abstract

Infectious disease is an important factor influencing the health and sustainability of both managed collection and free-ranging marine mammal populations. Fungal infections are important as primary pathogens, particularly of the respiratory, gastrointestinal and integumentary systems, and as complicating secondary infections.^2-8 Identification of fungal infections using standard culture methods can be an arduous process as many fungi are difficult or time consuming to grow and differentiate. The use of molecular diagnostic methods can assist in the rapid and accurate identification of a fungal pathogen enabling a prompt clinical response¹. In managed collections of marine mammals, the need for rapid diagnosis and initiation of appropriate treatment is of heightened importance given the time and expense of training and small population sizes. Therefore, the goal of this study was to develop a rapid polymerase chain reaction (PCR) based diagnostic assay for multiple fungal pathogens of clinical importance in marine mammals.

For this assay, real-time PCR primers were developed that amplify an approximately 260bp fragment of the 28s rRNA gene from a wide range of fungal pathogens in a variety of clinical samples including frozen and formalin-fixed paraffin embedded tissue. Species-specific minor groove binding probes with attached fluorophores have been designed to identify amplicons of the pathogens of interest for marine mammals including Aspergillus fumigatus, Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatiditis, Candida albicans and Candida glabrata. Internal positive controls include a passive internal reference dye and an exogenous DNA control. Probes were tested against confirmed isolates and clinical samples containing the organism in question and other fungal pathogens. None of the probes bound to non-target DNA (100% specificity). Because this assay utilizes generic primers, unknown samples can be run not just with the species-specific probes, but also with the primers and SYBR green for identification of other fungal pathogens within a sample. Fungal DNA amplified from clinical samples that does not cross-react with any of the specific probes could then be sequenced for identification. For example, zygomycete infections which are caused by a variety of different organisms, could be identified using this later methodology. Because the primers are generic, additional probes could be designed to detect other specific pathogens. This multi-plex PCR provides a new and rapid diagnostic tool for the identification of fungal pathogens within clinical and post-mortem samples. As this assay is specific for the pathogen (rather than the host), it also has utility for a wide variety of host organisms.

acknowledgments

This study was sponsored by a grant from the Office of Naval Research (Award No: N00014-06-1-0249). The authors also thank the U.S. Navy Marine Mammal Program, John G. Shedd Aquarium, Chicago Zoological Society's Brookfield Zoo and The Marine Mammal Center for submission of clinical samples and isolates used in validation of this assay.

References

1.  Chemaly RF, Procop GW. Molecular detection and characterization of fungal pathogens. In: Molecular Microbiology, Diagnostic Principles and Practice. Persing, D.H., Tenover, F.C., Versalovic, J., Yi-Wei, T., Uunger, E.R., Relman, D.A., and White, T.J., eds. ASM Press, Washington D.C., USA. pp. 551-9.

2.  Fauquier DA, Gulland FM, Trupkiewicz JG, Spraker TR, Lowenstine LJ. 1996. Coccidioidomycosis in free-living California sea lions (Zalophus californianus) in central California. J. Wildl. Dis. 32: 707-10.

3.  Joseph BE, Cornell LH, Simpson JG, Migaki G, Griner L. 1986. Pulmonary aspergillosis in three species of dolphin. Zoo Biol. 5: 301-8.

4.  Migaki G, Jones SR. 1983. Mycotic diseases in marine mammals, In: Pathobiology of marine mammal diseases, Vol. II. Howard, E.B., ed. CRC Press, Boca Raton, Florida, USA. pp. 1-25.

5.  Reidarson TH, McBain JF, Dalton LM, Rinaldi MG. 2001. Mycotic diseases, In:CRC Handbook of Marine Mammal Medicine, Dierauf, L.A. and Gulland, F.M.D, eds. CRC Press, Boca Raton, Florida, USA. pp. 337-55.

6.  Robeck TR, Dalton LM. 2002. Saksenaea vasiformis and Apophysomyces elegans zygomycotic infections in bottlenose dolphins (Tursiops truncatus), a killer whale (Orcinus orca), and pacific white-sided dolphins (Lagenorhynchus obliquidens). J. Zoo Wildl. Med. 33(4): 356-66.

7.  Sweeney JC, Migaki G, Vainik PM, Conklin RH. 1976. Systemic mycoses in marine mammals. J. Am. Vet. Med. Assoc. 169: 946-8.

8.  Zwick LS, Briggs MB, Tunev SS, Lichtensteiger CA, Murnane RD. 2000. Disseminated blastomycosis in two California sea lions (Zalophus californianus). J. Zoo Wildl. Med. 31: 211-4.