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Published online 31 May 2006
Published in J Environ Qual 35:1170-1180 (2006)
DOI: 10.2134/jeq2005.0449
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TECHNICAL REPORTS

Waste Management

Salmonella Survival in Manure-Treated Soils during Simulated Seasonal Temperature Exposure

Richard A. Holleya,*, Katia M. Arrusa,d, Kimberly H. Ominskib, Mario Tenutac and Gregory Blanka

a Department of Food Science, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada
b Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada
c Department of Soil Science, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada
d present address: Food Development Centre, 810 Phillips Street, Portage la Prairie, MB, R1N 3J9 Canada

* Corresponding author (rick_holley{at}umanitoba.ca)

Received for publication December 7, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Addition of animal manure to soil can provide opportunity for Salmonella contamination of soil, water, and food. This study examined how exposure of hog manure-treated loamy sand and clay soils to different simulated seasonal temperature sequences influenced the length of Salmonella survival. A six-strain cocktail of Salmonella serovars (Agona, Hadar, Heidelberg, Montevideo, Oranienburg, and Typhimurium) was added to yield 5 log cfu/g directly to about 5 kg of the two soils and moisture adjusted to 60 or 80% of field capacity (FC). Similarly, the Salmonella cocktail was mixed with fresh manure slurry from a hog nursery barn and the latter added to the two soils at 25 g/kg to achieve 5 log cfu/g Salmonella. Manure was mixed either throughout the soil or with the top kilogram of soil and the entire soil volume was adjusted to 60 or 80% FC. Soil treatments were stored 180 d at temperature sequences representing winter to summer (–18, 4, 10, 25°C), spring to summer (4, 10, 25, 30°C), or summer to winter (25, 10, 4, –18°C) seasonal periods with each temperature step lasting 45 d. Samples for Salmonella recovery by direct plating or enrichment were taken at 0, 7, and 15 d post-inoculation and thereafter at 15-d intervals to 180 d. Salmonella numbers decreased during application to soil and the largest decreases occurred within the first week. Higher soil moisture, manure addition, and storage in the clay soil increased Salmonella survival. Salmonella survived longest (≥180 d) in both soils during summer-winter exposure but was not isolated after 160 d from loamy sand soil exposed to other seasonal treatments. For all but one treatment decimal reduction time (DRT45d) values calculated from the first 45 d after application were ≤30 d and suggested that a 30-d delay between field application of manure in the spring or fall and use of the land would provide reasonable assurance that crop and animal contamination by Salmonella would be minimized.

Abbreviations: cfu, colony forming units • DRT, decimal reduction time • FC, field capacity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE use of animal manures as plant fertilizers replenishes nutrients and soil organic matter, supports the concept of agricultural sustainability, and is consistent with popular views regarding recycling of waste. With intensive animal agriculture, problems can arise when manure is applied (i) at rates in excess of those required to maintain soil nutrient and moisture balance, (ii) just before heavy rainfall, or (iii) seasonally outside periods of regional weather patterns that normally allow maximum retention of manure in the soil. Consideration must also be given to the probable presence of viable zoonotic bacteria and protozoan parasites which can re-infect animals or humans following ingestion of feed or food contaminated by untreated wastes. A variety of methods can be used to reduce the risk from pathogens in agricultural wastes including composting and thermal or chemical (alkaline) treatments, as well as anaerobic digestion (Guan and Holley, 2003b). Some procedures are more effective than others but all increase production costs. Since pathogens of concern (Salmonella, cytotoxigenic E. coli, Yersinia, Campylobacter, Cyclosporidium, and Giardia) die off at variable rates during manure storage or when applied to fields (Guan and Holley, 2003a), an alternative approach involves use of untreated stored manure-slurry applied to fields before planting or harvest at experimentally defined intervals that maximize pathogen reduction (Ingham et al., 2005). Studies characterizing pathogen survival in liquid manure storage reservoirs, however, have been less useful in defining intervals than studies on survival of pathogens in treated fields because fresh manure is continuously added to the reservoirs even just before manure application to fields (Hutchison et al., 2005c).

A number of studies have examined survival of zoonotic pathogens in soils with or without manure addition, but only a few used hog manure and even fewer studies have been done with Salmonella (Chandler and Craven, 1981; Baloda et al., 2001; Gessel et al., 2004; Hutchison et al., 2004; Côté and Quessy, 2005). At 4 to 6°C most pathogens were able to survive 30 d in soils and Salmonella serovars were relatively persistent. Among the pathogens of concern, Cryptosporidium survived best in frozen soil and E. coli O157:H7 and Salmonella survived better than others in warm (20–30°C) soil (Guan and Holley, 2003a).

Data describing the survival of Salmonella alone in soil or with hog, sheep, or cattle manure are shown in Table 1. Salmonella serovars were reported to survive about 300 d in soil treated with cattle or hog manure (Jones, 1986; Baloda et al., 2001). Temperature, moisture, soil type (clay or sand) and texture, exposure to sunlight, predation by protozoans and insect larvae, and the initial number of organisms present influenced the length of Salmonella survival in soil. Although factors influencing survival often interacted and complicated interpretation, in general, Salmonella survival was longer when larger numbers were initially present in heavier soils with higher moisture at cool temperatures.


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  		Table 1. Studies reporting the survival of Salmonella in soil.

 
Decimal (or 90%) reduction time (DRT) values are used to describe the decline in pathogen viability during exposure to suboptimal conditions (Guan and Holley, 2003b). However, their use to estimate the time for complete elimination of a bacterial population can be misleading if a small proportion of the population under study exhibits enhanced resistance to the stress examined (Hutchison et al., 2005a).

The present laboratory study was undertaken to examine the rate of Salmonella decline when initially present at identical levels in manure-treated soils exposed to specific temperature sequences to model seasonal change. It was of interest to determine whether survival or lethality dominated at the upper or lower ranges of temperature used.

In the present work survival of a group of Salmonella serovars was studied in loamy sand or clay soils exposed to temperature sequences simulating seasonal changes from spring to summer, summer to winter, or winter to spring in south-central Canada. Temperature sequences had four steps (from –18 to 30°), each of which lasted 45 d. Tests were conducted at two soil moisture levels with (or without) hog manure slurry applied at the soil surface or incorporated in the soil.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Experimental Design
A randomized five-factorial, three-block design test was performed. Factors included: soil type (loamy sand and clay); temperature sequence (–18, 4, 10, 25°C; 4, 10, 25, 30°C; or 25, 10, 4, –18°C); water content as a percent of field capacity at either 60 or 80%; method of manure-inoculum addition (Salmonella inoculated manure blended by mixing with the soil, inoculated manure applied on the soil surface, or inoculum added to soil without manure); and sampling time (0, 7, and 15 d and subsequently at 15-d intervals for 6 mo). Soil replicates consisted of samples obtained from three different locations in the same field which were each treated as a block. Controls consisted of soil samples without bacterial addition which were mixed at the ratio of 25 g manure/kg soil.

Detection of Salmonella by Enrichment
Twenty five grams of soil or manure were suspended in 225 mL of buffered peptone water (BPW) (BD/Difco, Sparks, MD) and incubated at 37°C for 24 h. A 1-mL aliquot was transferred to 99 mL of Rappaport-Vassiliadis (RV) broth (BD/Difco) and incubated at 42°C for 24 h. Loopfuls of RV broth culture were streaked on xylose-lysine-tergitol-4 (XLT-4) agar (BD/Difco) and brilliant green sulfa (BGS) agar plates (BD/Difco), and were incubated overnight at 37°C. Typical Salmonella colonies were transferred to triple sugar iron (TSI) (BD/Difco) and urea agar (BD/Difco) slants, and identities confirmed using agglutination with Salmonella somatic antisera (Salmonella latex test; Oxoid, Basingstoke, UK) and biochemical testing (Analytical Profile Index, API 20E strips; Biomerieux, Marcy l'Etoile, France). Salmonella serovars were serotyped at the National Microbiology Laboratory (Canadian Science Center for Human and Animal Health, Winnipeg, MB).

Manure Preparation
A composite sample of 50 L hog manure slurry from a nursery barn located in the regional municipality of La Broquerie, 45 km south east of Winnipeg, Manitoba, was collected after agitation for 24 h at the end of October 2004, during pump-out for soil application. Analyses conducted to examine the nutrient content of the manure are shown in Table 2. Selection of this manure type for use in tests for Salmonella survival was based on prior observations that Salmonella survived better in this type of manure than in manure from sow or feeder barns when stored at 4°C (>300 d) (Arrus et al., 2006). The manure slurry was stored at 4°C in tightly capped containers to prevent pH change. Three samples were examined for the presence of Salmonella following the procedure described above, and manure was used in experiments within 30 d.


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  		Table 2. Nutrient profile{dagger} of nursery barn manure.

 
Inoculum Preparation
Salmonella serovars used were human clinical isolates and were chosen because five of the six are among the ten most frequently isolated serovars of S. enterica from both human and non-human sources in Manitoba and Canada (Guan and Holley, 2003b). The sixth serovar, S. Oranienburg, was chosen because it is ubiquitously distributed in farm environments (mammals, plants, feed, food) worldwide. Salmonella serovars (S. Typhimurium 03-5608, serotype 4,5,12:i:1,2; S. Agona 03-0890, serotype 4,12:f,g,s:-; S. Hadar 03-4494, serotype 6,8: z10: e,n,x; S. Oranienburg 04-6532, serotype 6,7,14:m,t:-; S. Heidelberg S467, serotype 4,5,12:r:1,2; and S. Montevideo S1451, serotype 6,7:g,m,p,s:-) were obtained from the National Microbiology Laboratory (Canada) and maintained at –80°C until used. The bacterial cultures were activated by transferring loopfuls into 30 mL of trypticase soy broth (TSB) (BD/Difco) and incubated at 35 to 37°C for 24 h. Following two consecutive 24-h culture transfers, the cells were harvested by centrifugation at 2800 x g for 10 min. The pellets were resuspended in 30 mL sterile saline water (8.6 g/L NaCl). Cell concentrations were adjusted with sterile saline to obtain OD600 values in the range 0.255 to 0.300 for each Salmonella serovar (Ultrospec 2000; Pharmacia Biotech, Baie d'Urfe, QC, Canada), which corresponded to approximately 2 x 108 colony-forming units per mL (cfu/mL) based on direct plate counts (TSA, 24 h, 37°C). A six-serovar cocktail of Salmonella was prepared by mixing equal volumes of each standardized bacterial strain. This cocktail was used in the subsequent studies.

Soil Preparation
About 400 kg of each of the two soils used in this study, mapped as Reinfeld loamy sand and Marquette clay, was collected from Carman and Rosser, MB, respectively, and brought to the University laboratory. Soils were analyzed for physicochemical properties as noted in Table 3. Samples were air dried by spreading the soil on a fiberglass tarpaulin at 22°C for 4 d. Soil was sieved using a commercial 2-mm screen, manually mixed, and stored in commercial, 9-L white plastic pails (23.3-cm diameter x 24.6-cm height; Kay Containers, Winnipeg, MB, Canada) with snap lids until the start of the experiment. Pail lids were perforated with 4- x 6-mm holes. Water holding capacity (Cassel and Nielsen, 1986), bulk density of the soil in the field, and texture of all soils were assessed (Sheldrick and Wang, 1993). In addition, three samples of each soil type were examined for the presence of Salmonella using the enrichment method previously described.


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  		Table 3. Characterization of test soils.

 
The moisture level of soils was adjusted to 60 or 80% of field capacity by the addition of distilled water or liquid manure to soils (Cassel and Nielsen, 1986). To account for soil bulk density differences, either 4.4 kg of loamy sand or 3.6 kg of clay soil were added to each pail to reach a height of ≥20 cm. Soil moisture was restored to its original level on a biweekly basis using distilled water additions calculated on the basis of weight loss. The amount of liquid manure added to soil was 25 g/kg of the total soil present. This corresponds to an application rate of 37 000 L/ha incorporated to a 30-cm depth, which is commonly used regionally and in Minnesota (Gessel et al., 2004). Each treatment consisted of three pails of soil repeated in triplicate as described below.

For the manure surface treatment, an amount of manure representing 25 g/kg of the soil in each pail was mixed with 1 kg soil from the pail plus the standardized Salmonella cocktail (in distilled water) added at a rate of 1 mL/kg soil in the total treatment to achieve a final level of 5 log cfu/g soil. After mixing in a separate sterile plastic container with gloved hands, the inoculated manure-soil was added back to the surface of the soil in the original pail.

For mixed manure-soil treatments, the weighed soil from each pail was removed and mixed thoroughly (using gloved hands) with manure (25 g/kg) plus 1 mL Salmonella inoculum/kg soil. Controls contained 25 g manure/kg soil without addition of Salmonella.

For treatments without manure addition, weighed soil was transferred from its pail to a separate sterile container where 1 mL/kg Salmonella inoculum (in distilled water) was added and mixed thoroughly with gloved hands.

After mixing, the soil samples were returned to their respective pails and the bulk density of the soil was adjusted by compressing the soil with a sterile plastic lid until it reached a height of 20 cm. The initial Salmonella inoculation level of 105 cfu/mL was verified by colony counts on XLT-4 agar using a spiral plater (Autoplate 400; Spiral Biotech, Norwood, MA). Containers were covered with perforated lids to allow for some aeration and were stored under temperature-controlled conditions. Three 180-d temperature sequences representing winter to summer (–18, 4, 10, 25°C), spring to summer (4, 10, 25, 30°C), and summer to winter (25, 10, 4, –18°C) seasonal periods were used for environmental exposure of soils, with a 45-d period for each temperature interval in the simulated season. At the end of each period, pails were directly moved to another incubator pre-set at the next designated temperature.

Assessment of Salmonella Survival in Inoculated Soil during Storage
Recovery of viable Salmonella was examined at 0, 7, and 15 d post-inoculation and thereafter at 15-d intervals to 180 d. Samples were collected to 10 cm using a 2-cm-diameter soil auger. When incubated at –18°C, soil subsamples were collected using an electric drill equipped with an alcohol sterilized 1.1-cm-diameter auger bit. Subsamples of soil (25 g) were placed in filter bags (Whirlpak; Nasco, Fort Atkinson, WI) and combined with 50 or 75 mL of sterile saline resulting in a 1:3 (w/v) or 1:4 dilution, respectively. Bags were shaken vigorously for 1 min and 50 or 250 µL were spread on XLT-4 agar using the spiral plater. Inoculated plates were incubated at 37°C for 24 or 48 h. Colonies exhibiting typical Salmonella characteristics were counted. The detection limit for bacterial recovery was 12 cfu/g. When bacterial growth decreased below the detection limit (no recovery), enrichment as previously described was used to confirm Salmonella absence or presence. Salmonella were monitored until two consecutive soil samples at different storage times were negative following enrichment. About 400 isolates from XLT-4 agar and from enrichment samples were serotyped at the National Microbiology Laboratory.

Statistical Analysis
Salmonella survival data were examined using an analysis of variance (ANOVA) procedure (SAS Institute, 1990). Differences among treatment means were compared using Tukey's tests. Data were transformed to log10 numbers before analysis. When Salmonella was not detected by direct plating, a value of 1.0 log cfu/g was assigned for calculation of the mean and standard deviation.

The length of Salmonella survival in treatments was expressed as the decimal reduction time or DRT (days required for 90% reduction in viability). The DRT was calculated according to the formula: DRT = –1/{alpha}, where {alpha} corresponded to the slope of the linear regression line fitted to the data points until that time when Salmonella was unable to be quantified. A DRT45d was also calculated, and it represented the DRT observed following the first 45-d exposure at the initial temperature of each test sequence.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The nutrient content of the hog nursery manure used in this study was within the average range reported for manure stored in open lagoons in the Manitoba area and is presented in Table 2. The Reinfeld soil had a loamy sand texture with higher bulk density as well as lower organic carbon and field capacity than the Marquette clay soil (Table 3). Manure collected from the nursery barn and used in these tests initially contained Salmonella. The isolates were confirmed as S. Albany serotype 8:z4,z24:- and S. Albany 8,20:z4,z24:- by the National Microbiology Laboratory (Canada).

Salmonella was not detected in any of the soil samples tested before manure application, but S. Albany 8:z4,z24:- (one of the adventitious manure contaminants) was recovered for up to 7 d from non-inoculated loamy sand soil amended with manure and stored at 4°C. However, this organism was not detected in soil samples at any time when stored at –18 or 25°C. The other S. Albany serotype (8,20:z4,z24:-) originally present in the nursery barn manure was not found in any stored samples.

Evaluation of treatment effects on survival of Salmonella in soil was complicated by numerous statistically significant interactions between and among factors studied (Table 4). Because each of the factors examined interacted with one or more factors affecting Salmonella survival, the data on survival are presented in a separate table for each soil studied (Tables 5 and 6). This facilitated the analysis of the data and allowed inspection of fewer factors at the same time. The decimal reduction time (DRT) was determined for each type of soil, the method of manure application, and soil temperature regimen. The DRT45d was calculated since results obtained showed that the greatest reduction occurred within the initial 45-d storage period of each sequence of temperatures tested.


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  		Table 4. Significant interactions of factors that influenced the survival of Salmonella serovars in inoculated soils.

 

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  		Table 5. Survival (log cfu/g) of Salmonella{dagger} in Reinfeld loamy sand soil (60 and 80% field capacity{ddagger}) stored at different temperature regimens.§

 

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  		Table 6. Survival (log cfu/g) of Salmonella{dagger} in Marquette clay soil (60 and 80% field capacity{ddagger}) stored at different temperature regimens.§

 
Consistently, levels of Salmonella decreased with soil storage time. Overall, the greatest decrease in Salmonella occurred during the first week of storage regardless of the treatment and temperature. Numbers of surviving Salmonella cells at this interval among the temperature sequences used were significantly different (p ≤ 0.05). As the simulated spring-summer season progressed it showed the slowest rate of Salmonella reduction (averaging 2.6 log cfu/g) and was followed by the summer-winter sequence (2–4 log cfu/g) during the first 6 wk. The simulated winter-summer seasonal sequence yielded the most rapid initial decline in cell numbers.

Salmonella survived longest (≥180 d) in both loamy sand and clay during the simulated summer-winter season (25, 10, 4, –18°C) but was not isolated after 165 d from loamy sand soil stored during the other simulated seasonal periods. In soils without added manure, Salmonella survival was longer in clay than the loamy sand during the seasonal temperature sequences starting with 4 or –18°C (spring or winter, respectively).

Manure application enhanced the survival of Salmonella in soil. It was also found that the method of manure application significantly (p ≤ 0.05) influenced the survival of Salmonella in soils. Although there were exceptions, when bacteria were applied to the surface greater survival of Salmonella was found than in treatments where manure and inoculum were mixed throughout the soil. As expected, it was found that Salmonella survived significantly (p ≤ 0.05) better in Marquette clay soil than in the more coarsely textured Reinfeld soil. In both soils, higher soil moisture appeared to favor survival of Salmonella.

The influence of soil temperature at manure application on Salmonella reduction in manure-treated soil was substantial. Reduction of Salmonella in soil reached an average of 2.7 log cfu/g in samples initially stored at 4°C for 45 d and 60% FC while an average reduction of 4.4 log occurred in samples stored at 25°C for 45 d (Tables 5 and 6). A lower storage temperature and a higher soil moisture content favored Salmonella survival. At 80% FC and 4°C, numbers of Salmonella in the presence of manure reached the lower detection limit at 90 to 105 d regardless of soil type (Tables 5 and 6). In contrast, Salmonella reached the lower detection limit within 30 to 75 d in soil incubated at 25°C with 60% FC. In soil samples stored at –18°C and 60% FC, Salmonella levels were reduced 5 log cfu/g during the first 45 d. The DRT45d ranged from 11 to 15 d in soil samples that received manure. In contrast, the DRT45d for Salmonella in soils without manure addition, initially stored at –18°C, was <0.5 d and levels reached the detection limit in 15 d.

The Salmonella isolates recovered from inoculated loamy sand and clay soils are shown in Table 7. Salmonella Oranienburg, S. Hadar, S. Agona, and S. Heidelberg survived up to 180 d of storage, whereas the S. Montevideo and S. Typhimurium serovars in the inoculated cocktail were not recovered from any of the soil samples.


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  		Table 7. Salmonella serovars{dagger} recovered from inoculated Reinfeld loamy sand and Marquette clay stored at different temperatures{ddagger} and at different field capacities (FC).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Salmonella has been reported to survive up to 300 d in soil inoculated with cattle or hog manure slurry (Baloda et al., 2001; Jones, 1986) and 231 d in soil with composted cattle manure (Islam et al., 2004). Other work indicated that S. Typhimurium survived 119 d in sandy soil and loamy sand (Natvig et al., 2002). In the present study, Salmonella survived more than 180 d in manure-amended loamy sand and clay soils stored during simulation of a summer-winter seasonal temperature sequence. Salmonella Albany, originally present in the manure, was isolated from manure-amended soil up to, but not beyond, 7 d. Factors affecting Salmonella survival are evaluated individually below.

Influence of Temperature
Temperature was shown to have a significant (p ≤ 0.05) effect on survival of Salmonella in soil. For example, during the first 45 d of storage, survival was higher at 4 than 25°C. This result is in agreement with previous findings by Cools et al. (2001) and Zibilske and Weaver (1978) who reported that hog manure application during the colder months increased survival of inoculated bacteria in soil. Similar results were reported by Tamasi (1981) and Islam et al. (2004). In the present study Salmonella levels declined relatively quickly in soil samples stored at –18°C, reaching the lower detection limit from ≤30 to ≤120 d depending on the manure treatment.

Initial sample storage at –18°C, which was used to simulate winter conditions, decreased Salmonella levels by ≤4 log cfu/g within 7 d. No freeze-thaw treatments of soil with inoculated Salmonella were used in the present study, but such exposure is known to have a negative effect on Salmonella survival in manure-amended soil (Natvig et al., 2002; Tannock and Smith, 1972). Ray et al. (1972) indicated that slow thawing of Salmonella Anatum cells resulted in injury to more than 90% of the cells present. In addition, Natvig et al. (2002) reported reductions from 0.5 to >4.3 log cfu/g, depending on the number of freeze-thaw cycles the soil underwent. In the present study, Salmonella were not detected quantitatively after 30 d of storage in any of the inoculated soil samples without manure application stored at –18°C. However, Salmonella were isolated from these samples by enrichment after storage for 165 to 180 d regardless of soil type. Manure addition slowed the initial decline in Salmonella viability at –18°C, but had little effect on Salmonella persistence in soil during gradual temperature shifts to 25°C.

There were 2.6 to 5 log cfu/g reductions during the first 45 d when soils were stored at 25°C during the simulated summer-winter season. Nevertheless, Salmonella was consistently isolated from samples in these treatments of ≤180 d of storage (45 d after reaching –18°C). In samples exposed to the spring-summer regimen, Salmonella exhibited a slower initial decline (1–3 log cfu/g) at 4 than –18°C but as temperature increased to 10 and 25°C further reductions of ≤3 log cfu/g occurred. After reaching 30°C, Salmonella was only occasionally recovered by enrichment from the clay loam soil. In contrast with the seasonal effects of temperature on Salmonella viability reported here, Hutchison et al. (2004) reported no differences in initial reductions of pathogens due to the season. However, the authors pointed out that there had only been a 5°C difference for the first 3 wk of their summer and winter field trials. Results from the present study clearly showed that the summer-winter temperature regimen yielded a larger Salmonella reduction during the first month of storage than the spring-summer temperature sequence. However, the length of Salmonella survival was greatest in the summer-winter regimen (≥180 d) in all soils with or without manure. In addition, while exposure of Salmonella-contaminated soils to the winter-summer regimen resulted initially in 4 to 5 log cfu/g reductions at –18°C, viable Salmonella were present when manure was incorporated for 165 to 180 d in loamy sand and clay soils at 60% FC.

Even though survival of Salmonella during a fall-spring temperature sequence was not evaluated, based on the results of the other temperature regimens it can be inferred that temperatures present during the fall would prolong Salmonella survival in soil. This effect is evident in the later temperature steps of the simulated summer-winter season. It is important to note that freeze-thaw cycles were not modeled in these experiments and would be expected to shorten the survival of Salmonella in field situations (Natvig et al., 2002).

Influence of Manure Application
The influence of manure in soil on the survival of Salmonella has been reported (Avery et al., 2004; Hutchison et al., 2004; Jiang et al., 2002; Platz, 1980; Mallmann and Litsky, 1951). Overall, manure was shown to increase the number of viable pathogens in soil and this was thought to be due to an increase in nutrient availability (Dazzo et al., 1973), nitrogen (Gagliardi and Karns, 2000), and organic matter (Tate, 1978). Freitas et al. (2003) demonstrated that organic matter in liquid hog manure rapidly decomposed and was used as a source of energy and nutrients for biomass production by soil microorganisms. In the present study, treatments containing manure slurry showed significantly (p ≤ 0.05) greater survival of Salmonella. This was especially evident in samples that underwent slow thawing (room temperature for 1–2 h) after storage at –18°C where injury would be expected to be high. Nutrient availability provided by manure addition may have allowed cell repair leading to larger numbers of Salmonella being recovered by direct plating of manure-containing samples. It has been reported that Salmonella may require 1 to 2 h at 25°C for repair in an environment with nutrients (Ray et al., 1972). Differences in Salmonella survival in soil amended with manure from different species (hog, chicken) have also been reported (Dowe et al., 1997). Soil amended with chicken manure supported better survival of Salmonella than hog manure.

The method of manure-inoculum application significantly (p ≤ 0.05) influenced the survival of Salmonella in treated soils. In sandy soil (Table 5) there was a significant (p ≤ 0.05) difference in Salmonella survival between surface-applied manure and thoroughly mixed manure treatments. Greater Salmonella survival was found when the slurry was surface-spread compared with results when the inoculum was mixed throughout the soil. Although surface application on the clay soil yielded higher numbers of survivors than the mixed application (Table 6), the values were not significantly (p > 0.05) different. It is important to recognize that because of practical constraints, surface application in this laboratory study involved some incorporation of manure within the soil surface, while in field trials surface application might not involve any or could include delayed incorporation with less significant effects on soil physicochemical properties (Hutchison et al., 2004). Further, design of the present experiments did not include exposure to sunlight which would reduce bacterial survival.

Greater declines in bacterial numbers have been found when animal wastes are spread on the soil surface (Hutchison et al., 2004). In part, this could be due to the detrimental action of sunlight (UV) exposure (Zaafrane et al., 2004; Tannock and Smith, 1972) and temperature or moisture fluctuations as well as atmospheric drying (Hutchison et al., 2005a, 2005b; Gessel et al., 2004). Incorporating manure into soil was reported to double Salmonella survival compared to surface application (Platz, 1980). The surface treatment in the present study involved incorporation of manure-bacterial inoculum to a soil depth of about 5 cm, and while comparable with surface application in some field trials (Côté and Quessy, 2005), it would be atypical of a field situation since soil samples were placed in vented, covered containers where moisture levels were regularly maintained at 60 or 80% FC.

Influence of Salmonella Serovars Present
Among the six Salmonella serovars tested in these experiments, S. Agona, S. Hadar, S. Heidelberg, and S. Oranienburg were repeatedly recovered from inoculated soils from ≤130 to 180 d. However, S. Typhimurium and S. Montevideo were not found in any treatments. The former organism has been used in many studies characterizing Salmonella survival in agricultural environments and it was surprising that it was not recovered from inoculated soils here. It is possible that these serovars were present in Salmonella positive samples, but if they were not present in the largest number, chances for their recovery would have been reduced. Among a group of five Salmonella serovars different from those used in the present study, S. Montevideo survived best in soil and on tomatoes (Guo et al., 2002). Thus, the ability of these pathogens to survive in soil environments is dependent to some extent on the individual characteristics of the Salmonella serovars present.

Influence of Soil Conditions
Irrespective of temperature or sampling times, Salmonella survived significantly (p ≤ 0.05) better in clay than in loamy sand soil. This may have been due to the higher organic matter content (x4) and moisture capacity (x1.4) of the clay soil. The protective effects of moisture and organic matter content of soils on the survival of bacteria have been described in several reports (Cools et al., 2001; Dowe et al., 1997; Tate, 1978; Dazzo et al., 1973; Tannock and Smith, 1972). Generally, soils with a higher FC such as Marquette clay, used in the present study, exhibited higher microbial survival by making water available to microorganisms over longer periods compared to the sandy soils. Platz (1980) also noted that clay soils not only held more water than sandy soils, but the moisture was not as easily lost, which increased bacterial survival.

Moisture has been described as one of the more important factors influencing bacterial survival (Mubiru et al., 2000; Platz, 1980) and soils with higher moisture content favored survival of Salmonella. During the present study in the simulated summer-winter season at 60% FC the DRT for Salmonella ranged from 7 to 17 d in loamy sand but was 29 to 32 d in clay soil. At 80% FC the DRT values were increased and ranged from 13 to 36 and 31 to 79 d for loamy sand and clay, respectively. The influence of FC on Salmonella DRT was not as great in the other two seasonal treatments. Lower storage temperature with higher soil moisture content also favored Salmonella survival. Soil stored at 4°C with 80% FC provided the best survival during the first 45 d of incubation in both soils, with DRT45d values ranging from 14 to 35 d.

The natural microflora of soil also plays a role in the elimination of pathogens (Platz, 1980; Tamasi, 1981). Under warmer conditions indigenous microorganisms flourish (Cools et al., 2001; Jiang et al., 2002; Platz, 1980), becoming more competitive for nutrients and by the production of antimicrobials; however, at 4°C growth may slow or stop and this would mean that competitive organisms present might fail to produce significant levels of antimicrobials (Jiang et al., 2002). This may explain, in part, the greater decline of Salmonella at warmer temperatures during the first weeks of storage. In addition, the endogenous metabolic rate of organisms is higher at higher temperatures, and in marginal environments where nutrient supply is limiting bacteria can die more rapidly because of nutrient exhaustion. It is possible that toxic products formed from nitrite and/or nitrate in manure may have affected Salmonella survival initially in manure-treated soil (Tamasi, 1981).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Salmonella survived better in Marquette clay than in the Reinfeld loamy sand soil and higher soil moisture also facilitated Salmonella survival. Manure application enhanced survival of Salmonella in soil and this was likely due to an increase in nutrient availability. In contrast with results from other studies, Salmonella in the present work survived better when applied on the surface of soil but these samples were not exposed to drying, the effects of UV light, or freeze-thaw cycles. The simulated season of manure application played an important role in Salmonella survival. Simulated summer application of manure provided a large initial reduction of viable Salmonella during the first month; however, it also increased the length of time Salmonella survived (≥180 d). Based on the results from other temperature sequences tested, it can be inferred that fall application of manure would also allow lengthy survival of Salmonella in treated soil. Low temperatures reached in soil in the prairie region during fall application (5°C) would likely cause low initial reduction of Salmonella numbers. A further decrease of soil temperature during winter would prolong Salmonella viability in soil. Naturally occurring freeze-thaw cycles during fall and early spring would be expected to substantially shorten Salmonella viability (Natvig et al., 2002). Direct exposure of soils to –18°C was highly lethal to the inoculated organisms but a small residual population of viable Salmonella was recovered in some treatments up to 180 d.

The decimal reduction time calculated in this study indicated that at normal field conditions ≥30 d after manure application, there would be a 90% reduction of viable Salmonella in manure-treated soil. Since the level of Salmonella in manure is between <3 and 600 organisms/mL (Hutchison et al., 2005b; Hill and Sobsey, 2003) and is normally applied to soil (Gessel et al., 2004) at a rate of 25 g/kg, the risk of environmental contamination and recycling of Salmonella in animals would be minimized by following a 30-d delay between field application of manure and use of the treated land. With this provision, either fall or early spring application of manure should be suitable.


   ACKNOWLEDGMENTS
 
We thank Scott Dick and Cliff Loewen, Elite Swine (Landmark, MB), for supplying manure slurry for the experiments. We also thank David Woodward and Helen Tabor from the National Microbiology Laboratory (Canadian Science Center for Human and Animal Health) for confirming and serotyping Salmonella isolates recovered from manure and soil. Technical assistance of Namita Goswami, Georgina Mejia, and Heather Maskus is gratefully acknowledged. Cooperation of Alvin Iverson from the Carman Research Station and Scott Corbett from the University of Manitoba by supplying the soil used for this study was gratefully appreciated. Financial support was received from the Manitoba Manure Management Initiative, Inc.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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R. Holley, J. Walkty, G. Blank, M. Tenuta, K. Ominski, D. Krause, and L.-K. Ng
Examination of Salmonella and Escherichia coli Translocation from Hog Manure to Forage, Soil, and Cattle Grazed on the Hog Manure-treated Pasture
J. Environ. Qual., October 23, 2008; 37(6): 2083 - 2092.
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