University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Environmental Protection Agency Papers U.S. Environmental Protection Agency 2012 Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution Hodon Ryu U.S. Environmental Protection Agency Jingrang Lu U.S. Environmental Protection Agency Jason Vogel Oklahoma State University, [email protected] Michael Elk U.S. Environmental Protection Agency Felipe Chávez-Ramírez Gulf Coast Bird Observatory See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/usepapapers Part of the Earth Sciences Commons, Environmental Health and Protection Commons, Environmental Monitoring Commons, and the Other Environmental Sciences Commons Ryu, Hodon; Lu, Jingrang; Vogel, Jason; Elk, Michael; Chávez-Ramírez, Felipe; Ashbolt, Nicholas; and Santo Domingo, Jorge, "Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution" (2012). 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Authors Hodon Ryu, Jingrang Lu, Jason Vogel, Michael Elk, Felipe Chávez-Ramírez, Nicholas Ashbolt, and Jorge Santo Domingo This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ usepapapers/107 Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution Hodon Ryu,a Jingrang Lu,b Jason Vogel,c Michael Elk,a Felipe Chávez-Ramírez,d Nicholas Ashbolt,b and Jorge Santo Domingoa National Risk Management Research Laboratorya and National Exposure Research Laboratory,b U.S. Environmental Protection Agency, Cincinnati, Ohio, USA; Oklahoma State University, Stillwater, Oklahoma, USAc; and Gulf Coast Bird Observatory, Lake Jackson, Texas, USAd While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no meth- ods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was devel- oped, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detec- tion ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was rela- tively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with different free-range hosts) being negative. Of the presumed crane-impacted 20 ;553 ؍ of the nontarget fecal samples (i.e., n 95% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were %88 ,(16 ؍ water samples (n -Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-posi .(165 ؍ positive (n tive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics. he Platte River is a major river in the state of Nebraska in the bility of a putative crane-specific assay to environmental water TUnited States and has many beneficial uses to humans, such as samples collected from different geographic locations. We also groundwater recharge for the drinking water supply, irrigation, studied the microbial composition of snow geese, as this water- and recreation activities. This river is the preferred transient roost- fowl species uses the Platte River as a roosting site during its winter ing habitat of various migratory birds, including the endangered migration. whooping crane, sandhill cranes, snow geese, Canada geese, white-fronted geese, mallard ducks, and pintail ducks, among MATERIALS AND METHODS others. Sandhill cranes are among the most numerous migratory Sample collection and DNA extraction. Most sandhill crane (Grus ca- birds in the region, with an estimated 500,000 birds seasonally in nadensis, n ϭ 95) and all snow goose (Chen caerulescens, n ϭ 22) excreta the area (http://www.nwf.org/Wildlife/Wild-Places/Platte-River were collected as fresh droppings on sand bars in a Platte River watershed .aspx). Sandhill cranes primarily use the Central Flyway migration (latitude, 40.78°N; longitude, 98.48°W). The sandhill crane samples were collected on five different sampling dates: 15 March 2010 (n ϭ 6), 18 route, coming into the Platte River in early spring, which coin- March 2010 (n ϭ 16), 25 March 2010 (n ϭ 16), 5 April 2010 (n ϭ 26), and cides with resuming human recreational activities in the river ba- 21 March 2012 (n ϭ 31). A sandhill crane sample and 11 whooping crane sin. This is important, as migratory birds have the potential to be (G. americana) samples collected in Texas (1 March 2012) were also pro- seasonal sources of waterborne fecal bacteria, including pathogens cessed in this study. In addition, 71 samples from 12 captive cranes (col- (9, 11, 29). In fact, sandhill cranes were identified as the fecal lected in March 2012) were analyzed: black-crowned crane (Balearica source in a recent campylobacteriosis outbreak in Alaska associ- pavonina, n ϭ 1), black-necked crane (G. nigricollis, n ϭ 5), Brolga crane ated with the consumption of peas (8), underscoring the impor- (G. rubicunda, n ϭ 3), Eurasian crane (G. grus, n ϭ 1), gray-crowned crane (Balearica regulorum, n ϭ 1), hooded crane (G. monacha, n ϭ 4), red- tance of monitoring microbial water quality in waterfowl-im- ϭ ϭ pacted waters. crowned crane (G. japonensis, n 4), sandhill crane (G. canadensis, n 11), Siberian crane (G. leucogeranus, n ϭ 10), wattled crane (Bugeranus Fecal indicator bacteria (FIB) such as Escherichia coli and en- carunculatus, n ϭ 1), white-naped crane (G. vipio, n ϭ 3), and whooping terococci are commonly used to determine the microbial quality crane (n ϭ 27). Five individual samples from sandhill cranes and snow of environmental waters. While measuring FIB can be used to geese were used to develop 16S rRNA gene clone libraries. Crane fecal establish the overall level of fecal pollution, traditional FIB detec- clone sequences were subsequently used to develop a crane-specific assay tion methods are uninformative for determining the primary fecal (the Crane1 assay). A total of 553 nontargeted fecal samples from 20 source(s) in polluted waters (30). Specifically, in the Platte River different hosts were collected and used to test host specificity. To evaluate there are other potential fecal contributors, such as cattle and wildlife. Several avian markers have been developed (7, 19, 20, 21), but to our knowledge, there is no crane-specific assay. This is in Received 19 December 2011 Accepted 28 March 2012 part due to the lack of data on the microbial composition of crane Published ahead of print 6 April 2012 excreta. To address this, 16S rRNA gene fecal clone sequences Address correspondence to Jorge Santo Domingo, [email protected]. from sandhill crane were analyzed to characterize microbial pop- Copyright © 2012, American Society for Microbiology. All Rights Reserved. ulations and to identify potentially crane-specific markers for use doi:10.1128/AEM.07923-11 in fecal source tracking. Additionally, we investigated the applica- 4338 aem.asm.org Applied and Environmental Microbiology p. 4338–4345 June 2012 Volume 78 Number 12 Development of a Novel Crane qPCR Assay the applicability of the developed assay with environmental water sam- tion protocol involved incubation at 50°C for 2 min, followed by 95°C for ples, a total of 43 water samples were collected in 1-liter sterilized plastic 10 min and then 40 cycles of 95°C for 15 s and 61°C for 1 min with a bottles from three locations (upstream, within, and downstream of the fluorescence read, followed by a melt-curve analysis from 60 to 90°C in crane roosting area) in the Central Platte River, Nebraska, during the 0.1-degree increments. The qPCR assays were performed using a 7900 HT sandhill crane migratory season (i.e., between January 2010 and May Fast real-time sequence detector (Applied Biosystems, Foster City, CA). 2010). Water samples were collected on Sunday night or Monday morn- All reaction mixtures were prepared in triplicate in MicroAmp Optical ing, when effluents from power plants were not discharged. A total of 138 96-well reaction plates with MicroAmp Optical caps (Applied Biosystems, water samples were also collected from the Arecibo watershed (Puerto Foster City, CA). PCR data were analyzed using ABI’s Sequence Detector Rico) presumed to be primarily impacted by cattle, human, and wildlife software (version 2.2.2). PCR signals were recorded as presence/absence (but not impacted by migratory birds) and used as putative negative- of data and signal quantity values. Duplicate serial dilutions of crane fecal Ϫ Ϫ control samples. Water and fecal samples were shipped overnight on ice DNA (10 8 to 10 12 g/reaction) were used to generate standard curves. packs or dry ice to the EPA laboratory (Cincinnati, OH). Triplicates of Percent amplification efficiencies were calculated by the instrument man- 100-ml water samples were filtered onto polycarbonate membranes ufacturer’s instructions (Applied Biosystems). No-template controls were (0.4-␮m pore size, 47-mm diameter; GE Water and Process Technologies, used to check for cross contamination (two per PCR plate).