A non-lethal technique for detecting the chytrid fungus Batrachochytrium dendrobatidis on tadpoles

Abstract

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling. KEY WORDS: Batrachochytrium dendrobatidis · Tadpole · Detection · PCR · Technique · Chytridiomycosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [77][78][79][80][81][82][83][84][85] 2006 et al. 2004, Knapp & Morgan 2006, and are killed for histological diagnosis. This limits studies of amphibian disease, particularly those that span life-history stages.The polymerase chain reaction (PCR) is the basis for a highly sensitive, non-destructive diagnostic technique for sampling post-metamorphic amphibians. An animal's skin is scraped or swabbed lightly with a finely pointed, slightly abrasive object, such as a wooden toothpick or cotton surgical swab, to remove the outermost skin cells, which are then screened for Bd DNA using real-time Taqman PCR assays (Boyle et al. 2004). Researchers studying chytridiomycosis in amphibians have used the swabbing technique for about 4 yr, and it is a generally accepted protocol used by several laboratories (Briggs & Vredenberg 2004, Livo 2004, Kriger et al. 2006. We evaluate the sensitivity and feasibility of swabbing tadpole mouthparts for Bd diagnosis as a non-lethal alternative to histology. The project arose from a desire to learn more about the rare Ramsey Canyon leopard frog Rana subaquavocalis in southeastern Arizona, where Bd has been detected at several sites and tadpoles often remain at sites after frogs have become scarce or absent (K. J. Field pers. obs.). Wildlife managers are rightly reluctant to test for Bd by killing large numbers of tadpoles in small, vulnerable populations. Our question was: Could we detect Bd reliably and non-lethally in tadpoles using quantitative PCR? MATERIALS AND...

First survey for the amphibian chytrid fungus Batrachochytrium dendrobatidis in Connecticut (USA) finds widespread prevalence

“…While cotton-tipped, wire swabs (e.g. Medical Wire & Equipment MW 100−100) are a common collection tool, toothpicks have been used successfully in previous studies to collect Bd from amphibians (Retallick et al 2006, Woodhams et al 2006, Retallick & Miera 2007, Hyman & Collins 2012. Despite the popularity of wire swabs, there is no evidence demonstrating that wooden toothpicks are any better or worse at collecting Bd.…”

“…It is important to note, however, that metrics of zoospore load are associated with substantial variation related to sampling protocol. Different researchers swab differently -some swab more thoroughly or with greater pressure than those with a lighter hand or less skill (Retallick et al 2006) -but there is probably a species, life stage, or even an individual host effect. Recent research found that zoospore load variation could not be explained solely by species, nor habitat, climate, or elevation; zoospore load may be entirely an individual host characteristic (Gründler et al 2012).…”

“…DNA from swabs was extracted according to Retallick et al (2006) with a minor modification: we used 100 µl of PrepMan Ultra (Applied Biosystems) rather than 40 µl. Bd DNA was detected via qPCR according to Boyle et al (2004) and Garland et al (2010).…”

Chytridiomycosis in frogs of Mount Gede Pangrango, Indonesia

“…Tadpoles from 4 of the 13 species encountered (Table 1), namely Microhyla achatina, M. palmipes, Megophrys montana and Philautus aurifasciatus, were not examined because of the natural absence of an oral disk in the first 3 species and the absence of a tadpole stage in the last species. Examination of tadpole mouthparts has been used to indicate the likelihood of chytridiomycosis in several species (Knapp & Morgan 2006, Retallick et al 2006. Only one tadpole that showed abnormalities in its mouthparts was swabbed for PCR testing.…”

Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection

“…Sampling was performed by gently stroking flat wooden toothpicks (see Retallick et al 2006) across the toes, webbing, feet, thighs, sides and abdomen of the frogs (Briggs & Vredenberg available at http://amphibiaweb.org/ chytrid/swab_instruction.pdf, Livo 2004 at http:// wildlife.state.co.us/NR/rdonlyres/710BBC95-2DCF-4CF9-8443-D4561 DBC3B 69/0/PCRsampling2004.pdf), with a minimum of 20 strokes per individual. Toothpicks were then stored in 2 ml Axygen screw-cap tubes (with rubber o-rings) filled with 1 ml 70% ethanol.…”