Abstract

Chytridiomycosis, a skin disease caused by the chytrid fungus Batrachochytrium dendrobatidis, has been implicated as the proximate cause of many amphibian population declines worldwide. In Central America, amphibian mortality events due to chytridiomycosis have occurred in a predictable pattern and have been associated with significant losses in amphibian biodiversity.¹ Because the effects of chytridiomycosis cannot be ameliorated in wild populations, there is an urgent movement towards development of ex-situ survival assurance colonies.² A prototype for amphibian survival assurance colonies is the El Valle Amphibian Conservation Center (EVACC) located at the El Nispero Zoo in El Valle de Anton, Panama. EVACC will ultimately house up to 40 native species and is located at an epicenter of amphibian mortality events due to chytridiomycosis in Panama.

Until very recently, diagnostic testing for chytridiomycosis in Panamanian amphibians required shipment of samples to remote laboratories in the United States or elsewhere and results were often significantly delayed by transportation time and government permitting requirements. In late June and early July of 2006, we evaluated the use of a mobile molecular laboratory capable of processing samples for conventional polymerase chain reaction (PCR) as a means of rapidly diagnosing chytridiomycosis in a rural location close to an active amphibian mortality event. The mobile laboratory consisted of a miniature thermocycler (capable of 16 PCR reactions per run), agarose gel electrophoresis equipment, UV transilluminator and microcentrifuge all assembled in a single foam-lined case by the MJ Research Company (now Bio-Rad, Hercules, CA, USA). Equipment was transported as checked baggage on a commercial airline. A published conventional PCR reaction for amplifying a 300 bp product specific for Batrachochytrium dendrobatidis was used.³ Modifications for use in field conditions included a rapid protocol for preparation of template DNA from amphibian skin swabs (PrepMan Ultra, Applied Biosystems, Foster City, CA, USA); pre-packaged reaction tubes with room temperature stable beads containing Taq polymerase, dNTPs and MgCl (PuRe Taq Ready-To-Go PCR beads, GE Healthcare, Piscataway, NJ, USA); and use of pre-cast agarose gels containing ethidium bromide (Reliant FastLane Gel System, Cambrex Bioscience, East Rutherford, NJ, USA). For use, the laboratory was assembled in a kitchen within a small house and utilized a single standard electrical outlet. Positive and negative control samples were processed with each run of the PCR equipment.

Over a 2-wk period samples from over 25 species and 217 individual animals were analyzed and included 144 samples from 10 field sites in the El Valle, Panama region and 63 samples from field-collected animals housed in EVACC that had been treated for presumptive chytridiomycosis using itraconazole baths. Of field-collected samples 36/144 (25%) were presumptively positive for Batrachochytrium DNA. In samples from field-collected animals previously treated for chytridiomycosis there was only a single frog with a weak positive result (1/63; 1.6%). Within these samples, 4 animals with both pre- and post-treatment swabs had positive samples at the time of capture and negative samples following itraconazole treatment.

Findings suggest that the mobile laboratory was successful at providing rapid preliminary diagnosis of chytridiomycosis at an in-country location close to a site of an amphibian mortality event. In addition, sampling of animals previously treated for presumptive chytridiomycosis allowed some evaluation of the effectiveness of the itraconazole treatment used in processing field-collected animals. Based on this experience, use of mobile molecular laboratory equipment may be useful in a variety of international wildlife disease investigations in which rapid in-country laboratory support may not be available.

Despite the advantages of rapid diagnosis, there are also significant caveats to use of mobile molecular laboratory equipment in a rural setting. A major concern is the possibility of contamination of reactions with non-sample source DNA with subsequent false-positive reactions. Negative controls used in this project were consistently negative until the last 2 PCR reactions (32 samples) performed suggesting that contamination may have become a factor late in the effort. An additional significant concern is that positive PCR results for any pathogen do not prove that the detected agent is responsible for an observed mortality event. In fact, preliminary positive PCR results may provide misleading information because subsequent comprehensive diagnostic investigation (to include complete necropsy and histopathology) may indicate an alternative cause of the mortality event.

Literature Cited

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