Science-based evaluation of risks associated with wildlife exposure for contamination of irrigation water by Salmonella.

Although Salmonella enterica is distributed throughout the environment and can be recovered from various wildlife reservoirs, the risks to human health imposed by these sources are not clear. Different Salmonella strains appear to be associated with different animal host species and represent a range of disease potential in humans. We are proposing to define genetic diversity among strains recovered from wildlife sources and determine their relationship to strains recovered from water and sediment sources in irrigation ponds in a produce production region of the southeastern United States. The disease potential of these strains will be evaluated by integrating various strain collections using PCR-based rapid diagnostics, CDC PulseNet fingerprinting analysis, and DNA sequencing of selected virulence-associated genes. This project will link eventually with an FDA effort to establish genetic markers that define the regional distribution of Salmonella for the purpose of source tracking. The results will shed light upon wildlife-associated risks, thereby providing science based data for implementation (or rejection) of agricultural management practices that are designed to reduce exposure of irrigation water to wildlife. Outcomes could also provide more rapid and accurate assessment of food products implicated in outbreaks and prevent the broad-based recalls that impact an entire industry nationwide.

Although irrigation water is a recognized risk factor for contamination of produce, the impact of wildlife as a source for foodborne pathogens remains uncertain. Some growers are using ground water as their sole irrigation reservoir or have cleared vegetation in an attempt to exclude wildlife from these reservoirs. However, these strategies may reduce biodiversity and thus increase the potential for survival of pathogens in irrigation ponds. In order to address controllable risks for contamination of produce by potential vectors for pathogens it is necessary to understand the distribution and virulence potential of these pathogens in wildlife. The proposed research will leverage results from two previously funded CPS projects, namely “Science-based evaluation of regional risks for Salmonella contamination of irrigation water at mixed produce farms in the Suwannee River watershed” (Wright, PI) and “Evaluation of amphibians and reptiles as potential reservoirs of foodborne pathogens and risk reduction to protect fresh produce and the environment” (Jay-Russell, PI). These studies investigated some of the same irrigation ponds on multiple small farms with various vegetable and fruit crops in the upper Suwannee River Watershed in Georgia. This region was selected because it is a "hot spot" for environmental sources of salmonellosis with case rates that were 1.5 times higher than the national average (CDC, 2009). We found Salmonella densities directly correlated with water temperature, but we found no correlation of Salmonella distribution with fecal coliforms or E. coli, suggesting that contamination is not associated with warm-blooded animals (Wright et al., CPS Annual Report). Salmonella was isolated from water and/or sediment at all ponds examined (n=10) and from toad (37.5%), turtle (40%), and bivalve (18.4) samples taken from a subset (n=5) of these ponds (Jay-Russell, CPS Final Report). Proposed research will merge genetic analyses from the above studies with our recently described database of isolates derived from other regions and clinical infections (Rajabi et al., 2011). Results will build a regional DiversiLab rep-PCR library that will be validated by other genetic approaches, including pulsed field gel electrophoresis and multi-locus sequence typing. Research goals will be achieved through the following objectives: 

1) Determine genetic relatedness of Salmonella strains isolated from wildlife relative to isolates from irrigation water and sediments in the same geographic region vs. strains from other sources in our database. 

2) Validate genetic diversity and evaluate virulence potential by PFGE using the CDC PulseNet protocol (Jay-Russell) and by allelic variation of selected genes. This research will address some of the target research question in sections 1.2 and 1.3 of the proposal guidelines and help to establish regional genetic profiles for Salmonella strains. The PIs provide expertise in Salmonella biology and ecology, pathogen detection, molecular typing, modeling systems, and agricultural extension. Studies will eventually link to a nationwide database being developed by FDA for evaluation of the geographic distribution of Salmonella strains. The ultimate goal will be to develop predictive models that can be used to evaluate the risk of pathogens in irrigation water in order to determine agricultural practices that will reduce disease risk.

1.) Determine genetic relatedness of Salmonella strains isolated from wildlife relative to isolates from irrigation water and sediments in the same geographic region vs. strains from other sources in our database. 

2.) Validate genetic diversity and evaluate virulence potential by pulsed field gel electrophoresis using the PulseNet protocol (Jay-Russell) and by allelic variation of selected genes.

• We discovered a novel “cross-streaking” method that provides cost effective detection and enumeration of Salmonella from aquatic reservoirs with specificity and sensitivity comparable to PCR methods. 

• Salmonella strains derived from wildlife from irrigation ponds in Georgia represent a genetically diverse population. 

• There was no association of a particular genotype with a particular species, pond, or season. 

• Although 50% Salmonella strains from irrigation pond water or sediment showed genetic similarity to strains from clinical origin, strains from wildlife were mostly (87%) clustered with strains from environmental sources. Unfortunately, the use of fecal contamination indicators frequently does not reflect distribution of foodborne pathogens such as Salmonella enterica, and ensuring food safety may require direct detection and enumeration of pathogens in agricultural settings. Herein, we summarize novel methods that may facilitate these efforts, and filtration combined with IMS may result in even greater sensitivity for evaluation of environmental samples. We recommend further study before the introduction of stringent measures to control wildlife access to irrigation water. Although we water and sediment samples frequently contained Salmonella with genetic similarity to clinical strains, in general, strains from amphibians and reptiles did not appear to serve as a reservoir for these strains. Currently, a consortium of FDA, CDC, and USDA researchers, as well as various public health and academic institutions are building a whole genome sequence database that will eventually permit public access the literally thousands of strains from different geographic locations, food product, agricultural, veterinary, and clinical sources. The prospect of WGS for a “GenomeNet” approach not only hold promise from more rapid and accurate source tracking during outbreak, but will potentially help to define virulence potential of the wide repertoire of diverse Salmonella populations for more informed and science-based policy decisions and management strategies.