Salmonella Enteritidis Phage Type 30 from a Single Almond Orchard over a 5-Year Period

In 2001, Salmonella Enteritidis phage type (PT) 30 was isolated from drag swabs of 17 61-ha almond orchards on three farms linked to an outbreak of salmonellosis associated with consumption of raw almonds. The objective of this study was to evaluate the long-term persistence of Salmonella Enteritidis PT 30 in one of the almond orchards associated with the outbreak. Swabs (gauze saturated with full-strength sterile evaporated skim milk and attached to string) were pulled along the orchard floor in a standardized manner for 55 m. At each sample time, two pooled samples (four swabs each) were collected from each orchard quadrant. Swabs were enriched for Salmonella using a delayed secondary enrichment procedure developed for isolation of Salmonella from poultry houses. Suspect Salmonella isolates were selected, confirmed, serotyped, and phage typed, and pulsed-field gel electrophoresis (PFGE) patterns were determined after cleavage with Xbal and BlnI. Salmonella was recovered infrequently from pooled samples collected from January through July (3 of 56 samples, 5.3%). In general, Salmonella isolation frequency per sample time increased during and immediately after the harvest, when large amounts of dust were generated in or near the orchard: August, 4 (20%) of 20 samples; September, 13 (20%) of 64 samples; October, 27 (42%) of 64 samples; November, 4 (25%) of 16 samples; and December, 2 (25%) of 8 samples. All 53 Salmonella isolates during the 5 years were identified as Salmonella Enteritidis PT 30, and two PFGE patterns that differed by the presence of an approximately 40-kb fragment were identified. These data demonstrate the potential for long-term environmental persistence of Salmonella in almond orchards. Salmonella is common in the environment, but the primary habitat of this pathogen is the intestinal tract of birds, reptiles, animals, humans, and occasionally insects (11). The potential for populations of Salmonella to multiply in the environment in the absence of an animal host has been discussed (8, 27), but environmental persistence of Salmonella is more commonly attributed to its broad host range and continual reintroduction of the organism (27). The ability of Salmonella to survive for long periods in dried foods and feed (2, 25), animal production facilities (6), and water, soil, and manure (14) has been well documented. An outbreak of Salmonella Enteritidis phage type (PT) 30 infection in 2000 and 2001 was linked to consumption of raw almonds that were harvested in the fall of 2000 (10). Salmonella Enteritidis PT 30 was isolated from open and sealed bags of bulk almonds, and the implicated lot was traced to one California almond processor. In traceback investigations 7 to 8 months after the 2000 harvest, Salmonella Enteritidis PT 30 was isolated from drag swabs of almond orchard floors on three geographically linked farms * Author for correspondence. Tel: 530-757-5767; Fax: 530-752-4759; E-mail: [email protected]. t Present address: Department of Food Science, Cornell University, Ithaca, NY 4853-7201, USA. Present address: Department of Food Science and Human Nutrition, University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Expeimcnt Station Road, Lake Alfred, FL 33850, USA. in a 25-km 2 area. Samples of farm irrigation water, airplane spray equipment, migratory waterfowl, and rodents all were negative for Salmonella. None of the farms had used manure or biosolids in the previous 5 years, and no livestock or poultry farms were adjacent to or near the almond farms (3). A source for the contamination was not identified during the outbreak investigation, but the isolation of Salmonella from such a large geographic area suggested a widely distributed contamination source (3). These naturally contaminated almond orchards provided a unique opportunity to study the environmental persistence of Salmonella in this area. Salmonella Enteritidis PT 30 was isolated over a period of 5 years from a single almond orchard. MATERIALS AND METHODS A modified drag swab and a delayed secondary enrichment procedure developed for isolation of Salmonella Enteritidis from poultry houses (18, 21, 22) were used successfully to isolate Salmonella Enteritidis from almond orchards during the tracehack investigation of the salmonellosis outbreak that occurred in 2000 and 2001 (10). This same procedure was used to isolate Salmonella from the almond orchard examined in the present study. Unless otherwise noted, all media were obtained from Difco (Becton Dickinson, Sparks, Md.). Swab preparation. Drag swabs were prepared by folding two pieces of gauze (10 by 10 cm; Nu Gauze general-use sponges, Johnson & Johnson, New Brunswick, N.J.) in half and tying one end of a I -m piece of cotton string around the center. Swabs were J. Food Prot., Vol. 70, No. 8 SALMONELLA PERSISTENCE IN AN ALMOND ORCHARD 1785 TABLE 1. Weekly isolation of Salmonella Enteritidis PT 30 from one almond orchard during August, September, and October, 2002 through 2006