Abstract
Erysipelothrix rhusiopathiae, a known cetacean pathogen, is periodically implicated in fatal infections in a variety of cetaceans at zoological facilities worldwide.1 Manifestation of dermal lesions or septicemia is typically seen without definitive symptoms prompting studies focusing on the prevention of cetacean erysipelas. It has been suggested that one route of infection may be feed fish stocks which house the bacteria in the outer slime layer. Previous studies have identified the presence of E. rhusiopathiae on a variety of fish commonly used in feeding captive cetaceans, but traditional culture-based detection methods can take as long as 48 to 96 hours to complete.2 The use of the polymerase chain reaction (PCR) in identifying E. rhusiopathiae using unique sequences of 16s ribosomal DNA has been successful, but the assay has only been used to screen extracted DNA samples or suspected cultures of E. rhusiopathiae.3,4 We believe that the detection time could be reduced by adapting the previously reported PCR assay to detect the bacterium directly from fish slime samples and/or employing real-time PCR. The reduction of detection time may be helpful in preventing cetacean erysipelas since contaminated feed fish could be disinfected prior to being added into feeding stocks.
Fish slime samples (herring and capelin) were collected using BBL CultureSwabs®, diluted 1:2 in Tris-EDTA (TE) buffer, mixed and then spiked (10%, v/v) with a known positive E. rhusiopathiae broth culture (OD600 = 1.0). The samples were then centrifuged at 10,000 x g for one minute, the pellet was resuspended in sterile ddH2O and used in the standard PCR protocol previously reported.4 PCR product was detected for the spiked samples but not for original unspiked samples. Results for the real-time PCR on cultures of known E. rhusiopathiae cultures correlated to results using standard PCR with the exception of cultures of E. tonsillarum. No amplification was seen using standard PCR, but when using real-time PCR, product was seen indicating the primer set may not be species specific. At the time this abstract was prepared, realtime PCR had not been performed on the spiked slime samples and we were investigating the false positive results on the E. tonsillarum sample.
Acknowledgements
Thanks to Jeff Boehm and Brigita Harris from the Shedd Aquarium in Chicago for fish slime samples and the Rush University Medical Center Clinical Laboratory staff for assistance with real-time PCR support.
References
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2. Greenwell MG, JR Boehm, BM Harris. A One-Year Surveillance Program for Erysipelothrix rhusiopathiae: Methodology, Findings, and Recommendations. 33rd Annual Conference of the International Association for Aquatic Animal Medicine, 2002. 33: p. 148-152
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4. Palmer C, JC Jones (2005). The Use Of Colony PCR For The Rapid Detection Of Erysipelothrix rhusiopathiae. Proceedings of the 36th Annual Conference of the International Association for Aquatic animal Medicine. P. 141.