Die-off rates of human pathogens in manure amended soil under natural climatic conditions using novel sentinel chamber system.

Animal production in California produces over 300 million pounds of manure each day. There are various approaches to dispose of manure with the major route being to divert it to field applications as part of an overall nutrition management strategy. Although manure is rich in nutrients required to grow plant it also harbors virulent pathogens that can potentially contaminate crops such as leafy greens. To reduce the risk of pathogens the time period between application of the manure to the field and planting the crop is set. For example, a 45 day period is required from the application of composted manure until crops can be planted. The interval is set using data for pathogen die-off rates under standardized conditions. Yet, pathogen die-off rates are dependent on the climatic conditions (especially variations in weather conditions) that are difficult to recreate under laboratory conditions. Consequently, it is conceivable that pathogens may persist up to the point of crop planting and hence represents a significant food safety risk. To this end the proposed project will study the pathogen die-off rate under actual field conditions. Undertaking environmental trials is problematic due to the ethics of deliberate introduction of virulent pathogens into the environment. In addition, differentiating die-off from the washout of pathogens from the inoculated sites in the soil can results in inaccurate determination of persistence. The applicants have addressed both these issues by devising a novel vial based method in which pathogens are introduced into the soil. The vial contains the spread of pathogens but at the same time exposes the bacteria to the environmental stresses of the general microbiota. The study will establish the die-off rates of a range of relevant pathogens and determine the impact of climatic and soil characteristics. The data generated will enable models to be constructed to facilitate predictive die-off rates. The information will be useful to farmers who can more accurately predict the time interval between composted manure application and planting.

Field production of fruits and vegetables, herbs, and herbal products such as ginseng, frequently includes high application rates of organic materials such as manure and compost as part of crop nutrient management. For organic production systems, these are the only fertilizer materials acceptable for use. Many of currently recommended management practices, such as manure incorporation, are aimed at reducing the nutrient impacts of agriculture on surface and ground waters. However, it is not clear if some of the prescribed practices reduce or enhance risks of pathogen contamination of foods and water. Field studies show some contradictory effects and results are often highly variable because of the compounding issues of pathogen die-off and losses in runoff and/or leaching. The proposed field-based study will use a sentinel chamber (captive population) technique to investigate the die-off of pathogens following land application, as separate from transport issues. This information would identify not only how long pathogens are likely to remain in fields where a food crop is being grown, but also the risk of pathogen transport from one field to another or to an irrigation water source. The information is critical in formulating recommendations for management practices and assessing their effect on pathogen risk management, and the appropriateness of current recommended harvest withholding periods. In the proposed field-based study, sentinel chambers will enable the study of confined human pathogen populations exposed to the same field environmental conditions as the surrounding soil matrix. The study builds on a previous and an on-going study examining pathogen die-off rates under various manure storage management systems. The proposed study examines the effect of alternate application practices (seasonal timing and surface or incorporated application) on captive and bulk soil microbial populations, thus separating survival from transport factors to better inform the evaluation of recommended management practices. The project will be carried out by a consortium that includes academics (Drs. K. Dunfield and K. Warriner (UofG), industry (Soil Resource Group) and officials from the Ministry of Agriculture (OMAFRA). The deliverables from the research will be data to predict the die-off of relevant pathogens under contrasting conditions. Such data can be applied directly for improving manure management and thereby directly enhancing the food safety of fresh produce.

1) Establish a steering committee to advise on trial methodology and disseminate results to stakeholder groups. 

2) Verify the methodology of introducing and enumerating model pathogens into manure-amended manure. 

3) Undertake field trials to determine die-off rates of model pathogens under variable climatic conditions. 

4) Correlate pathogen die-off rates with soil type, season and climatic conditions.

The study determined the die off rates of STEC and Salmonella in manure (dairy and swine) amended soil (loam and sandy loam). The inoculated manure and soil mix was placed into vials that were placed at depth (15 cm) or on the surface of test plots. The trials were performed in the Spring (May) and Fall (October) with the temperature and soil moisture being monitored over the trial period. It was found that the die-off rates of STEC and Salmonella were variable so could not be fitted to linear or non-linear models. As a consequence the time for the population to decrease by 2 log cfu. By doing so it was found that the persistence of Salmonella was lower than that of STEC. Highest persistence of both pathogens was observed in loam soil at depth with lowest persistence on the surface of sandy soil. The rate of die-off of endogenous E. coli was lower than that of pathogens thereby suggesting it is a suitable metric for gauging the microbiological stats of soil. Although there was a relatively rapid die-off rates of STEC and Salmonella under field conditions persistent, low levels of sub-populations were observed over the trial period (90-120 days). Under laboratory conditions the die off rates STEC and Salmonella were lower compared to those observed in the field. Here, high temperature and moisture increase the die-off rate of Salmonella although STEC were less stable under low moisture. Generic E. coli exhibited higher die off rates in laboratory microcosms compared to STEC or Salmonella. The persistence of Clostridium difficile endospores was ribotype dependent with 027 being more persistent compared to 078 which is commonly found in the environment. Recommendations The study has demonstrated that Salmonella and STEC can persist over 120 days in certain soil types. Consequently, it possible that pathogens could be present even after the 90 day wait period before planting crops. However, it should be noted that majority of the cells within the bacterial population die-off within the initial 14 day period leaving a small residual population that persists over an extended time. Recommendation 1: Risk analysis should be performed on the hazard associated with low residual levels of pathogens with respect to contaminating crops or water courses. Recommendation 2: The physiology of sub-populations of pathogens (i.e. STEC and Salmonella) should be studied to determine is environmental fitness has been acquired at the cost of virulence. Recommendation 3: Algorithms should be developed based on predicting the die-off of pathogens under varying conditions of pH, temperature and soil type. The models developed can then predict the level of pathogen die-off under the measured climatic conditions. This would provide a more accurate estimate of the wait period from manure application to planting crops than using the arbitrary 90 or 120 day rule. Recommendation 4: Laboratory based soil microcosm models underestimate pathogen die off in the real environment and hence are suitable for risk assessment studies. Recommendation 5: Selective methods for enumerating pathogens in soil and manure samples are required to be developed. This is especially the case for Listeria monocytogenes were selective, quantitative methods are lacking. Recommendation 6: Further trials are required to verify that die-off models are applicable in different geographical areas. If not then further additional factors that contribute to the rate of pathogen die-off should be investigated.