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United States Department of Agriculture

Agricultural Research Service

Title: Microbial Populations Associated with Restricted Shell Eggs

item Jones, Deana
item Musgrove, Michael

Submitted to: National Egg Regulatory Officials Annual Meeting
Publication Type: Proceedings
Publication Acceptance Date: March 8, 2007
Publication Date: March 10, 2007
Citation: Jones, D.R., Musgrove, M.T. 2007. Microbial Populations Associated with Restricted Shell Eggs. National Egg Regulatory Officials Annual Meeting.

Technical Abstract: Restricted shell eggs are found in all shell egg processing facilities. The manner with which companies handle them can vary greatly. In most cases, restricted eggs are diverted to further processing. Some facilities do not rewash eggs, choosing instead to include dirts in restricted eggs. Eggs from inline facilities are generally processed within 24 h of lay. Nest run eggs can be of varying age before processing. Therefore, the age of the eggs and cleanliness of a lot of restricted eggs can vary greatly from facility to facility. After washing, restricted eggs can be packaged by various means. Many are placed on plastic or pulp flats and stacked directly on pallets which are wrapped in plastic for transport. The pallets are most often unfinished wood or molded plastic forms. Sometimes, restricted eggs are placed in cardboard cases before being palletized. The flats of restricted eggs can also be loaded onto nest run or other types of carts for transport to the breaker. There is no standard amount of time restricted eggs remain in a processing facility before being shipped to the breaker. Most often, these eggs are stored in the post-processing cooler. There are times (such as when processing volume is high) when they may be placed in nest run coolers. Most often, these pallets remain on the processing floor for the whole day before being placed in the cooler. Partial pallets are often brought back to the processing floor the next day to be completed. Through all this activity, restricted eggs can potentially be found in the processing environment in several locations. While the bacterial populations associated with these eggs is of particular concern to further processors and the corresponding regulatory agencies, it is also important to be mindful of the location of these eggs in a shell egg processing facility. Their movement through the facility may allow them to be a point of cross-contamination or source of aerosolized microorganisms. Materials and Methods A study was conducted to determine the levels of aerobic organisms and Enterobacteriaceae associated with the shell and in the contents of restricted shell eggs. Furthermore, samples were compared to determine the incidence of Salmonella, Campylobacter and Listeria. On a single day, three shell egg processing facilities were visited (off line, in line and mixed operation). All three facilities participate in the USDA Agricultural Marketing Service voluntary grading program. At each plant, two 30-egg pulp flats of restricted eggs were collected and transported to the laboratory on ice. This process was repeated for three consecutive weeks (replicates). After arriving at the laboratory, all flats were placed in a single cardboard half case and stored at 4ºC overnight. The next morning, eggs were cracked on the edge of a sterile glass beaker. The contents of six eggs were combined into a single sterile laboratory sample bag. The inside surface of the shells were rinsed with warm, sterile phosphate buffered saline (PBS) to remove any adhering albumen. Six shells were pooled into a sterile specimen cup, 60 ml of PBS were added and shells were macerated with a glass stirring rod for 1 minute according to the methods of Musgrove et al. (2005a). Contents pools were homogenized in a laboratory stomacher at normal speed for 1 minute. Ten pools each of contents and shells were formed for each plant during a replicate. Total aerobic microorganisms were enumerated by spiral plating 0.1 ml of shell diluent onto duplicate plate count agar plates. Aerobic counts were enumerated for contents by spread plating 0.25 ml of homogenized contents onto duplicate plate count agar plates. After 48 h at 37ºC, colonies were counted and recorded as cfu/ml. Enterobacteriaceae were enumerated by duplicate plating 1 ml of either shell diluent or homogenized contents into violet red bile glucose agar with overlay. Plates were incubated at 37ºC for 20-24 h. A random selection of Enterobacteriaceae isolates were identified according to the methods of Musgrove et al. (2004). Salmonella, Campylobacter and Listeria prevalence was determined according to the methods described in Jones et al. (2006). Presumptive Listeria were biochemically identified with Microgen Listeria ID strips. Results and Discussion Detected levels of total aerobic counts associated with the shells ranged from 2.4 – 5.6 log cfu/ml (Table 1). The average for all samples was 4.4 log cfu/ml. Aerobic counts in the egg contents ranged from 1.3 – 2.9 log cfu/ml (Table 1). The average plant values for all reps were within 1 log of each other. The overall average of all pools was 2.0 log cfu/ml. There were no differences between plants and replicates for levels of Enterobacteriaceae on the surface of the restricted eggs (Table 2). Detected levels ranged from 1.8 – 3.1 log cfu/ml. The average concentration was 2.4 log cfu/ml. Very low levels of Enterobacteriaceae were found in the egg contents pools (Table 2). While a significant difference exists between plants and replicates, this significance occurs due to the consistently low levels detected in positive contents pools. The prevalence of Enterobacteriaceae was 36.7 % in all of the contents pools. Of all 180 shell and contents pools, only two shell samples were positive for Salmonella. Both samples were from a single replicate at plant B. After serotyping (APHIS, Ames, IA), it was determined that both isolates were S. Heidelberg. A single Campylobacter isolate was found in a shell sample from plant B. Listeria was detected in 21 % of the total samples. Approximately 37 % of the shell pools were positive for Listeria, whereas, only 6 % of the contents pools were positive. There were no differences in prevalence of Listeria in the contents pools from the three plants (Table 3). Plant A had a greater frequency of Listeria associated with the shells of the restricted eggs (66.7 %) compared to plants B (13.3 %) and C (30.0 %). Of the Listeria isolates recovered (38 total) the following identifications were made: L. grayi (2.6 %); L. welshimeri (13.2 %); L. innocua (84.2 %). L. innocua was the only isolate found in the positive egg contents pools. Table 1. Effect of plant and replicate on total aerobic bacteria associated with the shells and contents of restricted eggs (log cfu/ml). Plant Rep 1 Rep 2 Rep 3 Shells A 4.2bc 5.0ab 5.1ab B 3.4cd 2.4d 4.3abc C 5.6a 4.2bc 4.9ab Contents A 1.5D 2.7AB 2.9A B 2.0BCD 2.4ABC 2.0BCD C 1.8CD 1.3D 1.4D a..d Means within the table with differing letters are significantly different (P < 0.05) A..D Means within the table with differing letters are significantly different (P < 0.01) Table 2. Effect of plant and replicate on Enterobacteriaceae associated with the shells and contents of restricted eggs (log cfu/ml). Plant Rep 1 Rep 2 Rep 3 Shells A 2.3 2.4 2.5 B 2.8 1.9 3.1 C 2.6 1.8 1.9 Contents A ND1 0.05bc 0.36ab B 0.24abc ND 0.33abc C 0.46a 0.01c ND 1 ND = none detected a..d Means within the table with differing letters are significantly different (P < 0.05) Twelve of the 30 genera of Enterobacteriaceae were identified from the random sampling of positive isolates. A total of 495 isolates were stored for identification. Three could not be revived from storage. Sixty-four were not identified by the biochemical methods utilized. The summary of the genera and species identification can be found in Table 4. Besides E. coli, the rest of the organisms identified are considered opportunistic human pathogens. This means they can have an infective effect when conditions are favorable such as at wound sites or as a secondary infection. Of the 428 identified isolates, no Salmonella was found. Most of these organisms are ubiquitous in nature. Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Pantoea, Proteus, and Serratia have been associated with feces of humans, animals and/or rodents

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