Abstract

Chytrid infection caused by Batrachochytrium dendrobatidis has been implicated in dramatic declines of anuran amphibians worldwide.^2,4,6-8 At present, microscopic examination of affected tissues is the method of choice to diagnose pathogenic chytrid (chytridiomycosis) infection,^3,7 and requires optimal sample selection and handling. Development of a polymerase chain reaction (PCR) method for detection of Batrachochytrium dendrobatidis (“chytrid”) in amphibians would increase sample throughput and the identification of poorly preserved (e.g., field) specimens. In addition, PCR detection would be useful in identifying early infection, for assessing therapeutic efficacy, and for monitoring environmental levels of Batrachochytrium dendrobatidis for the purpose of wild and captive amphibian population management. Finally, this method would be valuable in the assessment of disease risk prior to reintroduction of endangered amphibians and for improved characterization of the ecology of this organism in the wild. In the present study, PCR detection of Batrachochytrium dendrobatidis was compared with pathologic screening of tissue from wild specimens associated with amphibian die-offs.

Specimens were presented to the National Wildlife Disease Center in Madison, Wisconsin for pathologic evaluation in response to die-offs at the respective natural sites. Samples selected for initial PCR testing were the following: boreal toads (Bufo borealis, n=12) collected at four sites in Colorado, a bullfrog (Rana catesbeiana) from California, and an Idaho giant salamander (Dicamptodon aterrimus). Of these, five specimens were diagnosed with active chytridiomycosis histologically, two were from specimens where histopathologic examination was negative for chytridiomycosis, and five specimens were histologically inconclusive.

Because there is a strong agreement between taxonomic morphologic characteristics and sequence relationships from 18S ribosomal DNA for higher fungi, sequences from this region were selected for assay development.¹ During PCR primer selection, the Batrachochytrium dendrobatidis (GenBank accession AF164301) 18S sequence served as a reference to evaluate comparable sequences from closely related organisms using the alignment sequence tool (BLAST search, GenBank). Organisms were selected taxonomically,⁵ and included Batrachochytrium dendrobatidis (AF051932), Entophlyctis sp. (AF164257), Spizellomyces acuminatus (M59759), Powellomyces variabilis (AF164241), Spizellomyces kniepii (AF164237), Rhizophydium sp. (AF164264), and Nephrochytrium sp. (AF164295). These sequences were also aligned with the sequence for Xenopus tissue (X04025). Visual analysis of this alignment matrix identified four unique regions (amplifying a total of 1038 base pairs) within the approximately 1800 base pair long 18S rRNA gene from Batrachochytrium dendrobatidis. PCR testing was performed on hind foot toe web skin pieces from the 12 amphibians mentioned above. DNA was extracted from 50 mg of frozen tissue using DNeasy kits (Qiagen, Valencia, California), and 5 μl of elute was applied to each PCR reaction. Histologically positive and histologically negative skin from Bufo borealis were included as positive and negative controls, respectively. The water source used in sample preparation and testing was also included as a negative control.

Of the skin samples analyzed both by histopathology and PCR, five samples were positive and two specimens were negative, by each method. For the five specimens scored as “histopathologically inconclusive” due to poor sample quality, the PCR test detected the presence of chytrid in each. Interestingly, chytrid was detected in a sample from an Idahoan giant salamander (Dicamptodon aterrimus) from this group of specimens.

Initial results suggest that PCR detection for the presence of chytrid is as sensitive as histopathology but can also detect the agent in specimens of poorer quality. Although PCR positive samples were identified from die-offs, the relationship between the presence of chytrid and the cause of death could not be proven. Specificity testing of the PCR is continuing at this time.

In conclusion, the potential for PCR detection of chytrid in tissues and in the environment should allow a more complete understanding of the ecologic role of this agent in amphibian population dynamics. The need for a reliable PCR technique for chytrid risk assessment in the wild remains a major goal of this work.

Acknowledgments

The authors would like to thank Nick Douris, B.S. for technical assistance.

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