Advances in the development of diagnostic assays for the detection of the amphibian chytrid fungus (Genus Batrachochytrium)

Alex Hyatt, L. Berger, S. Hengstberger, D. Boyle and V. Olsen.

Australian Animal Health Laboratory, CSIRO, P.O. Bag 24, Geelong, Victoria, Australia 3022.
Email: alex.hyatt@li.csiro.au

Current diagnosis of Batrachochytrium relies on microscopic identification of sporangia in frog skin. The fungus can be detected in wet mounts of shedding skin and in histological skin sections. Examination of toe-clips has been used to detect infection in live frogs. Tadpoles need to be sacrificed in order to examine their mouthparts. Microscopy is highly sensitive for detecting chytrids in sick frogs but less sensitive for detecting low level infection in healthy frogs. In order to produce simple, rapid and sensitive diagnostic tests, we have generated panels of antibodies. Our polyclonal antibodies raised in sheep and rabbits are not specific to Batrachochytrium as they stain other chytridiomycete fungi, although they do not cross react with fungi from other phyla. These antibodies have been used to produce an indirect enzyme-linked immunosorbant assay (ELISA), a field test, and an immunoperoxidase stain for sections. These tests are useful in screening samples for Batrachochytrium. To produce an accurate species-specific test, we have generated a bank of monoclonal antibodies (MAbs). Preliminary data indicate that we have a large number with different staining characteristics. These antibodies will be adapted to the ELISA and to new field tests.

We have also refined freezing techniques whereby Batrachochytrium can be frozen, stored and routinely recovered. Finally, we have developed an "on-line diagnostic service" whereby images of samples are examined over the web in real time. At this stage the service functions by people sending us slides or specimens and then joining us "on-line" to examine. This 'network' is useful in trouble-shooting assays, identification procedures and describing histopathology (light microscopy). We hope that a national and international network will be established in the near future.

Whilst the above assays and services are mainly in the developmental stage, we welcome collaborations with scientists who would be willing to test them in their laboratory.