Sources and mechanisms of transfer of Salmonella in the production and postharvest tree nut environment.

Over the past decade, contamination of nuts with Salmonella has led to numerous recalls and two outbreaks in the U.S. However, sources of Salmonella in nut production and processing are not well understood. We hypothesize that contamination of almonds and pistachios with Salmonella is facilitated by movement of dusts in orchards and during initial post-harvest handling. We also speculate that airborne dust from animal feeding operations (dairies, feedlots) in close proximity to orchards may play a role in the spread of this pathogen. To test this hypothesis we will work closely with our collaborators in the almond, pistachio, and livestock industries to identify collaborating farms, hulling/shelling (almonds), and hulling/processing (pistachio) facilities. Environmental sampling and microbiological analyses will be used to characterize the microbial composition of bioaerosols and dust originating from livestock operations located in close proximity to almond and pistachio production areas in the California central valley; evaluate movement of microorganisms from livestock operations to nearby almond and pistachio orchards; and evaluate the microbial composition of bioaerosols and dusts at almond hullers/shellers and pistachio hulling facilities. Knowledge gained from this study will be shared with stakeholders to improve good agriculture practices that protect almond and pistachios from Salmonella contamination.

Foodborne disease outbreaks and recalls have been linked to consumption of contaminated raw and in-shell nuts (almonds, hazelnuts). In 2009 and 2010, type I recalls due to Salmonella contamination occurred in pecans, pistachios, macadamia nuts, hazelnuts, pine nuts and walnuts. The primary vectors and transport pathways leading to contamination by Salmonella remain unclear, but potential sources include livestock, local wildlife, and possible niches of self-sustaining Salmonella shown previously to replicate on the orchard floor under certain environmental conditions. We hypothesize that contamination of the almonds and pistachios by Salmonella occurs primarily through movement of bioaerosols and airborne dusts generated from concentrated animal feeding operations (dairies, feedlots) in close proximity to the orchards. To test this hypothesis we will work closely with our collaborators in the almond, pistachio, and livestock industries to identify collaborating farms, hulling/shelling (almonds), and hulling/processing (pistachio) facilities. The long-term goal of the project is to develop and implement science-based strategies to prevent off-orchard dissemination of Salmonella from livestock sources to almond and pistachio crops. We will accomplish our goal by completing the following objectives: Objective 1a. Characterize the microbial composition of bioaerosols and dust originating from livestock operations located in close proximity to almond and pistachio production areas in the California central valley. Objective 1b. Evaluate the movement of microorganisms from livestock operations to nearby almond and pistachio orchards compared with control orchards not in proximity to livestock operations. And Objective 2: Evaluate microbial composition of bioaerosols and dusts at a) almond hullers/shellers and b) pistachio hulling facilities

1. Objective 1a. Evaluate the microbial composition of bioaerosols and dust originating from livestock operations located in close proximity to almond and pistachio production areas in the California Central Valley. 

2. Objective 1b. Evaluate the movement of microorganisms from livestock areas to nearby almond and pistachio orchards compared with control orchards not in proximity to livestock operations. Standardized and validated bioaerosol collection and analytical techniques will be used to measure the occurrence, dispersion and transport of Salmonella and non-pathogenic indicator E. coli from livestock sources (solid stacks, lagoon, pen floors/bedding) to nearby almond and pistachio crops. Molecular subtyping approaches will be used to compare genetic relatedness and source track movement of strains from livestock operations to tree nut study sites. Pyrosequencing will be used on a subset of samples to evaluate the potential for this technique. 

3. Objective 2: Evaluate microbial composition of bioaerosols and dusts at a) almond hullers/shellers and b) pistachio hulling/processing facilities.

We made the following general observations: 

1. Microbial populations in air samples as measured by collection on agar were consistent throughout an orchard but differed with sample date. 

2. Before harvest amounts of dust were significantly greater on leaves collected from trees that were at the edge of the orchard than on leaves from trees further into the orchard. Differences in dust levels on leaves collected throughout the orchards were insignificant after harvest. This observation is likely due to shaking of the trees and subsequent sweeping (in the case of almonds) that takes place during harvest. 

3. Generic E. coli was detected in air from within the almond and pistachio orchards that were next to animal operations but not from the almond control orchard. 

4. Salmonella was isolated from some but not all samples of manure collected from the dairy or calf operation. Salmonella prevalence in manure and wastewater was consistent with other surveys of dairy operations. Salmonella was isolated at a single time point from each of the three orchards from pooled drag swabs and never from any of the other samples. For Almond Poultry and Pistachio Dairy Salmonella was found during harvest in the row closest to the animal operation. Salmonella was isolated in June from Pistachio Calf in all three of the rows examined. The Salmonella identified in the calf manure and lagoon samples (serovar Give) in February and April was the same as that identified in the corresponding pistachio orchard in June. 

5. The microbial communities identified by Illumina sequencing revealed that there were distinct bacterial populations associated with the air, leaf rinsate, and soil collected in the orchards. Bacterial populations in the leaf rinsate and air samples from Almond Poultry were significantly more diverse compared to the leaf rinsate samples from Almond Control. Almond Poultry leaf rinsate samples form unique clusters in PCA analysis and different bacterial families were associated with Almond Poultry. Collectively these data provide preliminary evidence that microbial populations in tree nut orchards may be altered by proximity to large-scale animal operations. However, further data from paired orchards (next to and at a distance from animal operations) are needed to characterize the significance of these altered microbial communities to the safety of tree nuts. Of the measurements taken the following would be recommended for further study: amounts of dust on leaves; pooled drag swabs in the orchards for Salmonella; E. coli in air samples; and microbial communities on leaf rinsates and soil.