Recirculating immunomagnetic separation and optimal enrichment conditions for enhanced detection and recovery of low levels of Escherichia coli O157:H7 from fresh leafy produce and surface water.

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (C(T) values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42 degrees C for 5 h provided better detection than enrichment at 37 degrees C. Extended incubation of surface water for 20 h at 42 degrees C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

Escherichia coli O157:H7 in feral swine near spinach fields and cattle, central California coast.

We investigated involvement of feral swine in contamination of agricultural fields and surface waterways with Escherichia coli O157:H7 after a nationwide outbreak traced to bagged spinach from California. Isolates from feral swine, cattle, surface water, sediment, and soil at 1 ranch were matched to the outbreak strain.

Incidence and tracking of Escherichia coli O157:H7 in a major produce production region in California.

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.

Clinical, epidemiologic, and environmental surveillance for ehrlichiosis and anaplasmosis in an endemic area of northern California.

Two forms of tick-borne leukocytotropic rickettsioses have been recognized in California since the mid-1990s: human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis and human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum. Between 1997 and 1999, two cases of HME and four cases of HGA were diagnosed in residents of southern Humboldt County, California. Environmental followup at case-patients' residences revealed dense populations of Ixodes pacificus ticks, particularly in grassy roadside areas. PCR evidence of A. phagocytophilum was detected in approximately 2.0% of I. pacificus; E. chaffeensis was not detected in any of 625 ticks tested. Serologic antibody to A. phagocytophilum was detected in two of 54 participants in a community epidemiologic study; one of these also had antibody to E. chaffeensis. Over 85% of study participants reported finding a tick on themselves in the preceding 12 mo. Residents of southern Humboldt County are at significant risk of tick bites and should take appropriate prevention measures to avoid infection with rickettsia and other tick-transmitted pathogens.