Patricia Orosz-Coghlan, Lawrence M. Sullivan and Charles P. Gerba
June 27, 2000
Barn owl pellets are increasingly being used as educational tools in elementary and junior high schools to teach food chain interactions of predaceous birds. Many educators incorporate class lectures with student participation in owl pellet dissection. The pellet is the compacted end product of the owl's digestive process that is regurgitated hours after the prey has been eaten. An owl can produce as many as two cigar-shaped pellets per day. Pellets consist of fur, teeth, bones, feathers, plant seeds and insect parts covered with a glossy film of saliva (1).
The most common source of pellets are barn owls (Tyto alba) due to their wide spacial distribution and their ability to adapt to nest boxes (2). When these pellets are obtained from commercial companies, they are normally fumigated or autoclaved to remove microbial pathogens and insects, but many educators will collect the pellets themselves from areas that are inhabited by owls. This can create a potential for humans to acquire an enteric infection from hand to mouth contact. Since little has been studied about the microbial aspects of owl pellets, the main goal of this project was to determine the presence of several enteric pathogens in untreated pellets: Salmonella, Yersinia enterocolitica, Campylobacterjejuni, Giardial Ciyptosporidium and enteric viruses. Test were also conducted for the fecal indicator bacteria, total coliforms and Escherichia coli. In addition, to determine microbial survival, the pellets were microwaved for 20 seconds at 960 Watts or ovenbaked for 40 minutes at 325oC. A total spore count was also performed on the pellets to assess the efficiency of treatment processes.
Materials and Methods
Since barn owls are less often found in southern Arizona (Arizona Game and Fish Department, personal communication), we solicited for pellets Via the internet. Pellets were shipped from North Carolina and California in zip-lock bags and refrigerated at 2-8oC. Freshness of pellets and collection techniques were not known.
Barn owl pellets were processed over a two month period from time of
Total coliforms and Escherichia coli were analyzed by use of the enzyme substrate test (3). This test simultaneously detects total coliform bacteria and E. coli by utilizing hydrolyzable substrates. Multi-well trays (IDEXX Laboratories, Inc., Westbrook, ME) were filled with 0.1 ml of the vortexed sample and 100 ml of sterile water, mixed with the substrate and incubated at 35oC for 24 hours. Wells displaying a yellow color were considered to be positive for total coliforms while those that fluoresced under ultra violet (UV) light were counted as E. coli. The concentration was assessed by the most probable number (MPN) using a table provided by the manufacturer. For confirmation of E. coli, several samples were plated on mFC agar (Difco, Detroit, Ml) by the spread plate method. Selected blue colonies were subcultured onto Tryptic Soy Agar (TSA) (Difco, Detroit, Ml) plates and placed on 20E API strips (biomerieux, Hazelwood, MO) for biochemical identification.
For Salmonella testing, a volume of 1 ml of the saline sample was placed in 10 ml of selenite cystine broth (Difco, Detroit, Ml) for selective enrichment and incubated at 35oC for 24 hours (4). After incubation, 0.1 ml of broth was plated onto Hektoen Enteric Agar (Difco, Detroit, Ml) and incubated overnight at 35oC. Colonies that were typical of Salmonella displayed a light green edge with a dark green to black center. Lysine Iron Agar (Difco, Detroit, Ml) slants were inoculated with any suspected Salmonella colony and incubated overnight. Tubes exhibiting a black precipitate (H2S production) were considered positive for Salmonella.
Yersinia enterocolitica and Campylobacter jejuni analyses were taken from "Laboratory Methods of Food Microbiology "(5 ). A volume of 0.1 ml of the saline mixture was plated on Yersinia Selective Agar containing Yersinia Antimicrobic Supplement CN (Oxoid, Hampshire, England). The plates were incubated for 48 hours at 25oC. Dark pink colonies with translucent borders were presumptively considered Yersinia enterocolitica positive. For confirmation, selective colonies were subcultured onto TSA and placed on 20E API strips.
For Campylobacter jejuni, 0.1 ml of the sample mixture was plated on Camplyobacter Agar Base (Oxoid, Hampshire, England) containing horse blood, Campylobacter Growth Supplement and Preston Campylobacter Selective Supplement (Oxoid, Hampshire, England). The plates were incubated for 48 hours at 37oC under microaerophilic conditions by sealing the plates in an anaerobic jar containing gas generator envelopes (Becton Dickinson, Sparks, MD) for a reduced oxygen atmosphere. Colonies that appeared tear-shaped, gray and flat with a glossy appearance were enumerated. Selective colonies were stained and observed for Gram-negative S-shaped rods. Furthermore, several colonies were placed on API Campy strips (biomerieux, Hazelwood, MO) for biochemical identification.
Bacillus spp. determination was performed by a total spore count (4). The saline sample mixture was heat shocked at 80oC for ten minutes in a water bath.
Afterwards, 0.1 ml of the sample mixture was plated onto TSA and incubated overnight at 35oC. For confirmation, selective colonies on the agar were stained and examined for Gram-positive rods with spores.
Several smears of the sample mixture were heat fixed onto a glass slide and stained by indirect immunofluorescence (Hydrofluor Combo, Strategic Diagnostics, Newark, DE) for detecting Giardia cysts and Cryptosporidium oocysts (adapted from Manual of Clinical Microbiology, 1995). These parasites fluoresce an apple green when viewed under a ultra violet microscope.
In order to detect enteric viruses, 1 ml of the saline mixture was placed into 25 cm2 flasks with a Buffalo Green Monkey (BGM) cell monolayer (3). The flasks were incubated for 14 days at 35oC and observed daily for cytopathic effects (CPE), the rounding and detaching of infected cells from the monolayer. Those exhibiting CPE were considered positive. To confirm any positive flasks, 1 ml from the infected flask was placed on fresh BGM cells for another 14 days.
Twenty-five pellets were baked at 325oF (163oC) in a dry heat oven and an additional twenty-five pellets were microwaved at 960 Watts for 20 seconds. The pellets were analyzed in the same way as the untreated pellets.
Twenty-five untreated owl pellets were analyzed for enteric pathogens. Pellets ranged in weight from 2.31 grams to 13.34 grams with an average weight of 4.33 grams. Total coliforms, E. coli and Bacillus spp. were found in most of the pellets (Table 1) with more than half of the pellets positive for total coliforms and E. coli. Total coliform counts ranged from 1.20 X 103 colony forming units (cfu) to greater than 4.2 X 105 cfu per gram. E. coli counts ranged from 2.3 x 102 to 1.4 x l05 cfu/gram. Eight selective colonies were confirmed on 20E API strips resulting in acceptable to very good identification profiles for E. coli. Bacillus spp. were observed in all 25 pellet samples with an average of 2.7 X 103 cfu/gram. Confirmation of Bacillus spp. was done by staining and observing for gram positive rods with spores. Y. enterocolitica agar plates displayed colonies that could only be considered presumptive. Six selective colonies were taken to 20E API strips but resulted in unacceptable profiles for this organism. Presumptive colonies ranged from 1.2 x 102 to 4.1 x 104 cfu/gram. C. jejuni agar plates exhibited colonies ranging from 2.3 x 102 to 4.1 X 103 cfu/gram. Several colonies were stained and Gram-negative, S-shaped rods were observed, but this organism could not be confirmed with the API Campy biochemical strips. Salmonella spp., Giardia cysts, Cryptosporidium oocysts and enteroaruses were not detected in any of the owl pellets.
Pellets that were treated by being baked in the oven for 40 minutes at 163oC (325oF) exhibited no microbial growth on any of the selective agars (Table 2). An additional heterotropic plate count on TSA resulted in negative growth as well. Pellets microwaved on high at 960 Watts for 20 seconds compared in microbial growth to the untreated pellets (Table 2). The only change observed is that fewer pellets had growth on C. jejuni agar plates.
Discussion and Conclusion
A majority of the owl pellets contained enteric organisms that have been implicated in human infections. E. coli counts were as high as 4.2 x 105 organisms per gram. Considering that the average weight of the untreated pellets was 4.33 grams, it would not be difficult for a person to ingest the infectious dose of greater than 106 total organisms (6). Although C. jejuni was not confirmed, the likelihood of finding these organisms in a healthy bird is high, as this organism is endemic in birds (7). The age of the barn owl pellets analyzed in this study was not known and older pellets may not harbor the same populations of organisms as fresher pellets. The lowest count for C. jejuni per gram of owl pellet was 2.3 x l02, almost half of the infectious dose of greater than 500 organisms per person (8).
The spores from Bacillus spp. are considered to be very resilient to heat or disinfectant treatment. Educators using owl pellets in their classroom may want to consider oven-baking the pellets for 40 minutes to maximize inactivation of microorganisms in the owl pellets. Microwave ovens may be a good way to decrease the number of microorganisms but they vary in their heating intensities. Further study should be done to gather information on optimum heating times for pellet sanitizing.
Although the age of the pellets was not know, the ability of the barn owl to cover their pellets with gelatinous saliva may allow the organisms to persist for the two month period of testing. In many cases, moisture is a crucial factor in maintaining bacterial survival in the environment. The pellets are also well-insulated with a thick coat of fur and feathers offering extra protection for microbes from dessication.
As precautionary measures for educators incorporating untreated owl pellet dissection with classroom studies, the use of gloves while working with the pellets is highly recommended, along with stressing to students to keep hands away from the mouth area. Untreated pellets should be baked to reduce exposure to potential enteric pathogens.
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