Evaluation of amphibians and reptiles as potential reservoirs of foodborne pathogens and risk reduction to protect fresh produce and the environment.

Our proposal will help the leafy greens produce industry determine if wild amphibians (frogs, toads) and reptiles (lizards, snakes) are potential carriers of E. coli O157:H7 and Salmonella in the central California coast produce production region. We will identify management practices and prevention strategies that reduce the risk of contamination of leafy greens and nearby waterways by these species. Statistical procedures and epidemiological methods will be used to complete three objectives: 1) determine if wild amphibians and reptiles are reservoirs of E. coli O157:H7 and Salmonella in the central California coast; 2) identify farm production practices, environmental factors and control strategies that reduce the risk of contamination from amphibian and reptile species in the leafy greens produce growing environment, and 3) extend knowledge of preventing produce contamination by amphibians and reptiles to the produce community. The science-based data from this study will support co-management to promote both food safety and environmental goals in the central California coast. Specifically, the results will improve pre-season and pre-harvest environmental assessments and interventions as required in the Leafy Green Marketing Agreement (LGMA) metrics, in particular those addressing animal intrusions.

Human foodborne outbreaks and recalls associated with the consumption of leafy green produce contaminated with bacterial pathogens such as E. coli O157:H7 and Salmonella continue to occur despite efforts to improve the microbial safety of these commodities (Lynch 2009). This is in part the result of not having clearly identified the primary vertebrate sources of enteric bacteria for these commodities and extending that information to the produce growing agricultural community. Furthermore, the uncertainty surrounding the relative importance of different wildlife species and their habitat in the transport of foodborne pathogens has resulted in perceived conflicts between food safety and environmental goals, especially in the central California coast. The overall goal of the proposed project is to conduct research that will provide a science-based approach that will reduce or eliminate bacterial contamination of leafy green produce by amphibians and reptiles while minimizing negative impacts on native wildlife and their habitat. We will accomplish our goal by completing three objectives: Objective 1: Determine if wild amphibians and reptiles are reservoirs of E. coli O157:H7 and Salmonella in the central California coast. We will identify the major species of amphibians and reptiles populating the leafy greens produce production environment and surrounding rangeland through intensive trapping and microbiological analysis. Objective 2: Identify farm production practices, environmental factors and control strategies that reduce the risk of contamination from amphibian and reptile species in the leafy greens produce growing environment. Through statistical analysis, we will identify specific environmental factors (e..g, riparian habitat, wetlands, vegetation strips, ponds) and management practices that are associated with reduced pathogen prevalence. Molecular genotyping will be used to source track and provide a comparison of the genetic relatedness of strains from these animals with strains from waterways where they are collected, as well as comparison with our extensive database of human and environmental strains. Objective 3: Extend knowledge of preventing produce contamination by amphibians and reptiles to the produce community. We will share the science-based knowledge gained from this study with growers, handlers, buyers, auditors, regulators, conservation groups, and other stakeholders to improve best practices relating to pre-season and pre-harvest environmental assessments and wildlife intrusion. The study hypothesis will be tested through a combination of epidemiological and microbiological approaches including intensive environmental sampling, field biology, bacteriologic analyses, source tracking using state-of-the-art molecular techniques, and statistical analyses. Upon completion, science-based data from this study will support both food safety and environmental stewardship. Specifically, the results will improve pre-season and pre-harvest environmental assessments and interventions as required in the Leafy Green Marketing Agreement (LGMA) metrics, in particular those addressing animal intrusions.

1. Determine if wild amphibians and reptiles are reservoirs of E. coli O157:H7 and Salmonella in the central California coast. We will identify the major species of amphibians and reptiles populating the leafy greens produce production environment and surrounding rangeland through intensive trapping and microbiological analysis. 

2. Identify farm production practices, environmental factors and control strategies that reduce the risk of contamination from amphibian and reptile species in the leafy greens produce growing environment. Through statistical analysis, we will identify specific environmental factors (e.g, riparian habitat, wetlands, vegetation strips, ponds) and management practices that are associated with reduced pathogen prevalence. Molecular genotyping will be used to source track and provide a comparison of the genetic relatedness of strains from these animals with strains from waterways where they are collected, as well as comparison with our extensive database of human and environmental strains. 

3. Extend knowledge of preventing produce contamination by amphibians and reptiles to the produce community. We will share the science-based knowledge gained from this study with growers, handlers, buyers, auditors, regulators, conservation groups, and other stakeholders to improve best practices relating to pre-season and pre-harvest environmental assessments and wildlife intrusion.

This is the first survey of foodborne pathogen occurrence in wild amphibians and reptiles in two diverse produce production regions in the United States. The findings will help inform the industry on potential reservoirs of zoonotic foodborne pathogens, and good agriculture practices to prevent microbial contamination from animal sources. We confirmed that common wild amphibian and reptile species in the central California coast and southeastern Georgia may shed Salmonella, but E. coli O157 and other STEC strains are rare. In California, Salmonella was not detected in pre-irrigation reservoirs where positive frogs and snakes were identified. This suggests that the animals are not contaminating the water, although longer-term studies are needed to confirm this finding. In contrast, Salmonella was isolated from both toads and turtles in pre-irrigation ponds in Georgia, and similar genotypes were identified. Notably, irrigation water stored in reservoirs is sourced from wells in California, whereas water from the ponds in Georgia originates from rain and surface water sources including irrigation runoff, which may increase the risk of these ponds serving as disease reservoirs (Rajabi et al, 2011). It is worth noting that the microbiological and chemical water quality parameters of water collected from tailwater (sediment) ponds and grassed ditches were potentially lower compared with other water sources at the California sites. Specifically, higher concentration of indicator bacteria (generic E. coli), dissolved oxygen, and chemicals (ammonium, nitrate, nitrogen) were found in these waterbodies. This may reflect the ability of these constructed sites to capture eroded soil and runoff, which has proven beneficial to the environment in agriculture settings (Tate et al, 2006; Knox et al, 2008). Additional studies are underway between UC Davis and NRCS to examine the ability of tailwater ponds and sediment basins to concentration foodborne pathogens in multiple produce production areas across the United States (R. Atwill, personal communication). In combination with our findings, the results may suggest improved management strategies to contain and remove Salmonella and other pathogens from farm environments, and reduce the risk of contamination of fresh produce due to runoff waters. In summary, the findings from this study emphasize the need to continue to follow food safety practices, especially those relating to animal intrusions and irrigation water quality. The results from this study will be shared with stakeholders in the produce industry, conservation, and regulatory communities. Specifically, the data can be used to improve pre-season and preharvest environmental assessments and interventions as required in the Leafy Green Marketing Agreement metrics, in particular. In Georgia, more in-depth studies of Samonella occurrence in the Suwannee watershed are underway to better understand the ecology of these ponds and development of mitigation strategies. Wildlife isolates from this study will be shared with CPS investigators at University of Florida to compare genetic relatedness with their Suwannee watershed Salmonella strains to provide a better understanding of the ecology of Salmonella in these irrigation systems.