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Isothermal recombinant polymerase amplification and CRIPSR (CAS12A) assay detection of Renibacterium salmoninarum as an example for wildlife pathogen detection in environmental DNA samples

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon ( Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum , is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20–40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Journal of Wildlife Diseases

Contaminant exposure and potential effects on terrestrial vertebrates residing in the National Capital Region network and Mid-Atlantic network

Part of the mission of the National Park Service is to preserve the natural resources, processes, systems, and associated values of its units in an unimpaired condition. Environmental contamination and pollution processes are well recognized stressors addressed by its management policies and plans. A recent study indicates that contemporary terrestrial vertebrate ecotoxicological data are lacking for 59 of 126 Park Service units located in coastal watersheds exhibiting serious water quality problems or high vulnerability to pollution. Based upon these findings, a more in-depth evaluation of contaminant threats and ecotoxicological data gaps related to terrestrial vertebrates was undertaken at 23 Inventory and Monitoring National Park units in National Capital Region and Mid-Atlantic Networks. Ecotoxicological data were compiled for each park unit through literature searches and meetings with Park Service personnel. Information on contemporary and on persistent legacy pollutants in air, water, soil, and terrestrial vertebrates (amphibians, reptiles, birds and mammals) were evaluated. To identify contaminant threats in proximity to the 23 Park Service study units, data was gathered on National Priority List Superfund sites, Section 303(d) Impaired Waterbodies, the number and relative toxicity of current use pesticides and herbicides, Toxic Release Inventory sites and discharge of priority pollutants, and Fish Consumption Advisories. A metric was derived that described the quality and quantity of existing data for each park, and in combination with known contaminant threats, park units in need of additional study were identified. Results demonstrated that over half of the Park Service study units are near Toxic Release Inventory sites discharging dioxins, polychlorinated biphenyls, lead or mercury into air or water, and fish consumption advisories are in effect at or near 22 of the 23 study units. Pesticide and herbicide use at the park units is minimal, with the exception of those units with significant agricultural leases. Despite highly regulated use, many of the pesticides and herbicides applied are believed to be highly toxic to amphibians, and some of the compounds are also highly toxic to birds. Only 70 reports were found that describe terrestrial vertebrate ecotoxicology data on or near the study units. Of the greater than 75,000 compounds in commerce in the United States, existing terrestrial vertebrate exposure and effects data in the pr esent study were limited to 58 legacy organochlorine pesticides, polychlorinated biphenyls and individual congeners, insecticides and rodenticides, metals, and some contemporary compounds (e.g., polybrominated diphenyl ether flame retardants, and alkylphenol and ethoxylate surfactants). Based upon these and other findings, ecotoxicological monitoring and research investigations of terrestrial vertebrates are warranted at several National Parks. These include Shenandoah National Park, Richmond National Battlefield, Chesapeake & Ohio Canal National Historic Park, Valley Forge National Historic Park, Hopewell Furnace National Historic Site, Monocacy National Battlefield, and Harpers Ferry National Historic Park. The types of investigations vary according to the species present at these parks and potential contaminant threats, but should focus on contemporary use pesticides and herbicides, polychlorinated biphenyls, mercury, lead, and perhaps, emerging contaminants including antibiotics, flame retardants, pharmaceuticals, and surfactants. Other management recommendations include additional training for natural resource staff members in the area of ecotoxicology, inclusion of terrestrial vertebrate contaminant monitoring and the Contaminant Assessment Process (U.S. Geological Survey Biomonitoring of Environmental Status and Trends Project) into the National Park Service Vital Signs Program, development of protocols for hand ling and toxicological analysis of dead or seemingly affected wildlife, consideration of some alternative methods and compounds for pest management and weed control, and use of non-toxic fishing tackle by visitors.

Maryland, Pennsylvania, Virginia

Environmental contaminant hazards to wildlife at National Capital region and Mid-Atlantic National Park Service units

Pollutant data for air, water, soil and biota were compiled from databases and internet sources and by staff interviews at 23 National Park Service (NPS) units in 2005. A metric was derived describing the quality and quantity of data for each park, and in combination with known contaminant threats, the need for ecotoxicological study was identified and ranked. Over half of NP units were near Toxic Release Inventory sites discharging persistent pollutants, and fish consumption advisories were in effect at or near 22 of the units. Pesticide and herbicide use was found to be minimal, with the exception of those units with agricultural leases. Only 70 reports were found that describe terrestrial vertebrate environmental contaminant data at or near the units. Of the >75,000 compounds in commerce, empirical exposure data were limited to merely 58 halogenated compounds, insecticides, rodenticides, metals, and some contemporary compounds. Further ecotoxicological monitoring and research is warranted at several units including Shenandoah National Park, Richmond National Battlefield Park, Chesapeake & Ohio Canal National Historical Park, Valley Forge National Historical Park, Hopewell Furnace National Historic Site, Monocacy National Battlefield, and Harpers Ferry National Historical Park. The types of investigations vary according to the wildlife species present and potential contaminant threats, but should focus on contemporary use pesticides and herbicides, polychlorinated biphenyls, mercury, lead, and perhaps antibiotics, flame retardants, pharmaceuticals, and surfactants. Other management recommendations include inclusion of screening level contaminant risk assessments into the NPS Vital Signs Program, development of protocols for toxicological analysis of seemingly affected wildlife, alternative methods and compounds for pest management, and use of non-toxic fishing tackle by visitors.

Book chapter

Gentamicin tissue concentration in various avian species following recommended dosage therapy

Plasma and tissue drug concentrations were compared in eastern bobwhite quail (Colinus virginianus virginianus) and pigeons (Columba livia) given gentamicin by IM administration at the dosage of 10 mg/kg, and in greater sandhill cranes (Grus canadensis tabida) and hybrid rosybill ducks (Netta sp) given the same antibiotic at a dosage of 5 mg/kg. Quail and cranes had significantly higher liver concentrations of gentamicin at 6 hours after injection than did pigeons and ducks. Cranes had significantly higher plasma concentrations than did ducks at 6 hours after injection. Compared with plasma values, gentamicin concentrations were significantly higher in the liver of cranes at 12 hours after injection, and in the kidneys at 18 hours.

American Journal of Veterinary Research

Pharmacokinetics and tissue concentrations of tylosin in selected avian species

Tissue and plasma concentrations and the biological half-life of tylosin in avian species of a variety of body sizes and metabolic rates were studied. The species chosen were eastern bobwhite quail (Colinus virginianus virginianus), pigeons (Columba livia), greater sandhill cranes (Grus canadensis tabida), and emus (Dromaius novaehollandiae). In the 1st phase of this study, tylosin was administered IM to quail, pigeons, and emus at a dosage rate of 25 mg/kg of body weight and to cranes at a dosage rate of 15 mg/kg. The average peak plasma concentrations of tylosin in quail, pigeons, cranes, and emus were 4.31, 5.63, 3.62, and 3.26 microgram/ml, respectively. These peak concentrations occurred at 0.5 to 1.5 hours after administration. The biological half-life of tylosin averaged 1.2 hours in quail, pigeons, and cranes, and was 4.7 hours in emus. In the 2nd phase of this study, tylosin concentrations in the tissues of quail, pigeons, and cranes were markedly higher than were plasma concentrations at corresponding sampling times. Six hours after antibiotic administration, tissue concentrations of tylosin in all species remained within the minimum inhibitory concentration for most pathogenic organisms. Dosage regimens of 25 mg of tylosin/kg 4 times daily for quail and pigeons, 15 mg/kg 3 times daily for cranes, and 25 mg/kg 3 times daily for emus would be needed to establish and maintain therapeutic tissue concentrations.

American Journal of Veterinary Research

Chronic toxicity of diphenhydramine hydrochloride and erythromycin thiocyanate to Daphnia, Daphnia magna, in a continuous exposure test system

Diphenhydramine hydrochloride (DH; Benadryl TM, an over-the-counter antihistamine) and erythromycin thiocyanate (ET; a commonly used macrolide antibiotic) are pharmaceutical compounds whose chronic toxicity to Daphnia magna had not been characterized. Continuous exposure to DH concentrations about 5 times greater than the maximum reported environmental concentration of 0.023 lg/L for 21 days or to ET concentrations about 40 times the maximum reported environmental concentration of 6 (mu or u)g/L for 21 days did not significantly impact D. magna survival and production. In this study the no observable effect concentration for DH was 0.12 (mu or u)g/L and for ET was 248 (mu or u)g/L.

Bulletin of Environmental Contamination and Toxico

Review of concepts useful for maintaining quality of male reproductive field samples for laboratory study

Investigations into cellular and molecular characteristics of male gametes obtained from fish in natural ecosystems require careful sample handling and shipping in order to minimize artifacts. Maintaining sample integrity engenders confident assessments of ecosystem health, whereby animal condition is often reflected by gamete biomarkers - indicators that respond in measurable ways to changes. A number of our investigations have addressed the hypothesis that biomarkers from fish along a pollution gradient are reflective of site location. Species biology and the selected biological endpoints direct choice of parameters such as: temperature, buffer osmolality, time in transit, fixation, cryoprotectants, protease inhibition, and antibiotic inclusion in extender. This paper will highlight case studies, and outline parameters and thoughts on approaches for use by field and laboratory researchers.

Conference Paper

Environmental presence and persistence of pharmaceuticals: An overview

Emerging contaminants (ECs) in the environment – that is, chemicals with domestic, municipal, industrial, or agricultural sources that are not commonly monitored but may have the potential for adverse environmental effects – is a rapidly growing field of research. The use of “emerging” is not intended to infer that the presence of these compounds in the environment is new. These chemicals have been released into the environment as long as they have been in production or, in the case of hormones and other endogenous compounds, since the rise of animal life. What is emerging is the interest by the scientific and lay communities in the presence of these chemicals in the environment, the analytical capabilities required for detection, and the subtle effects that very small concentrations of these chemicals appear to have on aquatic biota. In December 2006, Environmental Science & Technology devoted an entire special issue (volume 40, number 23) to the topic of ECs, illustrating the increased interest in the subject. Within the EGs, one particular class that has seen a substantial increase in research over the past 10 years is pharmaceuticals and personal-care products (PPCPs). This increased research interest can be demonstrated by several means, including requests for proposals from funding agencies, but the clearest indication of a focused effort to understand the introduction, transformation, and potential health and environmental effects of PPCPs and ECs, in general, is the number of published reports. This increase can be shown by examining six environmental journals that regularly publish PPCP-related papers – Chemosphere , Environmental Science & Technology , Environmental Toxicology and Chemistry , Science of the Total Environment , Water Research , and Water Science and Technology . In 1998 there were 22 papers published on pharmaceuticals, antibiotics, or drugs in these 6 journals; by 2006, this number increased sixfold to 132 papers (Figure 1.1). This growth can be attributed to a number of factors. The presence of pharmaceuticals in surface-water samples from Europe and the United States was documented in several sentinel papers. These ground-breaking works encouraged other scientists to examine the rivers, streams, lakes, and reservoirs in their regions for such chemicals. In addition, the intense public attention paid to news reports on the environmental detections of these chemicals and possible effects of aquatic life has made this issue visible to the wastewater-treatment, drinking-water treatment, and regulatory communities. This has driven the funding bodies associated with these communities to fund studies or request proposals that address the presence, fate, and effects of PPCPs in aquatic systems. The release of the first comprehensive reconnaissance of pharmaceuticals and other wastewater contaminants in the United States provides an example of the intense media interest in this topic. Within 6 days on online publication of this study, 72 newspapers across the United States had published articles describing the results, either locally written or based on international media syndicate reports. There also was substantial concurrent coverage by local and national radio and television outlets, including the Cable News Network, ABC World News Tonight , and National Public Radio. A substantial fraction of these news stories may be attributable to press releases and media briefings prior to publication. However, the interest by television and print journalists in reporting the results of a peer-reviewed journal article to the general public was motivated by the recognition that describing the presence of PPCPs in water supplies would be of interest to the public. To better convey the results of the study published by Koplin et al. to the public, a separate general-interest fact sheet was published to summarize the important points of the study. Because PPCPs are commonly and widely used by individuals, there is likely a preexisting, personal identification with these compounds that does not occur for the wide range of other organic and inorganic contaminants whose presence in the environment has previously been described. This greater public “name recognition” makes itself known through the media to the regulatory and technical community and has prompted interest in sponsoring research that defines the composition and concentrations of PPCPs in potential sources and their fate and effects following relase into the environment. Independent of the drivers that potentially fuel the interest in studies of PPCPs, it is clear that PPCP research has grown beyond surface-water studies to examine issues such as: • Presence in other matrices, such as groundwater, landfill leachates, sediments, and biosolids. • Environmental transport and fate in surface water, groundwater, and soils amended with reclaimed water or biosolids. • PPCP source elucidation, such as wastewater treatment plant (WWTP) effluents, confined animal feeding operations (CAFOs), and aquaculture. • Removal during wastewater and drinking-water treatment. • Effects on aquatic ecosystems, terrestrial ecosystems, and human health. The chapters in this book provide an extensive examination of current environmental pharmaceutical research and are divided into three sections: “Occurrence and Analysis of Pharmaceuticals in the Environment,” “Environment Fate and Transformations of Veterinary Pharmaceuticals,” and “treatment of Pharmaceuticals in Drinking Water and Wastewater.” The purpose of this introductory overview chapter is to outline current (2004-2006) knowledge about the presence and concentration of PPCPs as described in the published literature. Previous reviews should be consulted for discussions on pre-2004 publications. Those reviews will provide the reader with a comprehensive introduction to the topic of PPCPs in the environment. This chapter describes the sources of PPCPs and other organic contaminants often associated with human wastewater into the environment, the range of concentrations present in various environmental compartments, and the potential routes of removal/sequestration. An overview of the sources and fate of veterinary pharmaceuticals will be discussed in Chapter 5, “Fate and Transport of Veterinary Medicines in the Soil Environment.”

Book chapter