scholarly journals Highly Sensitive Quantitative PCR for the Detection and Differentiation of Pseudogymnoascus destructans and Other Pseudogymnoascus Species

2013 ◽  
Vol 80 (5) ◽  
pp. 1726-1731 ◽  
Author(s):  
Megan M. Shuey ◽  
Kevin P. Drees ◽  
Daniel L. Lindner ◽  
Paul Keim ◽  
Jeffrey T. Foster
Keyword(s):  
North America ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Etiologic Agent ◽  
Pseudogymnoascus Destructans ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
White Nose Syndrome ◽  
Highly Sensitive ◽  
Management Plans

ABSTRACTWhite-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent,Pseudogymnoascus destructans(formerlyGeomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiatingP. destructansfrom closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific toP. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifyingP. destructans, otherPseudogymnoascusspp., andGeomycesspp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.

scholarly journals Phenotypic Divergence along Geographic Gradients Reveals Potential for Rapid Adaptation of the White-Nose Syndrome Pathogen,Pseudogymnoascus destructans, in North America

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Adrian Forsythe ◽  
Victoria Giglio ◽  
Jonathan Asa ◽  
Jianping Xu
Keyword(s):  
North America ◽  
Filamentous Fungus ◽  
Phenotypic Diversity ◽  
The United States ◽  
Ecological Niches ◽  
Eastern North America ◽  
Potential Candidate ◽  
Pseudogymnoascus Destructans ◽  
Content Type ◽  
White Nose Syndrome

ABSTRACTWhite-nose syndrome (WNS) is an ongoing epizootic affecting multiple species of North American bats, caused by epidermal infections of the psychrophilic filamentous fungusPseudogymnoascus destructans. Since its introduction from Europe, WNS has spread rapidly across eastern North America and resulted in high mortality rates in bats. At present, the mechanisms behind its spread and the extent of its adaptation to different geographic and ecological niches remain unknown. The objective of this study was to examine the geographic patterns of phenotypic variation and the potential evidence for adaptation among strains representing broad geographic locations in eastern North America. The morphological features of these strains were evaluated on artificial medium, and the viability of asexual arthroconidia of representative strains was investigated after storage at high (23°C), moderate (14°C), and low (4°C) temperatures at different lengths of time. Our analyses identified evidence for a geographic pattern of colony morphology changes among the clonal descendants of the fungus, with trait values correlated with increased distance from the epicenter of WNS. Our genomic comparisons of three representative isolates revealed novel genetic polymorphisms and suggested potential candidate mutations that might be related to some of the phenotypic changes. These results show that even though this pathogen arrived in North America only recently and reproduces asexually, there has been substantial evolution and phenotypic diversification during its rapid clonal expansion.IMPORTANCEThe causal agent of white-nose syndrome in bats isPseudogymnoascus destructans, a filamentous fungus recently introduced from its native range in Europe. Infections caused byP. destructanshave progressed across the eastern parts of Canada and the United States over the last 10 years. It is not clear how the disease is spread, as the pathogen is unable to grow above 23°C and ambient temperature can act as a barrier when hosts disperse. Here, we explore the patterns of phenotypic diversity and the germination of the fungal asexual spores, arthroconidia, from strains across a sizeable area of the epizootic range. Our analyses revealed evidence of adaptation along geographic gradients during its expansion. The results have implications for understanding the diversification ofP. destructansand the limits of WNS spread in North America. Given the rapidly expanding distribution of WNS, a detailed understanding of the genetic bases for phenotypic variations in growth, reproduction, and dispersal ofP. destructansis urgently needed to help control this disease.

scholarly journals Environmental transmission of Pseudogymnoascus destructans to hibernating little brown bats

2021 ◽  
Author(s):  
Alan C Hicks ◽  
Scott Darling ◽  
Joel Flewelling ◽  
Ryan von Linden ◽  
Carol U Meteyer ◽  
...  
Keyword(s):  
North America ◽  
Environmental Exposure ◽  
Environmental Samples ◽  
Molecular Techniques ◽  
Myotis Lucifugus ◽  
Pseudogymnoascus Destructans ◽  
Environmental Transmission ◽  
Early Stages ◽  
White Nose Syndrome ◽  
Little Brown Bats

Pathogens with persistent environmental stages can have devastating effects on wildlife communities. White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has caused widespread declines in bat populations of North America. In 2009, during the early stages of the WNS investigation and before molecular techniques had been developed to readily detect P. destructans in environmental samples, we initiated this study to assess whether P. destructans can persist in the hibernaculum environment in the absence of its conclusive bat host and cause infections in naive bats. We transferred little brown bats (Myotis lucifugus) from an unaffected winter colony in northwest Wisconsin to two P. destructans contaminated hibernacula in Vermont where native bats had been excluded. Infection with P. destructans was apparent on some bats within 8 weeks following the introduction of unexposed bats to these environments, and mortality from WNS was confirmed by histopathology at both sites 14 weeks following introduction. These results indicate that environmental exposure to P. destructans is sufficient to cause the infection and mortality associated with WNS in naive bats, which increases the probability of winter colony extirpation and complicates conservation efforts.

scholarly journals Experimental Infection ofTadarida brasiliensiswithPseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Michelle L. Verant ◽  
Carol U. Meteyer ◽  
Benjamin Stading ◽  
David S. Blehert
Keyword(s):  
Animal Model ◽  
Experimental Animal ◽  
Experimental Infection ◽  
Abundant Species ◽  
Experimental Animal Model ◽  
Pseudogymnoascus Destructans ◽  
Tadarida Brasiliensis ◽  
Content Type ◽  
White Nose Syndrome ◽  
Management Actions

ABSTRACTWhite-nose syndrome (WNS) is causing significant declines in populations of North American hibernating bats, and recent western and southern expansions of the disease have placed additional species at risk. Understanding differences in species susceptibility and identifying management actions to reduce mortality of bats from WNS are top research priorities. However, the use of wild-caught susceptible bats, such asMyotis lucifugus, as model species for WNS research is problematic and places additional pressure on remnant populations. We investigated the feasibility of usingTadarida brasiliensis, a highly abundant species of bat that tolerates captivity, as the basis for an experimental animal model for WNS. Using methods previously established to confirm the etiology of WNS inM. lucifugus, we experimentally infected 11T. brasiliensisbats withPseudogymnoascus destructansin the laboratory under conditions that induced hibernation. We detectedP. destructanson all 11 experimentally infected bats, 7 of which exhibited localized proliferation of hyphae within the epidermis, dermis, and subcutaneous tissue, similar to invasive cutaneous ascomycosis observed inM. lucifugusbats with WNS. However, the distribution of lesions across wing membranes ofT. brasiliensisbats was limited, and only one discrete “cupping erosion,” diagnostic for WNS, was identified. Thus, the rarity of lesions definitive for WNS suggests thatT. brasiliensisdoes not likely represent an appropriate model for studying the pathophysiology of this disease. Nonetheless, the results of this study prompt questions concerning the potential for free-ranging, migratoryT. brasiliensisbats to become infected withP. destructansand move the fungal pathogen between roost sites used by species susceptible to WNS.IMPORTANCEWhite-nose syndrome (WNS) is a fungal disease that is causing severe declines of bat populations in North America. Identifying ways to reduce the impacts of this disease is a priority but is inhibited by the lack of an experimental animal model that does not require the use of wild-caught bat species already impacted by WNS. We tested whetherTadarida brasiliensis, one of the most abundant species of bats in the Americas, could serve as a suitable animal model for WNS research. WhileT. brasiliensisbats were susceptible to experimental infection with the fungus under conditions that induced hibernation, the species exhibited limited pathology diagnostic for WNS. These results indicate thatT. brasiliensisis not likely a suitable experimental model for WNS research. However, the recovery of viable WNS-causing fungus from experimentally infected bats indicates a potential for this species to contribute to the spread of the pathogen where it coexists with other species of bats affected by WNS.

Prevalence and phenology of white-nose syndrome fungus Pseudogymnoascus destructans in bats from Poland

2014 ◽  
Vol 9 (4) ◽  
pp. 437-443 ◽  
Author(s):  
Konrad Sachanowicz ◽  
Arkadiusz Stępień ◽  
Mateusz Ciechanowski
Keyword(s):  
North America ◽  
Mass Mortality ◽  
Parasitic Fungus ◽  
Pseudogymnoascus Destructans ◽  
Southern Poland ◽  
White Nose Syndrome ◽  
Two Samples ◽  
Myotis Myotis ◽  
Myotis Daubentonii ◽  
Fungal Dna

AbstractPseudogymnoascus destructans (Pd), a parasitic fungus (being responsible for a disease known as white-nose syndrome, WNS) that caused mass mortality of cave-dwelling, hibernating bats in North America, appears to be native of Europe, where it also occurs on wintering bats, but no similar outbreaks of WNS have been recorded. Herein, we provide the first account on prevalence and phenology of P. destructans in Poland. Bats were counted once per month, from October or January to May (2010-2013), in an abandoned ore mine in southern Poland. Presence of P. destructans in two samples was confirmed by sequencing of isolated fungal DNA. Observations of phenotypically identical mycosis on bats hibernating at this site in March 2006 are likely to be the first known records of P. destructans from Poland. All Pd-suspected individuals were Myotis myotis with an exception of one Myotis daubentonii. The first Pd-suspected bats were noted in mid-February, but their number was the highest in March, what overlapped with maximum numbers of hibernating M. myotis. The prevalence in March was 7%–27% of M. myotis individuals. No mass mortality of bats was observed in the mine, with only three dead individuals found in the hibernaculum which hosted up to 130 bats, representing 6–7 species.

scholarly journals Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Megan E. Reller ◽  
J. Stephen Dumler
Keyword(s):  
Quantitative Pcr ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Epidemiologic Studies ◽  
Qpcr Assay ◽  
Spotted Fever Group ◽  
Analytical Sensitivity ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

scholarly journals Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water

2010 ◽  
Vol 76 (21) ◽  
pp. 7348-7351 ◽  
Author(s):  
Nicolas Radomski ◽  
Françoise S. Lucas ◽  
Régis Moilleron ◽  
Emmanuelle Cambau ◽  
Sophie Haenn ◽  
...  
Keyword(s):  
Surface Water ◽  
16S Rrna ◽  
Real Time ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Real Time Quantitative Pcr ◽  
Qpcr Method ◽  
Pcr Method ◽  
Real Time Qpcr ◽  
Mycobacterium Spp

ABSTRACT A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).

scholarly journals Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

2011 ◽  
Vol 78 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Adeline Tissier ◽  
Martine Denis ◽  
Philippe Hartemann ◽  
Benoît Gassilloud
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Quantitative Pcr ◽  
Campylobacter Jejuni ◽  
Tap Water ◽  
Culture Method ◽  
Cellulose Ester ◽  
Campylobacter Coli ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACTInvestigations ofCampylobacter jejuniandCampylobacter coliin samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection ofCampylobacterin 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) ofCampylobacterper 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with aC. colistrain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence ofC. jejuniandC. coligenomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination byCampylobacter.

scholarly journals Phylogenetics of a Fungal Invasion: Origins and Widespread Dispersal of White-Nose Syndrome

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Kevin P. Drees ◽  
Jeffrey M. Lorch ◽  
Sebastien J. Puechmaille ◽  
Katy L. Parise ◽  
Gudrun Wibbelt ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
North America ◽  
North American ◽  
Fungal Pathogen ◽  
Phylogenetic Study ◽  
Pseudogymnoascus Destructans ◽  
Long Distance ◽  
Recent Introduction ◽  
White Nose Syndrome ◽  
The North

ABSTRACT Globalization has facilitated the worldwide movement and introduction of pathogens, but epizoological reconstructions of these invasions are often hindered by limited sampling and insufficient genetic resolution among isolates. Pseudogymnoascus destructans , a fungal pathogen causing the epizootic of white-nose syndrome in North American bats, has exhibited few genetic polymorphisms in previous studies, presenting challenges for both epizoological tracking of the spread of this fungus and for determining its evolutionary history. We used single nucleotide polymorphisms (SNPs) from whole-genome sequencing and microsatellites to construct high-resolution phylogenies of P. destructans . Shallow genetic diversity and the lack of geographic structuring among North American isolates support a recent introduction followed by expansion via clonal reproduction across the epizootic zone. Moreover, the genetic relationships of isolates within North America suggest widespread mixing and long-distance movement of the fungus. Genetic diversity among isolates of P. destructans from Europe was substantially higher than in those from North America. However, genetic distance between the North American isolates and any given European isolate was similar to the distance between the individual European isolates. In contrast, the isolates we examined from Asia were highly divergent from both European and North American isolates. Although the definitive source for introduction of the North American population has not been conclusively identified, our data support the origin of the North American invasion by P. destructans from Europe rather than Asia. IMPORTANCE This phylogenetic study of the bat white-nose syndrome agent, P. destructans , uses genomics to elucidate evolutionary relationships among populations of the fungal pathogen to understand the epizoology of this biological invasion. We analyze hypervariable and abundant genetic characters (microsatellites and genomic SNPs, respectively) to reveal previously uncharacterized diversity among populations of the pathogen from North America and Eurasia. We present new evidence supporting recent introduction of the fungus to North America from a diverse Eurasian population, with limited increase in genetic variation in North America since that introduction.

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