Mating type determination within a microsatellite multiplex for the fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose disease in bats

Dool, Serena E.; Altewischer, Andrea; Fischer, Nicola; Drees, Kevin P.; Foster, Jeffrey T.; Fritze, Marcus; Puechmaille, Sébastien J.

doi:10.1007/s12686-018-1064-6

Cited by 8 publications

(10 citation statements)

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“…A third and non -mutually exclusive possibility for genotypic richness to be maintained in the long-term could be the presence of sexual reproduction which would introduce novel MLGs. While sexual reproduction has never been observed in P. destructans , the presence of both mating types in hibernacula in the native range (Palmer et al, 2014) as well as the relatively even distribution of both mating types (frequency of 0.69:1 for MAT1_1/ and MAT1_2 across 1099 SSI from Eldena, unpublished data) suggest it might occur there, though likely less frequently than asexual reproduction (Dool et al, 2020). Additionally, spores resulting from sexual reproduction are often more durable than clonal spores (see e.g., Gersuk et al, 2006; O’Gorman et al, 2009 for Aspergillus fumigatus) which could further drive the long-term persistence of P. destructans in hibernacula environments.…”

Section: Discussionmentioning

confidence: 99%

“…Given that a swab sample, whether collected from a bat or a wall, often contains multiple MLGs (Dool et al, 2020), it is important to first isolate single spores before proceeding with culture and DNA extraction. To do so, each sample was cultured and upon germination of spores (visible under the microscope within 2-4 days after plating), individual spores were physically separated into fresh petri dishes to obtain single-spore isolates (SSI).…”

Section: Methodsmentioning

confidence: 99%

“…The DNA was then extracted using a KingFisher Flex extraction robot (Thermo Scientific) and the MagMAX™ Plant DNA Isolation Kit (Thermo Scientific) utilizing magnetic-particle technology with the addition of 40 μM dithiothreitol (DDT) to lysis buffer A and 16.33 μM RNase A to lysis buffer B. To genotype isolates of P. destructans , 18 microsatellite markers (Drees et al, 2017b) were used in 4 PCR multiplexes as described in Dool et al (2020). Genotyping was carried out using an ABI 3130 Genetic Analyser (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

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Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of white-nose disease in bats

Fischer

Altewischer

Ranpal

et al. 2021

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Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host, for example in environmental reservoirs. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans, the causative agent of white-nose disease. We also characterised the modes and timing of transmission of the pathogen; key elements to better understand the disease dynamics. Using 18 microsatellite markers, we determined the genotypic and genic (based on allele frequencies) differentiation between 1,497 P. destructans isolates collected from nine closely situated hibernacula in North-Eastern Germany. One hibernaculum was the focus of intensive sampling in which both the bats and walls of the site were sampled at regular intervals over five consecutive winter seasons (1,062 isolates). We found significant genic differentiation between sites and few multi-locus genotypes shared across hibernacula (genotypic differentiation). This demonstrates that each hibernaculum has an essentially unique population of the fungus. This would be expected if bats purge viable P. destructans over the summer, preventing the mixing and exchange of the pathogen in maternity colonies, where bats from all of the studied hibernacula meet. Results from the intensively sampled site show higher measures of genotypic richness on walls compared to bats, the absence of genic differentiation between bats and walls, and stable relative abundance of multi-locus genotypes over multiple winter seasons. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of white-nose disease in bats

Fischer

Altewischer

Ranpal

et al. 2021

Preprint

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“…Given that a swab sample, whether collected from a bat or a wall, often contains multiple multilocus genotypes (hereafter simply referred to as “genotypes”), it is important to first isolate single spores before proceeding with culture and DNA extraction (Dool et al, 2020). To do so, each sample was cultured on dextrose‐peptone‐yeast agar (DPYA) following Vanderwolf et al (2016) and upon germination of spores (visible under the microscope within 2–4 days after plating), individual spores were physically separated and moved into new petri dishes to obtain single‐spore isolates (hereafter referred to as “isolates”).…”

Section: Methodsmentioning

confidence: 99%

“…To genotype isolates of P . destructans , 18 microsatellite markers (Drees et al, 2017) were used in four PCR multiplexes as described in Dool et al (2020). Genotyping was carried out using an ABI 3130 Genetic Analyser (Applied Biosystems) and GeneMapper Software v.5 (Applied Biosystems) was used for fragment analysis.…”

Section: Methodsmentioning

confidence: 99%

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of White‐Nose disease in bats

et al. 2021

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Development and Application of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Diagnosis of the Bat White-Nose Disease Fungus Pseudogymnoascus destructans

et al. 2022

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Pseudogymnoascus destructans (= Geomyces destructans) is a psychrophilic filamentous fungus that causes White-Nose Disease (WND; the disease associated with White-Nose Syndrome, WNS) in hibernating bats. The disease has caused considerable reductions in bat populations in the USA and Canada since 2006. Identification and detection of the pathogen in pure cultures and environmental samples is routinely based on qPCR or PCR after DNA isolation and purification. Rapid and specific direct detection of the fungus in the field would strongly improve prompt surveillance, and support control measures. Based on the genes coding for ATP citrate lyase1 (acl1) and the 28S-18S ribosomal RNA intergenic spacer (IGS) in P. destructans, two independent LAMP assays were developed for the rapid and sensitive diagnosis of the fungus. Both assays could discriminate P. destructans from 159 tested species of filamentous fungi and yeasts. Sensitivity of the assays was 2.1 picogram per reaction (pg/rxn) and 21 femtogram per reaction (fg/rxn) for the acl1 and IGS based assays, respectively. Moreover, both assays also work with spores and mycelia of P. destructans that are directly added to the master mix without prior DNA extraction. For field-diagnostics, we developed and tested a field-applicable version of the IGS-based LAMP assay. Lastly, we also developed a protocol for preparation of fungal spores and mycelia from swabs and tape liftings of contaminated surfaces or infected bats. This protocol in combination with the highly sensitive IGS-based LAMP-assay enabled sensitive detection of P. destructans from various sources.

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Mating type determination within a microsatellite multiplex for the fungal pathogen Pseudogymnoascus destructans, the causative agent of white-nose disease in bats

Cited by 8 publications

References 19 publications

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of white-nose disease in bats

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of white-nose disease in bats

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of White‐Nose disease in bats

Development and Application of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Diagnosis of the Bat White-Nose Disease Fungus Pseudogymnoascus destructans

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