Abstract

Erysipelothrix rhusiopathiae is a known pathogen of cetaceans and infections have periodically resulted in the deaths of cetaceans at facilities worldwide.¹ Due to the rapid demise of animals with E. rhusiopathiae septicemia and the lack of definitive symptoms, many studies have focused on the prevention of cetacean erysipelas. One possible means could be the monitoring of feed fish stock for the presence of the bacterium. Previous studies have identified the presence of E. rhusiopathiae on a variety of fish commonly used in feeding captive cetaceans, but detection methods can take as long as 48 to 96 hours to complete.² The use of the polymerase chain reaction (PCR) in identifying bacterial species using unique sequences of 16s ribosomal DNA have been successful in confirming the identity of E. rhusiopathiae.³ But up until now, the PCR was performed on DNA isolated from cultured bacterial cells resulting in long lag times between initial inoculation and final PCR results. The development of a PCR protocol to accurately identify the presence of E. rhusiopathiae in a more timely manner could be quite helpful in the prevention of cetacean erysipelas since contaminated feed fish could be disinfected prior to being added into feeding stocks.

With many species of bacteria including Escherichia coli and Mycobacterium tuberculosis, PCR performed on plated colonies have been successful thereby reducing the assay time previously needed to isolated genomic DNA.^4,5 It was hypothesized that the same type of procedure could be adapted using E. rhusiopathiae specific PCR primers on intact bacterial cells. The PCR protocol described by Takeshi et al. was validated using isolated genomic DNA from a known isolate of E. rhusiopathiae.³ The protocol was then repeated using intact bacterial cells from broth culture and from plated colonies of the same isolate of E. rhusiopathiae. PCR products of the anticipated size were amplified using genomic DNA, dilutions of bacterial broth cultures and plated colonies as template. PCR was positive for cultures with OD600 measurements as low as 0.035 with detection of approximately 15 CFU and for single colonies taken from agar plates. This indicates that this type of PCR assay may be useful in decreasing the time needed for the detection and verification of E. rhusiopathiae in the slime coat of feed fish. While this study used cultured cells, it is hypothesized that the PCR may be sensitive enough to allow detection of bacterial directly from slime coat samples further reducing the time needed for detection.

References

1.  Dunn J. Bacterial and mycotic diseases of cetaceans and pinnipeds., in CRC Handbook of Marine Mammal Medicine: Health, Disease, and Rehabilitation, L.A. Dierauf, Editor. 1990, CRC Press: Boston. p. 73-87.

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.

3.  Takeshi K, et al. Direct and rapid detection by PCR of Erysipelothrix sp. DNAs prepared from bacterial strains and animal tissues. Journal of Clinical Microbiology., 1999. 37: p. 4093-4098.

4.  Lewin A, et al. Use of a quantitative TaqMan-PCR for the fast quantification of mycobacteria in broth culture, eukaryotic cell culture and tissue. J Vet Med B Infect Dis Vet Public Health, 2003. 50(10): p. 505-9.

5.  Fode-Vaughan KA, et al. Detection of bacteria in environmental samples by direct PCR without DNA extraction. Biotechniques, 2001. 31(3): p. 598, 600, 602-4, passim.