Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species

White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces 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 differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. 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 identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. 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

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations

Genomics of Brucellosis in Wildlife and Livestock of the Greater Yellowstone Ecosystem

Brucellosis, a disease caused by the bacterium Brucella abortus, has recently been expanding its distribution in the Greater Yellowstone Ecosystem (GYE), with increased outbreaks in cattle and rising seroprevalence in elk (Cervus elaphus) over the past decade. Genetic studies suggest elk are a primary source of recent transmission to cattle. However, these studies are based on Variable Number Tandem Repeat (VNTR) data, which are limited in assessing and quantifying transmission among species. The goal of this study was to (i) investigate the introduction history of B. abortus in the GYE, (ii) identify B. abortus lineages associated with host species and/or geographic localities, and (iii) quantify transmission across wildlife and livestock host species and populations. We sequenced B. abortus whole genomes (n= 207) derived from isolates collected from three host species (bison, elk, cattle) over the past 30 years, throughout the GYE. We identified genetic variation among isolates, and applied a spatial diffusion phylogeographic modeling approach that incorporated temporal information from sampling. Based on these data, our results suggest four divergent Brucella lineages, with a time to most recent common ancestor of ~130 years ago, possibly representing a minimum of four brucellosis introductions into the GYE. Two Brucella lineages were generally clustered by geography. Evidence for cross-species transmission was detected among all species, though most events occur within species and herds. Understanding transmission dynamics is imperative for implementing effective control measures and may assist in identifying source populations responsible for past and future brucellosis infections in wildlife and outbreaks in livestock

Global phylogenomic diversity of Brucella abortus: spread of a dominant lineage

Brucella abortus is a globally important zoonotic pathogen largely found in cattle hosts and is typically transmitted to humans through contaminated dairy products or contact with diseased animals. Despite the long, shared history of cattle and humans, little is known about how trade in cattle has spread this pathogen throughout the world. Whole genome sequencing provides unparalleled resolution to investigate the global evolutionary history of a bacterium such as B. abortus by providing phylogenetic resolution that has been unobtainable using other methods. We report on large-scale genome sequencing and analysis of B. abortus collected globally from cattle and 16 other hosts from 52 countries. We used single nucleotide polymorphisms (SNPs) to identify genetic variation in 1,074 B. abortus genomes and using maximum parsimony generated a phylogeny that identified four major clades. Two of these clades, clade A (median date 972 CE; 95% HPD, 781–1142 CE) and clade B (median date 150 BCE; 95% HPD, 515 BCE–164 CE), were exceptionally diverse for this species and are exclusively of African origin where provenance is known. The third clade, clade C (median date 949 CE; 95% HPD, 766–1102 CE), had most isolates coming from a broad swath of the Middle East, Europe, and Asia, also had relatively high diversity. Finally, the fourth major clade, clade D (median date 1467 CE; 95% HPD, 1367–1553 CE) comprises the large majority of genomes in a dominant but relatively monomorphic group that predominantly infects cattle in Europe and the Americas. These data are consistent with an African origin for B. abortus and a subsequent spread to the Middle East, Europe, and Asia, probably through the movement of infected cattle. We hypothesize that European arrival to the Americas starting in the 15th century introduced B. abortus from Western Europe through the introduction of a few common cattle breeds infected with strains from clade D. These data provide the foundation of a comprehensive global phylogeny of this important zoonotic pathogen that should be an important resource in human and veterinary epidemiology

Use of multiple sequencing technologies to produce a high-quality genome of the fungus pseudogymnoascus destructans, the causative agent of bat white-nose syndrome

White-nose syndrome has recently emerged as one of the most devastating wildlife diseases recorded, causing widespread mortality in numerous bat species throughout eastern North America. Here, we present an improved reference genome of the fungal pathogen Pseudogymnoascus destructans for use in comparative genomic studies

Use of multiple sequencing technologies to produce a high-quality genome of the fungus pseudogymnoascus destructans, the causative agent of bat white-nose syndrome

White-nose syndrome has recently emerged as one of the most devastating wildlife diseases recorded, causing widespread mortality in numerous bat species throughout eastern North America. Here, we present an improved reference genome of the fungal pathogen Pseudogymnoascus destructans for use in comparative genomic studies

Distribution of Biosurfactant-Producing Bacteria in Undisturbed and Contaminated Arid Southwestern Soils

Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R(2)A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils