Poultry litter as potential source of pathogens and other contaminants in groundwater and surface water proximal to large-scale confined poultry feeding operations.

Manure from livestock production has been associated with the contamination of water resources. To date, research has primarily focused on runoff of these contaminants from animal operations into surface water, and the introduction of poultry-derived pathogenic zoonoses and other contaminants into groundwater is under-investigated. We characterized pathogens and other microbial and chemical contaminants in poultry litter, groundwater, and surface water near confined poultry feeding operations (chicken layer, turkey) at 9 locations in Iowa and one in Wisconsin from May and June 2016. Results indicate that poultry litter from large-scale poultry confined feeding operations is a likely source of environmental contamination and that groundwater is also susceptible to such poultry-derived contamination. Poultry litter, groundwater, and surface water samples had detections of viable bacteria growth (Salmonella spp., enterococci, staphylococci, lactobacilli), multi-drug resistant Salmonella DT104 flost and int genes, F+ RNA coliphage (group I and IV), antibiotic resistance genes (ARGs; blaDHA, blaOXA-48, blaTEM, blaCMY-2, tetM), phytoestrogens (biochanin A, daidzein, formononetin), and a progestin (progesterone). In addition, mcr-1 (a colistin ARG), was detected in a groundwater sample and in another groundwater sample, antibiotic resistant isolates were positive for Brevibacterium spp., a potential signature of poultry in the environment. Detectable estrogenicity was not measured in poultry litter, but was observed in 67% of the surface water samples and 22% were above the U.S. Environmental Protection Agency trigger level of 1 ng/L. The transport of microbial pathogens to groundwater was significantly greater (p < 0.001) than the transport of trace organic contaminants to groundwater in this study. In addition to viable pathogens, several clinically important ARGs were detected in litter, groundwater, and surface water, highlighting the need for additional research on sources of these contaminants in livestock dominated areas.

The presence of antibiotic resistance genes in coastal soil and sediment samples from the eastern seaboard of the USA.

Infections from antibiotic resistant microorganisms are considered to be one of the greatest global public health challenges that result in huge annual economic losses. While genes that impart resistance to antibiotics (AbR) existed long before the discovery and use of antibiotics, anthropogenic uses of antibiotics in agriculture, domesticated animals, and humans are known to influence the prevalence of these genes in pathogenic microorganisms. It is critical to understand the role that natural and anthropogenic processes have on the occurrence and distribution of antibiotic resistance in microbial populations to minimize health risks associated with exposures. As part of this research, 15 antibiotic resistance genes were analyzed in coastal sediments and soils along the eastern seaboard of the USA using presence/absence quantitative and digital polymerase chain reaction assays. Samples (53 soil and 192 sediment samples including 54 replicates) were collected from a variety of coastal settings where human and wildlife exposure is likely. At least one of the antibiotic resistance genes was detected in 76.4% of the samples. Samples that contained at least five or more antibiotic resistance genes (5.7%) where typically hydrologically down gradient of watersheds influenced by combined sewer outfalls (CSO). The most frequently detected antibiotic resistance target genes were found in 33.2%, 34.4%, and 42.2% of samples (target genes blaSHV, tetO, and aadA2, respectively). These data provide unique insight into potential exposure of AbR genes over a large geographical region of the eastern seaboard of the USA.

Isolation, characterization and molecular identification of a novel aquareovirus that infects endangered fountain darter Etheostoma fonticola.

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

Environmental signatures and effects of an oil and gas wastewater spill in the Williston Basin, North Dakota.

Wastewaters from oil and gas development pose largely unknown risks to environmental resources. In January 2015, 11.4ML (million liters) of wastewater (300g/L TDS) from oil production in the Williston Basin was reported to have leaked from a pipeline, spilling into Blacktail Creek, North Dakota. Geochemical and biological samples were collected in February and June 2015 to identify geochemical signatures of spilled wastewaters as well as biological responses along a 44-km river reach. February water samples had elevated chloride (1030mg/L) and bromide (7.8mg/L) downstream from the spill, compared to upstream levels (11mg/L and <0.4mg/L, respectively). Lithium (0.25mg/L), boron (1.75mg/L) and strontium (7.1mg/L) were present downstream at 5-10 times upstream concentrations. Light hydrocarbon measurements indicated a persistent thermogenic source of methane in the stream. Semi-volatile hydrocarbons indicative of oil were not detected in filtered samples but low levels, including tetramethylbenzenes and di-methylnaphthalenes, were detected in unfiltered water samples downstream from the spill. Labile sediment-bound barium and strontium concentrations (June 2015) were higher downstream from the Spill Site. Radium activities in sediment downstream from the Spill Site were up to 15 times the upstream activities and, combined with Sr isotope ratios, suggest contributions from the pipeline fluid and support the conclusion that elevated concentrations in Blacktail Creek water are from the leaking pipeline. Results from June 2015 demonstrate the persistence of wastewater effects in Blacktail Creek several months after remediation efforts started. Aquatic health effects were observed in June 2015; fish bioassays showed only 2.5% survival at 7.1km downstream from the spill compared to 89% at the upstream reference site. Additional potential biological impacts were indicated by estrogenic inhibition in downstream waters. Our findings demonstrate that environmental signatures from wastewater spills are persistent and create the potential for long-term environmental health effects.

Molecular Detection of Avian Influenza Virus from Sediment Samples in Waterfowl Habitats on the Delmarva Peninsula, United States.

Avian influenza viruses (AIV) affect many species of birds including waterfowl and may persist in sediment in aquatic habitats. Sediment samples were collected from two areas representative of prime migration and overwintering waterfowl habitat in Dorchester County, Maryland in the fall and winter of 2013-2014. Samples were screened for the presence of AIV via reverse transcriptase-quantitative PCR targeting the matrix gene. Although 13.6% of sediment samples were positive for the AIV matrix gene across all collection dates and locations, differences in detection were noted with location and collection season. Percentage of AIV-positive sediment samples recovered corresponded to trends in waterfowl abundance at collection sites both temporally and spatially. These findings provide further support for the assertion that the presence of AIV in the aquatic environment is likely affected by the total number, site-specific density, and array of waterfowl species.

Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. national wildlife refuge waters: A reconnaissance study.

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

Reproductive health indicators of fishes from Pennsylvania watersheds: association with chemicals of emerging concern.

Fishes were collected at 16 sites within the three major river drainages (Delaware, Susquehanna, and Ohio) of Pennsylvania. Three species were evaluated for biomarkers of estrogenic/antiandrogenic exposure, including plasma vitellogenin and testicular oocytes in male fishes. Smallmouth bass Micropterus dolomieu, white sucker Catostomus commersonii, and redhorse sucker Moxostoma species were collected in the summer, a period of low flow and low reproductive activity. Smallmouth bass were the only species in which testicular oocytes were observed; however, measurable concentrations of plasma vitellogenin were found in male bass and white sucker. The percentage of male bass with testicular oocytes ranged from 10 to 100%, with the highest prevalence and severity in bass collected in the Susquehanna drainage. The percentage of males with plasma vitellogenin ranged from 0 to 100% in both bass and sucker. Biological findings were compared with chemical analyses of discrete water samples collected at the time of fish collections. Estrone concentrations correlated with testicular oocytes prevalence and severity and with the percentage of male bass with vitellogenin. No correlations were noted with the percentage of male sucker with vitellogenin and water chemical concentrations. The prevalence and severity of testicular oocytes in bass also correlated with the percent of agricultural land use in the watershed above a site. Two sites within the Susquehanna drainage and one in the Delaware were immediately downstream of wastewater treatment plants to compare results with upstream fish. The percentage of male bass with testicular oocytes was not consistently higher downstream; however, severity did tend to increase downstream.

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar.

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (4 day) exposures using three doses each of 17 α-ethinylestradiol (EE2), 17 ß-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and 1 year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embryos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting plasma Vtg is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2, and plasma T3 was decreased at the highest dose of EE2. Our results indicate that all life stages are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild.

Characterization of a new myxozoan species (Myxozoa: Myxobolidae: Myxosporea) in largescale stonerollers (Campostoma oligolepis) from the Mobile River Basin (Alabama).

Myxobolus stanlii sp. n. was described from largescale stonerollers ( Campostoma oligolepis ) from the Mobile River Basin in Alabama. The parasite was described using critical identifying morphological features, and the 18S small subunit ribosomal RNA (SSU rRNA) gene sequence. The spore body was ovoid, 10.03 ± 0.7 (7.5-11.0) µm long and 8.8 ± 1.5 (6.3-11.3) µm wide in frontal view. Spore thickness was 6.3 ± 2.7 (6.2-8.6) µm in sutural view. Polar capsules were pyriform, of equal size, and oriented in plane with the sutural ridge. Polar capsules were 2.45 ± 1.5 (range 2.1-4.3) µm in width and 4.6 ± 2.7 (range 4.5-6.9) µm in length. Based on the SSU rRNA gene sequence of Myxobolus stanlii sp. n. is most closely related to M. pseudodispar.

A redescription of Myxobolus inornatus from young-of-the-year smallmouth bass (Micropterus dolomieu).

During investigations of young-of-the year smallmouth bass ( Micropterus dolomieu ) mortalities in the Susquehanna River, Pennsylvania, U.S.A. and affected tributaries, raised areas were noted in the muscle in the vicinity of the caudal peduncle. The raised areas were caused by plasmodia of a myxozoan parasite. Spores found within plasmodia were similar to those of Myxobolus inornatus previously described from the caudal peduncle of fingerling largemouth bass ( Micropterus salmoides ) in Montana. Here, M. inornatus is redescribed based on histologic comparisons and spore measurements. The addition of spore photographs, line drawings, a voucher specimen, and partial small-subunit ribosomal (rSSU) DNA gene sequence are new in this study. This is also the first description of M. inornatus from smallmouth bass. The plasmodia of M. inornatus were grossly observed at the base of the caudal and dorsal fins and were 280.3 ± 33.5 (range 77.1-920.3) µm long and 320.6 ± 41.0 (range 74.85-898.4) µm wide. In some instances, plasmodia of M. inornatus were large enough to rupture the epidermis or were associated with misaligned vertebrae. The slightly pyriform spores were 11.3 ± 0.2 (range 8.6-17.4) µm in length and 8.6 ± 0.2 (range 7.1-13.7) µm wide with an iodinophilous vacuole and a sutural ridge with 8 to 10 sutural folds. The SSU rDNA gene sequence places M. inornatus in a sister group with Myxobolus osburni .

Mortality of centrarchid fishes in the Potomac drainage: survey results and overview of potential contributing factors.

Skin lesions and spring mortality events of smallmouth bass Micropterus dolomieu and selected other species were first noted in the South Branch of the Potomac River in 2002. Since that year morbidity and mortality have also been observed in the Shenandoah and Monocacy rivers. Despite much research, no single pathogen, parasite, or chemical cause for the lesions and mortality has been identified. Numerous parasites, most commonly trematode metacercariae and myxozoans; the bacterial pathogens Aeromonas hydrophila, Aeromonas salmonicida, and Flavobacterium columnare; and largemouth bass virus have all been observed. None have been consistently isolated or observed at all sites, however, nor has any consistent microscopic pathology of the lesions been observed. A variety of histological changes associated with exposure to environmental contaminants or stressors, including intersex (testicular oocytes), high numbers of macrophage aggregates, oxidative damage, gill lesions, and epidermal papillomas, were observed. The findings indicate that selected sensitive species may be stressed by multiple factors and constantly close to the threshold between a sustainable (healthy) and nonsustainable (unhealthy) condition. Fish health is often used as an indicator of aquatic ecosystem health, and these findings raise concerns about environmental degradation within the Potomac River drainage. Unfortunately, while much information has been gained from the studies conducted to date, due to the multiple state jurisdictions involved, competing interests, and other issues, there has been no coordinated approach to identifying and mitigating the stressors. This synthesis emphasizes the need for multiyear, interdisciplinary, integrative research to identify the underlying stressors and possible management actions to enhance ecosystem health.

Intersex (testicular oocytes) in smallmouth bass from the Potomac River and selected nearby drainages.

Intersex, or the presence of characteristics of both sexes, in fishes that are normally gonochoristic has been used as an indicator of exposure to estrogenic compounds. In 2003, during health assessments conducted in response to kills and a high prevalence of skin lesions observed in smallmouth bass Micropterus dolomieu in the South Branch of the Potomac River, the presence of immature oocytes within testes was noted. To evaluate this condition, a severity index (0-4) was developed based on the distribution of oocytes within the testes. Using gonad samples collected from 2003 to 2005, the number of histologic sections needed to accurately detect the condition in mature smallmouth bass was statistically evaluated. The reliability of detection depended on the severity index and the number of sections examined. Examining five transverse sections taken along the length of the gonad resulted in a greater than 90% probability of detecting testicular oocytes when the severity index exceeded 0.5. Using the severity index we compared smallmouth bass collected at selected sites within the South Branch during three seasons in 2004. Seasonal differences in severity and prevalence were observed. The highest prevalence and severity were consistently noted during the prespawn-spawning season, when compared with the postspawn season. In 2005, smallmouth bass were collected at selected out-of-basin sites in West Virginia where fish kills and external skin lesions have not been reported, as well as at sites in the Shenandoah River, Virginia (part of the Potomac drainage), where kills and lesions occurred in 2004-2005. The prevalence of testicular oocytes is discussed in terms of human population and agricultural intensity.

A new bacilliform fathead minnow rhabdovirus that produces syncytia in tissue culture.

A pathogenic bacilliform virus 130-180 nm in length and 31-47 nm in diameter was isolated from moribund fathead minnows (Pimephales promelas) exhibiting hemorrhages in their eyes and skin. A cytopathic effect of multifocal syncytia was observed in the epithelioma papulosum cyprini cell line after a 48 h incubation at 20 degrees C. A similar cytopathic effect was also observed in other cell lines tested, but not in bluegill fry, koi fin, or Chinook salmon embryo cells. The filterable agent was inactivated by exposure to 50 degrees C for 10 min, 20% ether, 2 and 50% chloroform, pH 3, and pH 10, was unaffected by 5'-iodo-2 deoxyuridine, and appeared bacilliform and occasionally bullet-shaped by electron microscopy. These results are consistent with those of rhabdoviruses. Immunodot blots performed with antisera against selected fish rhabdoviruses, an aquareovirus, and a birnavirus were all negative. River's postulates were fulfilled in fathead minnows, but the agent did not replicate or cause disease in other cyprinids or salmonids during challenge experiments. Hepatic, splenic, and renal lesions were observed during histological analysis of diseased fish from viral challenges and from the original case. Structural proteins resolved via SDS-PAGE had molecular weights similar to those reported in lyssaviruses of the family Rhabdoviridae; however, syncytia formation is not a typical cytopathic effect of rhabdoviruses. This virus, has tentatively been named the fathead minnow rhabdovirus (FHMRV) and is most similar to the members of the family Rhabdoviridae, but atypical properties like syncytia formation may justify the assignment to a novel taxon.