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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aims: To monitor the decay of E. coli O157 in soil (loamy sand) on a scout campsite following an outbreak in humans.

Methods and Results: Samples of soil and sheep faeces were collected from the campsite and tested for the presence of E. coli O157 by immunomagnetic separation (IMS) after enrichment in buffered peptone water + vancomycin at 42°C for 6 h. Enumeration of target was carried out by direct plating onto sorbitol MacConkey agar plates supplemented with cefixime and tellurite (CTSMAC) incubated at 37°C for 24 h. Low numbers (< 100 g−1) were estimated by the most probable number (3-tube MPN) technique.

Conclusions: Survival was observed for 15 weeks.

Significance and Impact of the Study: A number of laboratory studies have followed the decay of E. coli O157 in soil, animal faeces and water. This study follows (for the first time) the decay of the organism in soil after an outbreak associated with sheep. It demonstrates the long-term persistence of the organism in the environment and the results will be potentially important in performing risk assessments for both human and animal infection.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Escherichia coli O157 is a major food-borne pathogen in humans causing increasing concern world-wide as the number of incidences continue to rise (Jones 1999). The consumption of meat (Ahmed and Donaghy 1998Chapman et al. 2000) and dairy products (Upton and Coia 1994) have been linked to human infection and contact with animals has been shown to be a significant risk factor (Reilly et al. 2000). The current incidence rate of E. coli O157 in Scotland is approximately 6 per 100 000 population which is threefold greater than the rest of the UK. There have been a number of E. coli O157 outbreaks in a relatively small area some 20 miles north of Aberdeen, mostly associated with the consumption of cheese made from unpasteurized milk (Anon 1999). The location in this report, the New Deer Agricultural Showground falls within this geographical area. This region is extensively used for agricultural purposes but there remains no other indication or explanation as to why the locality appears to present a greater risk of human infection from E. coli O157 than other rural areas.

In May 2000, a scout camp attended by 337 people was held at the New Deer Agricultural Showground. Twenty scouts aged between 8 and 20 were later confirmed as having E. coli O157 with dates of onset suggestive of a point source outbreak. Investigations revealed that the field had been grazed by approximately 300 sheep prior to the camp and subsequent testing of 28 animals revealed 17 shedding E. coli O157. The Outbreak Control Team instigated microbiological analyses and E. coli O157 was isolated from soil, sheep faeces, standing water and scout climbing frames at the field. Isolates of E. coli O157 from animal, environmental and human sources were indistinguishable by pulsed field gel electrophoresis. Heavy rainfall during the camping period caused localized flooding and mud and faecal material was widespread. The likely route of E. coli O157 transmission was via hands contaminated with mud. Further investigations showed extensive contamination of all areas within the field. Strachan et al. (2001) modelled the transfer of E. coli O157 from sheep to the soil and subsequently to humans and demonstrated a low infective dose (4–24 organisms) which outlines the risk associated with recreational use of agricultural land.

A summer agricultural show was due to be held in the campsite field some 6 weeks after the scout camp weekend and the presence of contaminated soil and residual faecal material posed a potential risk to attendees. We investigated the long-term survival of E. coli O157 in the field, a hitherto difficult task as the highly infectious nature of this pathogen has prevented its experimental introduction outside laboratory containment facilities. Maule (1997) demonstrated prolonged survival (130d) of E. coli O157 in soil in laboratory conditions and other studies (Fenlon et al. 2000; Ogden et al. 2001) have compared the survival of commensal E. coli with E. coli O157 and have followed the fate of non-pathogenic strains in the environment after the application of slurry to agricultural sites. Fenlon et al. (2000) showed that survival of atoxigenic E. coli O157 spiked into loam and clay soils could exceed 20 weeks. Hence the aim of the current study was to monitor the New Deer field to determine survival of E. coli O157 shed by sheep.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Field samples

Soil (loamy sand) samples (top 2·5 cm including grass) and sheep faecal material were collected weekly after the outbreak but prior to the summer show, and monthly thereafter. Material was placed aseptically in sterile plastic bags, stored at 4°C in an insulated container and transferred to the laboratory within 2 h. Samples were tested on the day of arrival. A rain-gauge was installed as other work (Fenlon et al. 2000) suggested leaching of E. coli is greatest following rainfall events.

Long-term survival of E. coli O157 in soil was performed after the infected sheep were returned to the field. To prevent re-infection of soil from the sheep, an area (2 m × 3 m) was fenced in a position not liable to re-contamination by surface water run-off. There were no visible signs of faecal material (other than residual sheep faeces) from wild animals (e.g. rabbits) or birds.

Sheep samples

Faecal samples from sheep were collected three weeks after the scout camp by digital sampling per rectum. The number of sheep tested was approx. 10% of the entire flock and those sampled were selected randomly.

Microbiological analyses

Samples (25 g) were enriched in buffered peptone water (Oxoid CM509) supplemented with vancomycin (8 mg l−1) at 42°C for 6 h and 1-ml aliquots removed to determine the presence/absence of E. coli O157 by immunomagnetic separation (IMS) using 0·02 ml CaptivateTM O157 immunomagnetic beads (International Diagnostics Group, Bury, Lancs, UK). Beads were recovered after 3 washes in buffer (PBS + 0·05% Tween 20), resuspended in 0·1 ml buffer, spread onto two sorbitol MacConkey agar plates supplemented with cefixime and potassium tellurite (Mast SV48) (CTSMAC) and incubated at 37°C for 24 h. Non sorbitol fermenting colonies were confirmed as E. coli O157 by latex agglutination (Oxoid DR620). Numbers of E. coli O157 were estimated by direct plating onto CTSMAC and by the most probable number (3-tube MPN) technique with each dilution tested for the presence/absence of target by the IMS method described above.

Presence of pathogenicity markers, VT1 (Russman et al. 1995), VT2 (Mariani-Kurkdjian et al. 1993) and eaeA (Willshaw et al. 1994) genes were investigated in strains isolated at the beginning and end of the study.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Table 1 shows the results of E. coli O157 analysis on a range of samples collected at random across the whole campsite and indicates the extensive contamination of E. coli O157 one week after the scout camp. A limited number of samples were enumerated for E. coli O157 which indicated low levels of contamination in the soils at approx. 3 g−1 and levels between 3 and 1·1 × 104 g−1 in the faeces (data not presented).

Table 1.   Field analysis for E. coli O157 one week after the scout camp (sampled June 6th) Thumbnail image of

Table 2 shows analyses from freshly collected sheep faeces taken 3 weeks after the outbreak. The distribution of pathogen concentrations ranged between absent and (in one lamb) > 106 g−1. While the majority of ewes were positive (12/15), most lambs tested showed no presence of E. coli O157 (11/13).

Table 2.   Range of E. coli O157 numbers in New Deer sheep faeces (sampled 29th June) Thumbnail image of

Table 3 shows the number of soil and faeces samples positive for E. coli O157 during the period prior to the summer show. Associated rainfall is indicated. Numbers of E. coli O157 were < 10 g−1 in soil and between < 1 and 1 × 103 g−1 in faeces.

Table 3.   weekly sampling of residual faeces and soil for E. coli O157 with associated rainfall Thumbnail image of

The presence/absence of E. coli O157 in the six month period following the summer show is given in Table 4 and shows its continued presence for approx. 15 weeks. Enumeration was not attempted as the IMS procedure (which included a 6-h enrichment) produced small numbers (< 10) of target colonies indicating low levels of E. coli O157 in each sample. Throughout this period, there was significantly greater than average rainfall but levels of E. coli O157 appeared to remain relatively constant within the soil at below direct plating threshold levels.

Table 4.   monthly testing of soils for E. coli O157 with associated rainfall Thumbnail image of

There was no loss of pathogenicity genes in those strains tested isolated at the beginning and end of the 15-week survival period. Presence of VT2 and eaeA were observed in all cases and there was an absence of the VT1 gene.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This study demonstrates a survival of E. coli O157 in soil of approx. 105 d which is somewhat shorter than the laboratory based data (130 d) generated by Maule (1997). Leaching by long periods of heavy rain would have contributed to the decline of E. coli O157 in this study and may in part explain the difference. Gagliardi and Karns (2000) suggested the presence of manure (as seen in this study) enhanced the survival of E. coli O157 in untilled soil, due possibly to enhanced microsite habitat. Gagliardi (personal communication) indicated plant roots, particularly Rye grass, support E. coli O157 for longer periods and at higher numbers than soil alone. The possibility exists that E. coli O157 isolated within the fenced area were deposited in sheep faeces some time before this work began (e.g. at the time of the scout camp some 14 weeks earlier) and therefore our estimate of 15 weeks survival may be low. Long-term survival may also be a function of the large numbers of E. coli O157 shed daily (80% of ewes were positive plus the presence of a high quantity shedding lamb) while infected sheep were grazing.

From laboratory inoculated studies, Fenlon et al. (2000) showed survival of E. coli O157 was dependent on soil type with 5-log reductions observed in sandy soil within 8 weeks and similar reductions within 25 weeks in loam and clay soils. These soils were enclosed in sealed plastic bags stored at ambient temperatures (−5°C to + 20°C) but were not subject to leaching by rainfall. Ogden et al. (2001) showed the die off rate of E. coli O157 was slightly quicker than commensal E. coli and proposed the latter could be used to indicate the field behaviour of the pathogen. The overwhelming majority of such work relies on inoculating E. coli O157 into the substrate of concern at high concentrations (e.g. > 105g−1), thus permitting continued monitoring (as numbers decline) at above threshold levels. Inoculations at low levels (e.g. < 102 g−1), while being more realistic would quickly result in numbers becoming non-detectable without enrichment. Fenlon et al. (2000) applied low numbers (approx. 30 per 100 ml−1) of E. coli O157 to land in naturally contaminated slurry and their presence was only detected by enrichment for up to 7 d post application. These studies emphasize the need to monitor the fate of E. coli O157 in naturally contaminated soils.

The transfer of E. coli O157 from faeces to soil in this outbreak would have been facilitated by animal and human activity, during wet weather in particular. Some E. coli O157 may have been leached from the upper soil layers during the wet conditions at the time of this study but it was not possible to locate any adjacent water courses to test for E. coli O157. Remaining E. coli O157 in soil, detectable only by enrichment methods, may have been located in micropores not subject to leaching by the regular heavy rainfall.

Numbers of E. coli decay with time when applied to soil. Ogden et al. (2001) showed a decimal reduction time of approx. 16 d for E. coli in slurry applied to soil during spring and autumn periods in Scotland. The work reported here shows an extended survival not in accordance with the above data and demonstrates the ‘tail’ effect of exponential decay seen in many bacterial survival studies. The discrepancy may be due to ‘hot-spots’, i.e. localized areas containing high numbers of microbes originating from faecal material from animals shedding high numbers of E. coli O157. It should be noted that visible evidence of faeces did not extend beyond 8 weeks. However, it is possible that relatively high numbers of target bacteria adhered to soil in these areas and were collected for microbiological examination.

The majority of data on E. coli O157 in animals is from cattle and this study stresses the importance of sheep being significant hosts to the pathogen. Kudva et al. (1996) tested whether sheep (ewes) were a natural reservoir of E. coli O157 and found a transient incidence ranging from zero in November to 31% in June. Although numbers shed were not calculated, it was estimated that they were low as the target was not detected without enrichment. Escherichia coli O157 studies in cattle have most often been based on presence or absence assays. Zhao et al. (1995) showed individual cows in a herd to shed large variations of E. coli O157 numbers (0–105 g−1) but data from UK animals is minimal. The presence of a lamb shedding > 106 g−1 at New Deer may not be abnormal in NE Scotland and may account for the high incidence of human illness of E. coli O157 compared to elsewhere.

Comparisons were made between some virulence genes in strains isolated at the beginning and the end of this study. After 15 weeks in soil, E. coli O157 still possessed both the VT2 and the eaeA gene suggesting the potential for causing human illness. We did not investigate to what extent these genes were capable of expression but James and Keevil (1999) showed that limited oxygen (as might be experienced in subsoil environments) enhanced adherence and did not affect verocytotoxin expression. However, survival under starvation conditions can affect factors such as antigenicity (Hara-Kudo et al. 2000) and so it remains unclear as to the full pathogenic potential of long-term stressed E. coli O157, a subject requiring further investigation.

The outbreak at the New Deer campsite presented an opportunity to monitor the natural decay of E. coli O157 in soil outside laboratory conditions. Such data is essential to validate an increasing amount of information from both spiked E. coli O157 studies and work using marker organisms (e.g. commensal E. coli). Perhaps of more importance is the need for data for use in risk assessment exercises and for advice to users of recreational parks, etc. Decay rates in specific soils during different weather conditions will allow numbers of E. coli O157 in the soil to be estimated and reliable safety issues to be imposed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank the Food Standards Agency Scotland for funding this work and also thank members of the New Deer Agricultural Committee and the number of co-workers who contributed throughout.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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