Environmental detection of Batrachochytrium dendrobatidis in a temperate climate

Abstract

The aetiological agent of amphibian chytridiomycosis Batrachochytrium dendrobatidis is a primary cause of amphibian population declines. Current surveillance is based on the detection of B. dendrobatidis in its host but in vitro work suggests infective stages may survive in the abiotic environment for at least 3 mo. We describe here a surveillance system using filtration and quantitative PCR that can detect B. dendrobatidis in small (<1 l) volumes of water. After assessing the analytical sensitivity of the protocol for both water and sediment samples in the laboratory, we analyzed environmental samples from the Sierra de Guadarrama mountain range in Spain at locations associated with chytrid-related die-offs and at other sites across Spain. B. dendrobatidis was detected in samples from 64% of the ponds in the Sierra de Guadarrama and at 2 sites outside this region, showing that levels of amphibian exposure to B. dendrobatidis are spatially heterogeneous. In experimental microcosms, we detected B. dendrobatidis for up to 12 wk, though we found no evidence for an overall increase in biomass. Our results emphasise the need to further investigate the life cycle of B. dendrobatidis to more completely understand the epidemiology of this emerging pathogen. KEY WORDS: Chytridiomycosis · Batrachochytrium dendrobatidis · Environmental surveillance · Filtration Resale or republication not permitted without written consent of the publisherDis Aquat Org 77: [105][106][107][108][109][110][111][112] 2007 solid culture media (Longcore et al. 1999). Thus, zoospores may persist in the environment for long periods of time and there may be hitherto undetected saprophytic stages with the ability to multiply. To better understand the biology and to analyze the epidemiology of B. dendrobatidis, a technique that can detect the pathogen in the environment and outside the host is urgently required.Traditionally, zoosporic fungi are isolated with baiting methods using the bait a substrate for the fungus (Fuller 1987). This technique works well if the objective is to characterize an unknown fungal community, but is less effective if the objective is to isolate a particular species, especially if the target species is found at low densities or is poorly competitive. Previous attempts to obtain environmental samples of Batrachochytrium dendrobatidis by using traditional baiting techniques were not successful (Longcore et al. 1999, Livo, 2004.The screening of DNA samples with environmental quantitative PCR provides a powerful tool to quantitatively detect water-and soil-borne pathogens, and its applications have been demonstrated for pathogenic Candida cells (Brinkman et al. 2003) and for Perkinsus marinus, a serious pathogen of the oyster Crassostrea virginica (Audemard et al. 2004). Here, we describe the development of a method that combines a simple, hand-held filtration system, a commercially available DNA extraction kit and a highly sensitive quantitative real-time PCR assay to detect B. dendrobatidis in small volumes (<1 l)...

Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus

“…However, the concentration of zoospores we used was higher than that estimated from pond water in nature (Kirshtein et al, 2007;Walker et al, 2007). For example, Kirshtein et al, 2007 andWalker et al, 2007 estimated that zoospore density in ponds varied from 0.5 to 454 zoospores per liter, whereas our exposure protocol was 20 000 zoospores per liter for 24 h. A sampling of recent experimental papers reveals that individual amphibians were exposed to Bd densities in their experimental containers that ranged from 25 000 zoospores per liter (Rachowicz and Vredenburg, 2004) to 10 5 -10 6 zoospores per liter (Rachowicz et al, 2006;Retallick and Miera, 2007;Woodhams et al, 2007a). Further experimentation that varies level and duration of exposure to Bd zoospores in relation to population densities of antiBd bacteria is needed to determine under what conditions the cutaneous microbiota are protective.…”

“…In this experiment, we exposed juvenile frogs to a low concentration of Bd zoospores relative to most other investigators (Rachowicz et al, 2006;Retallick and Miera, 2007;Woodhams et al, 2007a). However, the concentration of zoospores we used was higher than that estimated from pond water in nature (Kirshtein et al, 2007;Walker et al, 2007). For example, Kirshtein et al, 2007 andWalker et al, 2007 estimated that zoospore density in ponds varied from 0.5 to 454 zoospores per liter, whereas our exposure protocol was 20 000 zoospores per liter for 24 h. A sampling of recent experimental papers reveals that individual amphibians were exposed to Bd densities in their experimental containers that ranged from 25 000 zoospores per liter (Rachowicz and Vredenburg, 2004) to 10 5 -10 6 zoospores per liter (Rachowicz et al, 2006;Retallick and Miera, 2007;Woodhams et al, 2007a).…”

Survival of the amphibian chytrid fungus Batrachochytrium dendrobatidis on bare hands and gloves: hygiene implications for amphibian handling

“…Subsequently, Kirshtein et al (2007) were able to detect between 19 and 454 zoospore equivalents l -1 in water collected from 4 different sites associated with amphibians in the USA which tested positive for B. dendrobatidis. Walker et al (2007) found 0.5 to 262 (median 31) zoospore equivalents l -1 in 64% of ponds at a site in Spain, with higher counts occurring in more turbid water. While washing with water is a cheap and convenient option, these hygiene practices have never been evaluated until now.…”

“…Although it is believed that B. dendrobatidis is a pathogen that occurs in the environment (Department of Environment and Heritage 2006), it has been difficult to isolate (Kirshtein et al 2007, Walker et al 2007, and no animal reservoir other than amphibians has been found (Rowley et al 2006(Rowley et al , 2007. Amphibians become infected when the water-borne zoospores of B. dendrobatidis invade their epidermis (Berger et al 1998, Longcore et al 1999.…”

Joint effects of habitat, zooplankton, host stage structure and diversity on amphibian chytrid

“…This 'UVR index' essentially assesses the relative exposure experienced by a Bd zoospore suspended in the water column (based on: [34,35]). This metric strongly correlates with UVR reaching depth z, L(z) (Pearson r ¼ 0.993, p , 0.0001).…”