Epidemiology of VTEC O157 and Other VTECs Likely to be Pathogenic to Man in Farm Wastes

Final report summary: <BR>
Possible routes of human infection of Verocytotoxin-producing Escherichia coli (VTEC) serogroup O157 include contaminated food or beverages, direct and indirect contact with farm animals and person-to-person. In particular, cattle have been identified as the main domestic animal reservoir of VTEC O157 and as such their manure can be a source of pathogens entering the environment and the food chain. Consequently, the epidemiology of VTEC 0157 and O26 in farmyard manure (FYM), slurry and dirty water on both dairy and beef cattle farms has been investigated. The overall aim of the project is to devise a set of recommendations that can be adopted by farmers in order to reduce the risk of human VTEC infection that is attributable to farm waste. To formulate such recommendations, several stages were followed and a multi-disciplinary approach was adopted.
<BR> <BR>
Review of cattle farm waste management practices in the context of the epidemiology of VTEC 0157 <BR>
A review was carried out so that the waste management information for UK beef and dairy farming and available data on the survivability of VTECs in the environment could be related. The information provided by this review is assembled in a format to enable the development of risk and mechanistic models of the epidemiology of VTECs in wastes on cattle farms, and to facilitate field studies to investigate the relative roles of different farm wastes and waste management systems in the survival and dissemination of VTECs within farm wastes. From this review, it was concluded that several aspects of livestock wastes management systems are able to sustain the survival of VTECs, leading to potential risks of transfer to humans who live or work on or visit farms, or other animals and to the broader environment. It was noted that some potential points of conflict exist where measures to avoid environmental pollution may conflict with the need to reduce the risks of pathogen transfer.
<BR> <BR>
Design environmental sampling strategy for the recovery of VTEC from cattle farm wastes <BR>
FOSS Enzyme Immunoassay, spiral plating on Harlequin agar and Chromagar O157, direct plating of dilutions of faeces on Harlequin agar, Chromagar O157 and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) and immunomagnetic separation (IMS) with subsequent plating on CT-SMAC were evaluated for screening of environmental samples for E. coli O157. O157-spiked waste samples were used to assess the specificity and sensitivity of the chosen method. In spiked samples, spiral plating on Harlequin agar detected counts of &#8805;5.4x103 cfu/g E. coli O157 in all replicates and &#8804;5.4x101 cfu/g E. coli O157 in some replicates. IMS detected lower numbers of the target organism. At counts >5.4x103/g, the majority of colonies growing on Harlequin agar plates were shown to be E. coli O157 by latex agglutination. However, below these levels, a decreasing proportion of the colonies were found to be E. coli O157. <BR> <BR>
Spiral plating of 1 in 20 and 1 in 100 dilutions of waste samples on Harlequin agar (for E. coli O157) or Rhamnose-MacConkey agar (for E. coli O26) followed by IMS where the target organism was not detected was chosen for enumerating farm environmental samples for VTEC. Ten colonies from each sample on spiral plates were tested by latex agglutination for the target organism to assess the proportion of the colonies which were positive. This was used to adjust the final count. Representative latex positive colonies were further characterised by serotyping, ‘phage typing and PCR for vt1, vt2 and eae genes. Spiral plating of 1 in 1000 dilutions of waste samples on Chromagar ECC was used to enumerate E. coli.
A standardised environmental sampling protocol was developed and evaluated. Protocols for livestock manure and environmental sampling, enumeration of faeces for E. coli and submission for further characterisation; telephone, screening visit, sampling visit and engineering questionnaires, data capture sheets and desk instructions were finalised.
<BR> <BR>
Environmental farm-based longitudinal study of VTEC in cattle farm wastes <BR>
25 farms were recruited and screened for E. coli O157 in 20 pooled faeces samples from young stock using IMS and plating on CT-SMAC. Six positive farms were enrolled into the longitudinal study and an engineering visit was conducted. One of these farms was also shown to be VTEC O26-positive. From each farm, up to 50 environmental samples (comprising 12 fresh farm wastes, 20 stored farm wastes, 12 dirty water and 6 surface of pasture samples) and up to 45 freshly passed faeces samples from young stock were collected on each visit. Farms were visited pre-turnout (December-March), at pasture (May-September) and after housing (November-December). <BR> <BR>
VTEC O157 was isolated from environmental samples from one farm at the first visit, 2 farms at the second visit and 5 farms at the third visit. Only farm 19 was detectably positive on all 3 sampling occasions. Farm 9 yielded no positive environmental samples on any visit. The results of the study were:
<ul> <LI> VTEC O157 was isolated predominantly from faeces (24/63 positive samples) and stored waste (34/63 positive samples) rather than dirty water (2/63) or pasture samples (3/63).
<LI> Counts were <200 colony forming units (cfu)/g in all but 9 positive samples where counts ranged from 0.4-28.4x103 cfu/g.
<LI> 41/72 representative isolates tested were eae and vt2-positive and 31/72 were eae and vt1 and vt2-positive.’Phage types identified were PT 2, 8, 21/28, 34 and 51
<LI> VTEC O26 was isolated from farm 15 at each visit from faeces samples. Counts in 3 positive samples collected on visits 2 and 3 were all <200 cfu/g. All were vt1 and eae positive.
<LI> E. coli counts and the proportion of samples positive were higher in faeces and stored waste samples than dirty water or pasture samples.
<LI> More E. coli and VTEC O157-positive samples were identified in the autumn months. When the stored waste was turned or stirred, there was a significant reduction in the number of positive VTEC O157 samples. VTEC O157 was also reduced when more than one waste store was used to prolong storage (without addition) before spreading. </ul>
In relation to the engineering visits, at 5 of the farms (one farm refused to participate), closed questionnaires and data collection sheets were designed to facilitate the collection of waste management data from the farms to be visited during the longitudinal study. Information on specific engineering aspects such as waste type, type of waste system and environmental factors were captured. From this, key conclusions of the engineering visits were as follows:
<UL> <LI> None of the 5 farms was grossly different in a systematic way from industry norms;
<LI> Management of livestock hygiene appeared to vary greatly between farms;
<LI> Engineering mass balance data showed that the material flows on the farms were well-understood by all of the farmers; <LI> No common waste management factors were observed that might have a direct link with epidemiology of VTEC O157. </ul>

Development of mathematical and risk models to evaluate the role of different farm wastes in the maintenance and epidemiology of VTEC on cattle farms <BR>
A farm-scale systems analysis model was developed that is capable of simulating almost any conventional type of cattle farm. The model considers many aspects of dairy and beef cattle farming such as the number of fields, yards, barns and the animals in each age group on the farm. It can also specify the characteristics of each enclosure, each group of animals, storage and movement of waste and movement of animals within and on and off the farm. Using this model, it was estimated that the proportion of farms that are positive can vary (depending on time of year and field type) between 0.55 to 0.83. Likewise, the median concentration ranged from 1.5 - 1× 105 cfu/m2 and at the 95th percentile concentration ranged from 8.5 - 1.5 × 106 cfu/m2. The model considered many potential interventions and these were used in the production of recommendations for farmers.
<BR><BR>
The VLA risk assessment model assesses the risk of human infection with VTEC O157 due to either direct or indirect contact with livestock waste. In particular, the effect of different farm waste management practices on this risk is evaluated. Specifically, the models investigated the impact on the human risk originating from slurry and dirty water spread onto land on dairy farms and the risk originating from FYM and dirty water spread onto land on beef farms.
Overall, based on the model results and assumptions, it was concluded that the risk of acquiring VTEC O157 human infection due to direct consumption of soil spread with farm waste is low (i.e. &#61603;0.09 with 95% certainty). Specifically, on average, on &#61603;1.4% of farms visited whereby spreading occurred prior to the visit, VTEC O157 human infection will arise from consuming soil contaminated with spread waste. The amount of VTEC O157 per gram of soil at the point of harvest of potatoes and sugar beet was also low (i.e. &#61603;2.6x10-8 for sugar beet and 33.35 for potatoes with 95% certainty). For beef farms specifically, this risk can be reduced by turning the manure heap, or by storing all FYM before spreading.
<BR><BR>
Recommendations to reduce environmental contamination with VTEC <BR>
The management of waste on farms significantly varies between farms, however there are recommendations/guidelines available to which farmers should adhere. Such recommendations on the many aspects of waste management often with particular reference to the transfer of food-borne pathogens on to crops and are not VTEC specific. Consequently, using a multidisciplinary approach, recommendations have been identified for VTEC O157, with the assumption that VTEC O26 will respond in a similar way to O157. Resulting from this, the following recommendations were identified:
<UL> <LI>
Use “partial housing” to reduce the amount of waste to be stored
<LI> Store FYM separately from the animals
<LI> Cover the midden to reduce the amount of leachate entering the dirty water store
<LI> Remove waste from housing/yards as often as possible
<LI> Clean the housing as well as possible
<LI> Store all waste types for as long as possible
<LI> Do not directly spread dirty water onto fields (i.e. store first)
<LI> Have an adequate storage facility for slurry and dirty water
<LI> Turn stored FYM
<LI> Stir stored slurry
<LI> Store FYM in a field heap prior to spreading
<LI> If possible, use more than one store for a type of waste (to allow storage without additions)
<LI> When spreading waste, take the soil type into consideration.
<LI> Duration between spreading and turn-out should be as long as possible.
</ul>
Overall, the recommendations from this project are in accordance to those that were in previous existence although some further work is required on some other potential recommendations. However, we can now be more certain about the scientific justification of these recommendations. It can therefore be concluded that farmers adhering to existing guidelines are managing their waste in such a way that minimises the risk of environmental contamination with VTEC. Consequently, by adhering to the existing guidelines, cattle farmers are reducing the risk of VTEC O157 spreading through their herd and maintaining a low risk of humans becoming infected with VTEC O157 due to direct or indirect exposure to cattle waste.
These recommendations will be disseminated back to farmers that participated in the study.
<P>