a dark-blue lamp (Philips PL-S 9W/08 Blacklight Blue, peak at 365-nm UV-A). Colony shape and GFP intensity enabled distinction between Salmonella serovar Typhimurium phenotypes 110 and 119. Colony counts were calculated as number of CFU gdw–1.

Transmission to and survival in soil.
To determine survival in manure-amended soil, a subset of 60 g of fresh weight (gfw) of manure was mixed with 540 gfw of each of the four soils (1:9). These mixtures were mixed thoroughly in plastic bags by hand and transferred to plastic pots (1 liter) similar to those used in the survival-in-manure part of the experiment. For E. coli O157:H7 this was done 56 days after inoculation with manure types GMH and GML, because the other manure types showed too low numbers of pathogens for further transition to soil at that time. With the Salmonella serovar Typhimurium experiment, pathogen levels allowed amending of the four soils with the two more contrasting manure types, GMH and SH, which was done after 28 days of survival in manure. The pots were incubated at 15°C in darkness. For each manure-soil combination there were three replicate pots. Soils for the noninoculated pots (blanks) were mixed in the same way as the manure blanks of the manure survival part of the experiment. Sampling of the inoculated pots to determine survival was done as described above (at time zero and after 2, 7, 13, 28, and 57 days).

Lettuce production.
Two weeks after the manure was mixed with soil, aliquots of 500 gfw of mixture were transferred to plastic pots; one seedling of iceberg lettuce (Lactuca sativa L cv. Dublin) was planted in each pot (3 replicate pots x 8 treatments = 24 plants on inoculated soil mixtures and 24 plants on noninoculated blanks), and the pots were placed in a completely randomized manner on a greenhouse bench (15°C; relative humidity, 60%). After 3 weeks, root samples (1 to 2 gfw) and shoot samples (on average three small leaves, 1 to 2 gfw) were checked for pathogen presence. Root samples were washed in sterile water twice to remove soil particles. Both root and leaf samples were ground with a pestle and mortar in 5 ml 0.1% proteose peptone (Oxoid) and crystal sand and plated (100 µl) directly on selective media as described above. To distinguish between the epiphytic and endophytic presence of pathogens, half of the samples were surface sterilized by being dipped in 1% AgNO3 for 10 s and washed two times in sterile water before grinding. Bulk soil samples were plated as described above.

Chemical measurements.
Chemical characteristics were determined before starting the experiment for each manure (Table 2) and soil type (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Chemical characteristics of six types of cattle manure, collected directly from cows fed the six diets described in


View this table:
[in this window]
[in a new window]
  		TABLE 3. Physical and chemical characteristics of four soils, collected as neighboring pairs in The Netherlands, used for mixing with pathogen-inoculated cattle manure which functioned as a substrate for growth of lettuce seedlings

(i) Manure.
Dried samples (40°C) were ground and analyzed for total carbon by the Dumas method followed by detection by a CHN1110 element analyzer (CE Instruments, Milan, Italy) and for fiber content (52). Total nitrogen content was determined by the Kjeldahl method (8), and ammonium content was determined in a solution of trichloroacetic acid with an Autoanalyzer II (Technicon Instrument Corporation, Tarrytown, NY). The pH was measured in a watery suspension with an Inlab pH level 1 (WTW GmbH, Weilheim, Germany).

(ii) Soil.
Total nitrogen and carbon were determined as for manure. Nitrate and ammonium contents in soil samples were determined with an Autoanalyzer II (Technicon Instrument Corporation, Tarrytown, NY) after addition of 0.01 M CaCl2 suspension. The pH of the soil samples was measured in this CaCl2 suspension with an Inlab pH level 1 (WTW GmbH, Weilheim, Germany).

Statistical analysis.
Microbial data (CFU counts) were log transformed, and these log numbers over time for each replica were fitted to the following logistic function by nonlinear regression (Gauss-Newton method): CFU(t) = a + [b/1 + e(c – dt)], where CFU(t) is the log number of CFU gdw–1 on day t, a is the lower asymptote, (a + b) is the upper asymptote, d is the slope parameter (referred to as the decline rate), and c is the position parameter (referred to as the location of the inflection point). The true location of the inflection point is given by c/d, and the true maximum decline rate at the inflection point is given by (b x d)/2 (SAS version 8; SAS Institute, Cary, NC). Time point zero was defined as the first sampling time, which occurred immediately after inoculation, and the upper and the lower asymptotes were kept constant at, respectively, 7 log CFU gdw–1 and 0. Time points which gave a CFU count of zero were included in the analysis with the value of 1 log CFU gdw–1, which was the detection limit. Significance of the fit was assessed by an F test (F = MSregression/MSresidual), and the goodness of fit was determined by calculating a pseudo-r2 [1 – (SSresiduals/SStotal corrected)], where MS is the mean square and SS is the sum of squares. For E. coli O157:H7, the number of days needed to reach the detection limit of 1 log CFU gdw–1 was calculated from the fitted decline function. Multivariate analysis of variance (significance level of 5%) followed by contrast analysis was conducted on the regression parameters c and d. From the second part of the multivariate analysis of variance (within-subject comparisons) the effects of roughage type and crude protein level on the decline rate (d) and the location of the inflection point (c) were assessed. Differences in decline rate between Salmonella serovar Typhimurium phenotypes were analyzed by two-sided t tests. Correlation tests were conducted to check for linear relationships between decline rate and chemical parameters of the manure. Stepwise multiple regressions were conducted to determine to what extent variation in chemical and biological parameters can explain variation in decline rates. Variables left in the regression model were significant at the 0.15 level, and models were restricted to a maximum of two parameters.

The decline of E. coli O157:H7 in manure-amended soil was analyzed by fitting survival data (log CFU gdw–1) of each replica to a simple exponential decline function, CFU(t) = N0 x est, where CFU(t) is the log number CFU gdw–1 on day t, N0 is the initial log number CFU gdw–1 on day 0, and s is the slope of the curve. Because all treatments showed an increase during the first 2 days (see Results), the log number CFU gdw–1 on day 2 was set to 100%. The subsequent log numbers CFU gdw–1 were relative to that on day 2. Slopes of the different treatments were compared by using two-sided t tests. When no CFU were detected, the value of the detection limit was used (0.5 log number CFU gdw–1). The decline of both phenotypes of Salmonella serovar Typhimurium was analyzed by fitting the survival data to the same logistic function as used for the data of survival in manure because of bad fits (no convergence or low pseudo-r2) to the exponential model.


arrow
RESULTS
 
Survival of E. coli O157:H7 in manure.
In all manure types, E. coli O157:H7 populations dropped directly after inoculation by approximately 1.5 log CFU gdw–1, followed by a period of around 16 days when it stabilized or increased by approximately 0.75 log CFU gdw–1 (GOL and SH) (Fig. 1). Thereafter, the pathogen declined continuously in all treatments. E.coli O157:H7 was not detected by plate counting after 84 days in both manures derived from a straw diet (SH and SL) and after 133 days in the other manure types.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Survival of E. coli O157:H7 (A), Salmonella serovar Typhimurium MAE 110 (B), and Salmonella serovar Typhimurium MAE 119 (C) in six different types of artificially inoculated cattle manure types resulting from three different roughage types with high (closed symbols and solid lines) and low (open symbols and dashed lines) levels of additional crude protein: high-digestible grass and maize silage (triangles), low-digestible grass silage (squares), and straw (circles).

Nonlinear logistic regression resulted in significant fits (P < 0.001) with high goodness-of-fit values for all six manure types (average pseudo-r2 over three replicas: GMH, 0.92; GOH, 0.92; SH, 0.89; GML, 0.82; GOL, 0.93; and SL, 0.95). The numbers of days needed to reach the detection limit of 1 log CFU gdw–1 according to the logistic fits for GMH, GOH, SH, GML, GOL, and SL were, respectively, 128 ± 8, 105 ± 8, 76 ± 5, 126 ± 18, 92 ± 12, and 71 ± 8. Roughage type had a significant effect (Wilks' lambda = 0.060; P < 0.001) on the course of decline (effect on combined variance of both estimated parameters). Moreover, roughage type had a significant effect on the slope of decline (P < 0.001), and crude protein level did not. Roughage type and crude protein level showed no interaction with respect to their effect on the decline rate. The location of the inflection point was not significantly influenced by roughage type or crude protein level. Decline rates in manures based on the same roughage type, but different crude protein levels, did not differ (Fig. 2). All three roughage types differed significantly from each other with respect to the rate of decline, irrespective of crude protein level. When the manure types from the high- and low-CP groups were aggregated to roughage type, E.coli O157:H7 declined faster in manure derived from a diet of straw (S) compared to low-digestible grass silage (GO) (P = 0.007), S compared to high-digestible grass silage plus maize silage (GM) (P < 0.001), and GO compared to GM (P = 0.027).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 2. Values of the estimated slope parameter for the survival of E. coli O157:H7 (A), Salmonella serovar Typhimurium phenotype MAE 110 (B), Salmonella serovar Typhimurium phenotype MAE 119 (C), and Salmonella serovar Typhimurium total counts (D) in six different types of artificially inoculated cattle manure types resulting from three different roughage types with high (H) and low (L) levels of additional crude protein: high-digestible grass and maize silage (GMH and GML), low-digestible grass silage (GOH and GOL), and straw (SH and SL). Error bars show standard errors of the means. Treatments with identical letters do not significantly differ.

The rate of decline (absolute value of slope) was positively correlated with pH (P = 0.003) and fiber content (acid detergent fiber [ADF], P = 0.032; neutral detergent fiber [NDF], P= 0.017) (Table 4). The GM manures had the lowest pHs and lowest decline rates, while the S manures had the highest pHs and the highest decline rate (Fig. 3). The GO manures had intermediate pHs and intermediate decline rates. The rate of decline showed a negative linear relationship with ammonium level (P = 0.024). Stepwise multiple regressions revealed that pH explained most of the variation in decline rate: slope (model r2 = 0.97) = –1.80 x 10–2 (pH; partial r2 = 0.91, P = 0.003) + 1.06 x 10–4 (dry matter content; partial r2 = 0.06, P= 0.056) + 7.03 x 10–2 (intercept). Alternatively, when excluding pH, the neutral detergent fiber (NDF) content was best at explaining the variation in decline rate: slope (model r2= 0.93) = –2.19 x 10–3 (NDF; partial r2 = 0.80, P = 0.016) + 7.05 x 10–4 (C/N ratio; partial r2 = 0.13, P = 0.093) – 3.35x 10–3 (intercept). The pH and NDF content were not significantly correlated (Table 4).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Pearson correlation coefficients between the absolute slope values of the fitted logistic decline curve for manure and chemical characteristics of the six types of manure (n = 18)



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 3. Relationship between pH (A) or NDF content (B) and the rates of decline of E. coli O157:H7 (closed symbols) and Salmonella serovar Typhimurium (open symbols) in six different types of artificially inoculated cattle manure resulting from three different roughage types with high (h) and low (l) levels of additional crude protein: high-digestible grass and maize silage (gmh and gml), low-digestible grass silage (goh and gol), and straw (sh and sl).

Survival of Salmonella serovar Typhimurium in manure.
The two phenotypes MAE 110 and 119 showed rather similar survival curves, and both clearly survived longer in all manure types than E. coli O157:H7 (Fig. 1). CFU counts dropped directly after inoculation with 0.5 log CFU gdw–1, followed by an increase of 0.5 log CFU gdw–1 within a few days. Thereafter, the pathogen declined continuously but at a lower rate than E. coli O157:H7. After 133 days, Salmonella serovar Typhimurium could still be detected at levels of 2 to 4 log CFU gdw–1 by the normal plating procedure, depending on the manure type.

As with E. coli O157:H7, nonlinear logistic regression resulted in significant fits (P < 0.001) with high goodness-of-fit values for phenotype MAE 110 (average pseudo-r2: GMH, 0.84; GOH, 0.84; SH, 0.89; GML, 0.71; GOL, 0.86; and SL, 0.85) and MAE 119 (average pseudo-r2: GMH, 0.90; GOH, 0.94; SH, 0.90; GML, 0.83; GOL, 0.86; and SL, 0.87). Salmonella serovar Typhimurium MAE 110 and 119 showed no difference in slope over all treatments (P = 0.223). Since both phenotypes behaved similarly, the effects of roughage type and CP level were assessed by summing the CFU counts of phenotypes 110 and 119. There was a significant multivariate effect of roughage type (Wilks' lambda = 0.300; P = 0.008) and CP level (Wilks' lambda = 0.516; P = 0.026) on the combined variance of both regression parameters but no significant effects of roughage type and CP level separately on the decline rate or the location of the inflection point. Contrast analysis legitimated the pooling of manure types based on the same roughage type but different CP levels (Fig. 2). When grouped by roughage type, Salmonella serovar Typhimurium declined significantly faster in the manure resulting from the straw (S) diet compared to the high-digestible grass silage plus maize silage (GM) diet (P = 0.020). The rate of decline was positively correlated with pH (P = 0.017) and fiber content (NDF, P = 0.005; ADF, P = 0.012) (Table 4 and Fig. 3). The rate of decline showed a negative linear relationship with ammonium level (P = 0.012) and dry matter content (P = 0.010). Stepwise multiple regressions revealed that NDF content explained most of the variation in decline rate: slope (model r2 = 0.97) = –2.97 x 10–4 (NDF; partial r2 = 0.91, P = 0.003) – 2.46 x 10–3 (pH; partial r2 = 0.06, P = 0.114) + 0.01081 (intercept). Alternatively, when excluding neutral detergent fiber content and the parameters with which it was significantly correlated (ADF and dry matter content) (Table 4), the pH was best at explaining the variation in decline rate: slope (model r2 = 0.95) = –4.98 x 10–3 (pH; partial r2 = 0.81, P = 0.015) – 2.08 x 10–4 (C/N ratio; partial r2 = 0.14, P = 0.056) + 1.56 x 10–2 (intercept). The pH and NDF content were not significantly correlated (Table 4).

Survival of E. coli O157:H7 in soil.
Survival of E. coli O157:H7 in the four soils amended with both manures derived from high-digestible grass silage plus maize silage diets (GMH and GML) varied between 2 and 56 days, depending on the soil (Fig. 4). Fitting the survival data to an exponential decline function resulted in good fits (average r2 over all treatments of 0.87 ± 0.17). The values of the estimated rate of decline are shown in Fig. 5. The kind of manure applied to the soil made no difference except for the conventionally managed loam soil, where rate of decline was higher when GMH was amended than when GML was amended (P = 0.012). E. coli O157:H7 declined significantly faster (P < 0.05) in all organically managed soils then in the conventionally managed neighboring soils, except for loam soil amended with GML. E. coli O157:H7 disappeared exceptionally rapidly in the organic sandy soil (Fig. 4 and 5).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. Survival of E. coli O157:H7 and Salmonella serovar Typhimurium in four different soils: organic sand (•), conventional sand ({circ}), organic loam ({blacktriangleup}), and conventional loam ({triangleup}). (A) Survival of E. coli O157:H7 in soils amended with manure GMH. (B) Survival of E. coli O157:H7 in soils amended with manure GML. (C) Survival of Salmonella serovar Typhimurium MAE 110 in soils amended with manure GMH. (D) Survival of Salmonella serovar Typhimurium MAE 110 in soils amended with manure SH. (E) Survival of Salmonella serovar Typhimurium MAE 119 in soils amended with manure GMH. (F) Survival of Salmonella serovar Typhimurium MAE 110 in soils amended with manure SH.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 5. Values of the estimated slope parameter for the survival of E. coli O157:H7 (A) and of Salmonella serovar Typhimurium MAE 110 (B) and MAE 119 (C) in four different soils: organic sand (OS), conventional sand (CS), organic loam (OC), and conventional loam (CC). For E. coli O157:H7, these four soils were amended with manure type GMH (1) and GML (2) and fitted to an exponential-decline model, while for Salmonella they were amended with GMH (1) or SH (2) and fitted to a logistic decline model as with the survival in manure. Error bars show standard errors of the means. Treatments with identical letters do not significantly differ.

The rate of decline in soils was positively correlated with total nitrogen content (r = 0.86, P = 0.006), nitrate content (r= 0.81, P = 0.014), and total carbon content (r = 0.82, P = 0.012). Stepwise multiple regression first resulted in a model solely including the total nitrogen content: slope (model r2 = 0.80) = –2.15 x 10–1 (total nitrogen; partial r2 = 0.80, P = 0.105) + 2.29 x 10–1 (intercept). When the total nitrogen content and parameters correlated with it (nitrate content and total carbon content) were excluded, no variable was strong enough to enter the model, thus explaining a significant part of the variation in the decline rate of E. coli O157:H7 in soil.

Survival of Salmonella serovar Typhimurium in soil.
The density of Salmonella serovar Typhimurium declined more steadily than that of E. coli O157:H7, and Salmonella serovar Typhimurium was in most cases still detected at 56 days after application of the manure to the soils (Fig. 4). The decline rates of Salmonella serovar Typhimurium could not be compared with those of E. coli O157:H7 directly because a different decline model was used. The two Salmonella serovar Typhimurium phenotypes showed quite different patterns of decline rate over the treatments: the two phenotypes differed significantly from each other in decline rate in five of the eight treatments (P < 0.05) (Fig. 5). With Salmonella serovar Typhimurium phenotype 110, none of the manure-soil treatments was exceptional with respect to the decline rate. Phenotype 119 showed an exceptionally fast decline in conventional sand amended with manure GH and a relative slow decline in organic loam with GMH, compared to the other treatments. No consistent differences were found between organic and conventional soils.

The rate of decline of phenotype 110 in soils amended with SH was positively correlated with nitrate content (r = 0.95, P= 0.049), total nitrogen content (r = 0.95, P = 0.047) and total carbon content (r = 0.99, P = 0.007). The rate of decline in soils amended with GMH did not show any correlations with soil characteristics. The rate of decline of phenotype 119 showed no correlations with any of the chemical parameters. The variation in the rate of decline of phenotype 110 over all treatments was best explained by a model solely including the nitrate content: slope (model r2 = 0.99) = –4.30 x 10–4 (total nitrogen; partial r2 = 0.99, P = 0.071) – 3.03 x 10–2 (intercept). For phenotype 119 and the total Salmonella counts, no parameter entered the regression model.

Presence on or in lettuce.
Only one root sample of a lettuce crop grown on conventional loam amended with manure type GMH showed the presence of E. coli O157:H7 (1.5 log CFU gdw–1). Because this sample was not surface sterilized, it is not clear whether the pathogen was present in the rhizosphere, attached on the root surface, or internalized in the root tissue. None of the samples were positive for Salmonella serovar Typhimurium phenotypes 110 and 119.


arrow
DISCUSSION
 
The potential presence of human pathogens such as E. coli O157:H7 and Salmonella serovar Typhimurium in vegetables grown in soils enriched with manure is of growing concern. We simulated the lettuce production chain in three consecutive steps, monitoring the fate of E. coli O157:H7 and Salmonella serovar Typhimurium in manure, manure-amended soil, and lettuce. In this way the pathogens experience three different niches and two niche transitions, which is a more realistic setup compared to focusing on survival in one particular niche or determining the association of pathogens with vegetables by planting them directly on inoculated manure-amended soil. We investigated the effects of different cattle diets, soil types, and soil management types on pathogen survival in manure and soil.

We showed that the roughage type, but not the dietary crude protein level, influences the survival capabilities of both E. coli O157:H7 and Salmonella serovar Typhimurium. Decline of E.coli O157:H7 was faster in manure derived from a pure straw diet (higher pH and higher fiber content) than in manure derived from a high-digestible grass silage plus maize silage diet (lower pH and lower fiber content). The decline found in manure derived from a low-digestible grass silage diet was intermediate. Persistence of Salmonella serovar Typhimurium in manure was better than that of E. coli O157:H7. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although the decline was significantly faster in the manure derived from straw than in the manure from the grass silage plus maize silage diet. The decline rates of both pathogens were mainly determined by the pH and fiber content of the manure. After the first niche transition from manure to manure-amended soil, both pathogens declined further, and again E. coli O157:H7 declined faster than Salmonella serovar Typhimurium. E. coli O157:H7 declined exceptionally rapidly in the organically managed sandy soil. After survival in manure and manure-amended soils, the final and most likely more realistic bacterial loads in the soils used in this experiment did not result in the presence of E. coli O157:H7 or Salmonella serovar Typhimurium in or on the edible parts of lettuce.

The survival times of E. coli O157:H7 reported in this study, ranging between 56 and 133 days at 10°C, resemble earlier published persistence times of E. coli O157:H7 in bovine manure (5, 26, 28). Salmonella serovar Typhimurium clearly survived longer than E. coli O157:H7 and was still present after 133 days. Theoretical elimination times of Salmonella serovar Typhimurium of 151 days at 4°C, 85 days at 20°C, and 14 days at 37°C in bovine manure could be derived from linear regression equations (18). In general it is very difficult to compare survival studies, due to the variety of experimental setups used. Moreover, as we showed with this study, survival times depend not only on temperature but also on the manure composition, which is determined by the feeding regimen.

Cattle diet has been considered a potentially important factor in controlling the presence of E. coli O157:H7 and Salmonella in cattle, given that it likely affects gut microbial populations (34), but results are not unambiguous. Considerable attention has been paid to the controversial effect of cattle diet on pathogen shedding by the animal (11, 20, 25, 38, 45, 48). Roughage type may be important not only in controlling shedding but also with respect to pathogen survival in manure. We showed that the human pathogens E. coli O157:H7 and Salmonella are more persistent in manure derived from cattle fed a diet characterized by a higher energy and lower fiber content (high-digestible grass silage plus maize silage) than in manure derived from a diet characterized by a lower energy and higher fiber content (straw). Feeding hay to cattle may be a way to reduce shedding of acid-resistant E. coli (11). Diets high in grain are thought to create a more acidic rumen environment because the starch is incompletely digested and is fermented in the colon, which in turn should lead to the selection of more acid-tolerant E. coli (11, 38). It is known that both E. coli O157:H7 and Salmonella serovar Typhimurium possess several systems for surviving exposures to low pH and therefore can be considered to be quite acid resistant (6, 12). Extrapolating to pathogenic E. coli, the results reported by Diez-Gonzalez et al. (11) seem to be supported by some experimental studies (7, 45) and several epidemiological studies (10, 16, 37) which found a positive association between E. coli O157:H7 prevalence and the feeding of barley, corn silage, and grains. Salmonella prevalence in dairy heifers was also found to be lower when hay was fed (27). In contrast, some epidemiological studies (39, 47) and various studies using artificially inoculated animals seem to contradict the idea that more forage feeding (hay) compared to grain feeding is a mechanism to reduce selection for increased acid resistance and E. coli O157:H7 shedding by ruminants (15, 20, 25, 48).

Although conditions in excreted manure are likely to be different from those encountered in the rumen environment, our results seem to agree with the proposition that a high-energy diet containing grains/starch favors the proliferation and survival of E. coli O157:H7. We also showed the importance of a high fiber content of the diet and the resulting manure with respect to the elimination of human pathogens. This might be related to the combination of a relative slow release of readily available nutrients in manure with higher fiber content and the more copiotrophic nature of E. coli and Salmonella. In practice, feeding starch in the form of grains or maize is a common practice in dairy farming in order to fulfill the energy need of high milk production. However, there is a trend in more sustainable and organic dairy farming of feeding a diet with increased fiber content consisting of lower concentrations of cytoplasmic carbohydrates (sugars and starch) and more so-called cell wall carbohydrates (hemicellulose, cellulose, and lignin). This is often accompanied by a higher C/N ratio, consequently reducing nitrogen losses to the environment (49). According to our findings, this should result in lower survival of E. coli O157:H7 and Salmonella serovar Typhimurium and consequently in a lower risk of transfer of these pathogens into the vegetable production chain.

The land application of infected manure is a major transition for human pathogens, since soil can be considered to be a hostile environment for bacteria that have the gastrointestinal tracts of mammals as their primary habitat. Although pathogen levels gradually decline with increased storage time and after land application, it is recommended that an interval of at least 120 days (2) or even 6 months (31) should be observed between manure spreading and harvest of the crop. Our results for E.coli O157:H7 survival between 2 and 56 days in manure-amended soil are comparable with earlier reported survival times of 34 days in sandy loam soil amended with cow manure at a similar temperature and manure-to-soil ratio (23). Others reported longer E. coli O157:H7 survival times of between 154 and 217 days in soils amended with inoculated compost (21) and Salmonella serovar Typhimurium persistence of between 203 and 231 days (22). However, those studies relied on inoculating the substrate with relatively high densities (>105 CFU gdw–1). In the present study we started monitoring the fate of the pathogens in manure-amended soil after they declined to relatively low and more realistic levels in manure (approximately 102 CFU gdw–1 for E. coli O157:H7 and 104 CFU gdw–1 for Salmonella serovar Typhimurium). As with survival in manure, it must be stressed that comparison between studies is difficult, as different substrates and experimental setups are used. Persistence seems to depend on factors such as temperature (23), manure-to-soil ratio (23), and soil type (32). We showed that decline of E. coli O157:H7 was faster in the organically managed soil than in its conventionally managed neighbor in three out of four cases and was exceptionally fast in the organic sandy soil treatments. The latter may be more due to the relative high levels of nitrate, total nitrogen, and total carbon in this specific organic sandy soil. This might have increased the activity of the native microbial population, which decreased the competitive success of the introduced pathogen. The extremely fast decline in this particular soil was not observed for Salmonella serovar Typhimurium, which may have a higher competitive ability. More research with more pairs of soils is needed in order to differentiate between organic and conventional soils with respect to human pathogen suppression.

The third transition, the planting of lettuce, did not eventually result in the presence of E. coli O157:H7 or Salmonella serovar Typhimurium on or in the edible parts of iceberg lettuce. Some experimental studies demonstrated that these pathogens can become associated with vegetables (21, 22, 30, 54, 56). However, a wide variety of experimental setups were used (seedlings or seeds grown hydroponically or in soil), and most of these studies proved only surface contamination. Solomon et al. (41) showed that E. coli O157:H7 can enter the lettuce plant from contaminated manure through the root system and can migrate throughout the edible part of the plant. Recently, our laboratory also confirmed the possibility of internalization of E. coli O157:H7 and Salmonella serovar Typhimurium in iceberg lettuce grown hydroponically and in inoculated soil (E. Franz, A. A. Visser, A. D. van Diepeningen, M. M. Klerks, A. J. Termorshuizen, and A. H. C. van Bruggen, submitted for publication). However, the numbers of bacteria used in these studies were far greater than what may be found in an agricultural field. In the current experiment the pathogen densities in the bulk soil at the time the lettuce was planted were approximately 10 to 100 CFU gdw–1 for E. coli O157:H7 and 100 to 1,000 CFU gdw–1 for Salmonella serovar Typhimurium. These densities might be more realistic. Most likely, the population pressure was too low to allow the pathogens to enter the plants. Indeed, the results of Solomon et al. (41) showed an increased number of positive samples with increasing pathogen density of the inoculum (104, 106, and 108 CFU gdw–1).

This study showed for the first time the fate of E. coli O157:H7 and Salmonella serovar Typhimurium through subsequent niches: manure, manure-amended soil, and manure-amended soil with lettuce. The results indicate that the cattle feeding regimen must be recognized as an important factor determining the survival of these pathogens in manure. Since manure is the primary fertilizer in organic vegetable production and is frequently used in conventional production, these results are of importance with respect to microbial safety in vegetable production. Our results indicate that although manure is more frequently used in organic production, this does not automatically imply a higher risk of pathogen transfer to vegetable production. More work has to be done on how differences between organic and conventional farming may lead to differences in pathogen survival, not only in manure but in the whole farm ecosystem.


arrow
ACKNOWLEDGMENTS
 
This research was supported by the Technology Foundation STW, the Applied Science Division of NWO, the Technology Program of the Ministry of Economic Affairs, and the Dutch National Product Board for Horticulture.

We thank Pina Fratamico for providing the GFP-modified E. coli O157:H7, Ute Römling for providing the GFP-modified S. enterica serovar Typhimurium strains, and J. W. Reijs for giving us the opportunity to collect manure from his cattle feeding experiment. We further thank A. M. Semenov and A. J. Termorshuizen for constructive discussions and M. de Visser and H. D. Halm for the chemical analyses.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Biological Farming Systems Group, Department of Plant Sciences, Wageningen University and Research Center, Marijkeweg 22, 6709 PG Wageningen, The Netherlands. Phone: 31-317-478202. Fax: 31-317-478213. E-mail: Eelco.Franz{at}wur.nl. Back


arrow
REFERENCES
 
    1
  1. American Society for Microbiology. 2000. National Organic Program. [Online.] http://www.asm.org/Policy/index.asp?bid=3585.
    2. 2
  2. Anonymous. 2000. National Organic Program. T CFR part 205:203. U.S. Department of Agriculture, Washington, D.C.
    3. 3
  3. Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204-216.
    4. 4
  4. Beuchat, L. R. 1998. Survival of enterohemorrhagic Escherichia coli O157:H7 in bovine feces applied to lettuce and the effectiveness of chlorinated water as a disinfectant. J. Food Prot. 62:845-849.
    5. 5
  5. Bolton, D. J., C. M. Byrne, J. J. Sheridan, D. A. McDowell, and I. S. Blair. 1999. The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7. J. Appl. Microbiol. 86:407-411.[CrossRef][Medline]
    6. 6
  6. Booth, I. R., F. Thomson-Carter, P. Carter, S. Jordan, S. Park, L. Malcolm, and J. Glover. 1999. Acid tolerance of Escherichia coli—the sting in the tail? p. 27-38. In C. S Stewart, and H. J. Flint (ed.), Escherichia coli O157:H7 in farm animals. CABI Publishing, New York, N.Y.
    7. 7
  7. Boukhors, K., N. Pradel, J. P. Girardeau, V. Livrelli, A. M. Ou Saïd, M. Contrepois, and C. Martin. 2002. Effect of diet on Shiga toxin-producing Escherichia coli (STEC) growth and survival in rumen and abomasum fluids. Vet. Res. 33:405-412.[CrossRef][Medline]
    8. 8
  8. Bremner, J. M., and C. S. Mulvaney. 1982. Nitrogen total, p. 595-624. In A. L. Page, R. H Miller, and D. R. Keeney (ed.), Methods of soil analysis, 2nd ed., part 2. Chemical and microbiological properties. American Society of Agronomy, Soil Science Society of America, Madison, Wis.
    9. 9
  9. Chapman, P. A., C. A. Siddons, D. J. Wright, P. Norman, J. Fox, and E. Crick. 1993. Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infection in man. Epidemiol. Infect. 111:439-447.[Medline]
    10. 10
  10. Dargatz, D. A., S. J. Wells, L. A. Thomas, D. D. Hancock, and L. P. Garber. 1997. Factors associated with the presence of Escherichia coli O157:H7 in feces of feedlot cattle. J. Food Prot. 60:466-470.
    11. 11
  11. Diez-Gonzalez, F., T. R. Callaway, M. G. Kizoulis, and J. B. Russel. 1998. Grain feeding and the dissemination of acid-resistant Escherichia coli from cattle. Science 281:1666-1668.[Abstract/Free Full Text]
    12. 12
  12. Foster, J. W., and M. P. Spector. 1995. How Salmonella survive against the odds. Annu. Rev. Microbiol. 49:145-174.[CrossRef][Medline]
    13. 13
  13. Fratamico, P. M., M. Y. Deng, T. P. Strobaugh, and S. A. Palumbo. 1997. Construction and characterization of Escherichia coli O157:H7 strains expressing firefly luciferase and green fluorescent protein and their use in survival studies. J. Food Prot. 60:1167-1173.
    14. 14
  14. Fukushima, H., K. Hoshina, and M. Gomyoda. 1999. Long-term survival of Shiga-toxin producing Escherichia coli O26, O111, and O157:H7 in bovine feces. Appl. Environ. Microbiol. 65:5177-5181.[Abstract/Free Full Text]
    15. 15
  15. Grauke, L. J., S. A. Wynia, H. Q. Sheng, J. W. Yoon, C. J. Williams, C. W. Hunt, and C. J. Hovde. 2003. Acid resistance of Escherichia coli O157:H7 from the gastrointenstinal tract of cattle fed hay or grain. Vet. Microbiol. 95:211-225.[CrossRef][Medline]
    16. 16
  16. Herriot, D. E., D. D. Hancock, E. D. Ebel, L. V. Carpenter, D. H. Rice, and T. E. Besser. 1998. Association of herd-management factors with colonization of dairy cattle by shiga toxin-positive Escherichia coli O157. J. Food Prot. 7:802-807.
    17. 17
  17. Hilborn, E. D., J. H. Mermin, P. A. Mshar, J. L. Hadler, A. Voetsch, C. Wojtkunski, M. Swartz, R. Mshar, M. A. Lambert-Fair, J. A. Farrar, M. K. Glynn, and L. Slutsker. 1999. A multistate outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun lettuce. Arch. Intern. Med. 159:1758-1764.[Abstract/Free Full Text]
    18. 18
  18. Himathongkham, S., S. Bahari, H. Riemann, and D. Cliver, D. 1999. Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry. FEMS Microbiol. Lett. 178:251-257.[CrossRef][Medline]
    19. 19
  19. Horby, P. W., S. J. O'Brien, G. K. Adak, C. Graham, J. I. Hawker, P. Hunter, C. Lane, A. J. Lawson, R. T. Mitchell, M. H. Reacher, E. J. Threlfall, and L. R. Ward. 2003. A national outbreak of multi-resistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce. Epidemiol. Infect. 130:169-178.[CrossRef][Medline]
    20. 20
  20. Hovde, C. J., P. R. Austin, K. A. Cloud, C. J. Williams, and C. W. Hunt. 1999. Effect of cattle diet on Escherichia coli O157:H7 acid resistance. Appl. Environ. Microbiol. 65:3233-3235.[Abstract/Free Full Text]
    21. 21
  21. Islam, M., M. P. Doyle, S. C. Phatak, P. Millner, and X. Jiang. 2004. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. J. Food Prot. 67:1365-1370.[Medline]
    22. 22
  22. Islam, M., J. Morgan, M. P. Doyle, S. C. Phatak, P. Millner, and X. Jiang. 2004. Persistence of Salmonella enterica serovar Typhimurium on lettuce and parsley and in soils on which they were grown in fields treated with contaminated manure composts or irrigation water. Foodborne Pathog. Dis. 1:27-35.[Medline]
    23. 23
  23. Jiang, X., J. Morgan, and M. P. Doyle. 2002. Fate of Escherichia coli O157:H7 in manure-amended soil. Appl. Environ. Microbiol. 68:2605-2609.[Abstract/Free Full Text]
    24. 24
  24. Kearny, T. E., M. J. Larkin, and P. N. Levett. 1993. The effects of slurry storage and anaerobic digestion on survival of pathogenic bacteria. J. Appl. Microbiol. 74:86-93.
    25. 25
  25. Kudva, I. T., C. W. Hunt, C. J. Williams, U. M. Nance, and C. J. Hovde. 1997. Evaluation of dietary influences on Escherichia coli O157:H7 shedding by sheep. Appl. Environ. Microbiol. 63:3878-3886.[Abstract]
    26. 26
  26. Kudva, I. T., K. Blanch, and C. J. Hovde. 1998. Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64:3166-3174.[Abstract/Free Full Text]
    27. 27
  27. Losinger, W. C., S. J. Wells, L. P. Garber, and H. S. Hurd. 1995. Management factors related to Salmonella shedding by dairy heifers. J. Dairy Sci. 78:2464-2472.[Abstract]
    28. 28
  28. Maule, A. 2000. Survival of verocytotoxigenic Escherichia coli O157:H7 in soil, water and on surfaces. J. Appl. Microbiol. 88:71S-78S.
    29. 29
  29. Mukherjee, A., D. Speh, E. Dyck, and F. Diez-Gonzalez. 2004. Preharvest evaluation of coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers. J. Food Prot. 67:894-900.[Medline]
    30. 30
  30. Natvig, E. E., S. C. Ingham, B. H. Ingham, L. R. Cooperband, and T. R. Roper. 2002. Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine manure. Appl. Environ. Microbiol. 68:2737-2744.[Abstract/Free Full Text]
    31. 31
  31. Nicholson, F. A., M. L. Hutchinson, K. A. Smith, C. W. Keevil, B. J. Chambers, and A. Moore. 2000. A study on farm manure applications to agricultural land and an assessment of the risk of pathogen transfer into the food chain. A report to the Ministry of Agriculture Fisheries and Food.
    32. 32
  32. Nicholson, F. A., S. J. Groves, and B. J. Chambers. 2005. Pathogen survival during livestock manure storage and following land application. Bioresource Technol. 96:135-143.[CrossRef][Medline]
    33. 33
  33. Pell, A. N. 1997. Manure and microbes: public and animal health problem? J. Dairy Sci. 80:2673-2681.[Abstract]
    34. 34
  34. Rasmussen, M. A., T. L. Wickman, W. C. Cray, and T. A. Casey. 1999. Escherichia coli O157:H7 and the rumen environment, p. 39-49. In C. S. Stewart and H. J. Flint (ed.), Escherichia coli O157:H7 in farm animals. CABI Publishing, New York, N.Y.
    35. 35
  35. Römling, U., W. D. Sierralta, K. Eriksson, and S. Normark. 1998. Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promotor. Mol. Microbiol. 28:249-264.[CrossRef][Medline]
    36. 36
  36. Römling, U., M. Rohde, A. Olsén, S. Normark, and J. Reinköster. 2000. AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways. Mol. Microbiol. 36:10-23.[CrossRef][Medline]
    37. 37
  37. Rugbjerg, H., E. M. Nielsen, and J. S. Andersen. 2003. Risk factors associated with faecal shedding of verocytotoxin-producing Escherichia coli O157 in eight known-infected Danish dairy herds. Prev. Vet. Med. 58:101-113.[CrossRef][Medline]
    38. 38
  38. Russel, J. B., and J. L. Rychlik. 2001. Factors that alter rumen microbial ecology. Science 292:1119-1122.[Abstract/Free Full Text]
    39. 39
  39. Schouten, J. M., M. Bouwknegt, A. W. van de Giessen, K. Frankena, M. C. M. De Jong, and F. A. M. Graat. 2004. Prevalence estimation and risk factors for Escherichia coli O157:H7 in Dutch dairy farms. Prev. Vet. Med. 64:49-61.[CrossRef][Medline]
    40. 40
  40. Sivapalasingam, S., C. R. Friedman, L. Cohen, and R. V. Tauxe. 2004. Fresh produce: a growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food Prot. 76:2342-2353.
    41. 41
  41. Solomon, E. B., S. Yaron, and K. R. Matthews. 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Appl. Environ. Microbiol. 68:397-400.[Abstract/Free Full Text]
    42. 42
  42. Suarez, A., A. Güttler, M. Strätz, L. H. Staender, K. N. Timmis, and C. A. Guzmán. 1997. Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications. Gene 196:69-74.[CrossRef][Medline]
    43. 43
  43. Synge, B. A. 2000. Veterinary significance of verocytotoxin-producing Escherichia coli O157. World J. Microbiol. Biotechnol. 16:725-732.[CrossRef]
    44. 44
  44. Tauxe, R., H. Kruse, C. Hedberg, M. Potter, J. Madden, and K. Wachsmuth. 1997. Microbial hazards and emerging issues associated with produce; a preliminary report to the National Advisory Committee on Microbial Criteria for Foods. J. Food Prot. 60:1400-1408.
    45. 45
  45. Tkalcic, S., C. A. Brown, B. G. Harmon, A. V. Jain, E. P. O. Mueller, K. L. Parks, S. A. Martin, T. Zhao, and M. P. Doyle. 2000. Effects of diet on rumen proliferation and fecal shedding of Escherichia coli O157:H7 in calves. J. Food Prot. 63:1630-1636.[Medline]
    46. 46
  46. Unc, A., and M. J. Goss. 2004. Transport of bacteria from manure and protection of water resources. Appl. Soil. Ecol. 25:1-18.
    47. 47
  47. Vaessen, M. A., J. Veling, K. Frankena, F. A. M. Graat, and T. Klunder. 1998. Risk factors for Salmonella dublin infection on dairy farms. Vet. Q. 20:97-99.[Medline]
    48. 48
  48. Van Baale, M. J., J. M. Sargeant, D. P. Gnad, B. M. DeBey, K. F. Lechtenberg, and T. G. Nagaraja. 2004. Effect of forage or grain diets with or without monesin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle. Appl. Environ. Microbiol. 70:5336-5342.[Abstract/Free Full Text]
    49. 49
  49. Van Bruchem, J., F. Schuring, and F. Verhoeven. 2002. De koe moet weer als koe gevoerd worden, p. 7-9. In Mineralenproject Vel & Vanla, November 2002. NLTO Projecten B.V., Drachten, The Netherlands.
    50. 50
  50. Van Bruggen, A. H. C. 1995. Plant-disease severity in high-input compared to reduced-input and organic farming systems. Plant Dis. 79:976-984.
    51. 51
  51. Van Bruggen, A. H. C., and A. J. Termorshuizen. 2003. Integrated approaches to root disease management in organic farming systems. Aust. Plant Pathol. 32:141-156.[CrossRef]
    52. 52
  52. Van Soest, P. J. 1963. Use of detergents in the analyses of fibrous feeds. A rapid method for the determination of fiber and lignin. J. Assoc. Off. Agric. Chem. 46:829-835.
    53. 53
  53. Veling, J., H. Wilpshaar, K. Frankena, C. Bartels, and H. W. Barkema. 2002. Risk factors for Salmonella enterica subsp. Enterica serovar Typhimurium infection on Dutch dairy farms. Prev. Vet. Med. 54:157-168.[CrossRef][Medline]
    54. 54
  54. Wachtel, M. R., L. C. Whitehand and R. E. Mandrell. Association of Escherichia coli O157:H7 with preharvest lettuce upon exposure to contaminated irrigation water. J. Food Prot. 65:18-25.
    55. 55
  55. Wang, G., T. Zhao, and M. Doyle. 1996. Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces. Appl. Environ. Microbiol. 62: 2567-2570.[Abstract]
    56. 56
  56. Warriner, K., F. Ibrahim, M. Dickinson, C. Wright, and W. M. Waites. 2003. Interaction of Escherichia coli with growing spinach plants. J. Food Prot. 66:1790-1797.[Medline]

Applied and Environmental Microbiology, October 2005, p. 6165-6174, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.6165-6174.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Semenov, A. V., van Overbeek, L., van Bruggen, A. H. C. (2009). Percolation and Survival of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry. Appl. Environ. Microbiol. 75: 3206-3215 [Abstract] [Full Text]  
  • Texier, S., Prigent-Combaret, C., Gourdon, M. H., Poirier, M. A., Faivre, P., Dorioz, J. M., Poulenard, J., Jocteur-Monrozier, L., Moenne-Loccoz, Y., Trevisan, D. (2008). Persistence of Culturable Escherichia coli Fecal Contaminants in Dairy Alpine Grassland Soils. J. Environ. Qual. 37: 2299-2310 [Abstract] [Full Text]  
  • Barak, J. D., Liang, A., Narm, K.-E. (2008). Differential Attachment to and Subsequent Contamination of Agricultural Crops by Salmonella enterica. Appl. Environ. Microbiol. 74: 5568-5570 [Abstract] [Full Text]  
  • Crohn, D. M., Bianchi, M. L. (2008). Research Priorities for Coordinating Management of Food Safety and Water Quality. J. Environ. Qual. 37: 1411-1418 [Abstract] [Full Text]  
  • Klerks, M. M., van Gent-Pelzer, M., Franz, E., Zijlstra, C., van Bruggen, A. H. C. (2007). Physiological and Molecular Responses of Lactuca sativa to Colonization by Salmonella enterica Serovar Dublin. Appl. Environ. Microbiol. 73: 4905-4914 [Abstract] [Full Text]  
  • Franz, E., Klerks, M. M., De Vos, O. J., Termorshuizen, A. J., van Bruggen, A. H. C. (2007). Prevalence of Shiga Toxin-Producing Escherichia coli stx1, stx2, eaeA, and rfbE Genes and Survival of E. coli O157:H7 in Manure from Organic and Low-Input Conventional Dairy Farms. Appl. Environ. Microbiol. 73: 2180-2190 [Abstract] [Full Text]  
  • Ravva, S. V., Korn, A. (2007). Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7. Appl. Environ. Microbiol. 73: 2191-2198 [Abstract] [Full Text]  
  • Klerks, M. M., van Bruggen, A. H. C., Zijlstra, C., Donnikov, M. (2006). Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control.. Appl. Environ. Microbiol. 72: 3879-3886 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.
Agricola
Right arrow Articles by Franz, E.
Right arrow Articles by van Bruggen, A. H. C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»