Ecotoxicoparasitology of mercury and trace elements in semi-aquatic mammals and their endoparasite communities.

Many contaminants persist in the environment for decades or more, influencing ecosystem health. Environmental contamination with mercury (Hg) is a particular concern due to its ability to biomagnify in food webs and its lethal and sub-lethal effects in exposed organisms. Despite the known impacts of anthropogenic contamination, there remains a need for data on wildlife exposure to Hg and other contaminants, and the effects of exposure on wildlife health. The objectives of this study were to: 1) quantify differences in concentrations of mercury and other trace elements among three sympatric semiaquatic mammals of different assumed trophic position: North American river otter (Lontra canadensis), raccoon (Procyon lotor), and North American beaver (Castor canadensis), 2) compare trace element concentrations between animals captured on the Savannah River Site (SRS) in South Carolina, USA, where known inputs of Hg and other trace elements have occurred, and reference sites in South Carolina (SC) and Georgia (GA), USA, and 3) investigate the relationship between host trace element concentrations and endoparasite communities. River otters, beavers, and raccoons were sampled from the SRS, SC, and GA to quantify trace element concentrations in liver tissue and quantify endoparasite communities. Both species and sampling location were important factors determining hepatic trace element concentration, however, there was no consistent trend of elevated trace element concentrations among animals sampled on the SRS. Only Hg demonstrated biomagnification based on assumed trophic position, with river otters having the highest Hg concentrations among the sampled species. Additionally, the results suggest a possible relationship between host hepatic mercury concentration and endoparasite abundance, while hepatic selenium concentration may be related to endoparasite diversity. These findings further demonstrate how wildlife can accumulate anthropogenic contamination, although future research is needed to determine the mechanisms contributing to patterns observed between endoparasite communities and the contaminant concentrations of their mammalian hosts.

PBDEs and Dechloranes in Raccoons in the Midwestern United States.

Raccoons (Procyon lotor) are one of the most widespread and abundant mammals in North America. To evaluate the suitability of using raccoons as bioindicator species, we analyzed liver tissues (n = 32) collected from 2013 to 2015 for the presence of polybrominated diphenyl ethers (PBDEs) and Dechloranes. ∑PBDE concentrations ranged from 19.1 to 2125 ng/g lw (median = 98.0 ng/g lw) and did not differ between gender or age of raccoon. Dechloranes were detected in 38% of raccoons and ranged from 0.15 to 50.4 ng/g lw (median = 2.32 ng/g lw). The comparatively high PBDE concentrations, and presence of Dechloranes in the raccoons in our study suggest that biota in terrestrial habitats are still widely exposed to and susceptible to the bioaccumulation of current and emerging flame retardants.

Raccoons (Procyon lotor) as Sentinels of Trace Element Contamination and Physiological Effects of Exposure to Coal Fly Ash.

Anthropogenic pollutants disrupt global biodiversity, and terrestrial sentinels of pollution can provide a warning system for ecosystem-wide contamination. This study sought to assess whether raccoons (Procyon lotor) are sentinels of local exposure to trace element contaminants at a coal fly ash site and whether exposure resulted in health impairment or changes in the intestinal helminth communities. We compared trace element accumulation and the impact on health responses and intestinal helminth communities of raccoons inhabiting contaminated and reference sites of the U.S. Department of Energy's Savannah River Site (South Carolina, USA). Data on morphometry, hematology, histopathology, helminth community and abundance, and liver trace element burdens were collected from 15 raccoons captured adjacent to a coal fly ash basin and 11 raccoons from a comparable uncontaminated site nearby. Of eight trace elements analyzed, Cu, As, Se, and Pb were elevated in raccoons from the contaminated site. Raccoons from the contaminated site harbored higher helminth abundance than animals from the reference site and that abundance was positively associated with increased Cu concentrations. While we found changes in hematology associated with increased Se exposure, we did not find physiological or histological changes associated with higher levels of contaminants. Our results suggest that raccoons and their intestinal helminths act as sentinels of trace elements in the environment associated with coal fly ash contamination.

Mercury levels in raccoons (Procyon lotor) from the Warta Mouth National Park, northwestern Poland.

This is the first report on mercury (Hg) levels in the liver, kidney, skeletal muscle, and brain of raccoon in Europe. It studied Hg concentration in 24 raccoons from the Warta Mouth National Park, northwestern Poland by atomic absorption spectroscopy (AAS). The highest total Hg concentrations in the raccoon were found in the liver (maximum, 18.45 mg/kg dry weight), while the lowest in the brain (maximum, 0.49 mg/kg dw). In adult raccoons, Hg concentrations in the liver, kidney, and brain were higher than in immature individuals (p<0.001), while similar in skeletal muscle in both age groups. Our results are consistent with studies by other authors conducted in North America in areas with similar environmental conditions.

Changes in the immunolocalization of steroidogenic enzymes and the androgen receptor in raccoon (Procyon lotor) testes in association with the seasons and spermatogenesis.

The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3ßHSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3ßHSD expression, suggesting that 3ßHSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.

Bromethalin poisoning in a raccoon (Procyon lotor): diagnostic considerations and relevance to nontarget wildlife.

Submission of a raccoon (Procyon lotor) for necropsy following exhaustion at a California wildlife care center revealed minimal gross pathologic changes and only mild vacuolar changes in the white matter of the brain. Turquoise granular material was noted in the gastrointestinal tract and was submitted for toxicological testing along with portions of the brain, liver, kidney, and mesenteric and perirenal adipose tissues. Testing of the turquoise material for 7 anticoagulant rodenticides, strychnine, 4-aminopyridine, starlicide, and salts revealed none of these compounds; however, desmethylbromethalin was detected by high-performance liquid chromatography-tandem mass spectrometry. Other tissues were subsequently analyzed; the mesenteric and perirenal adipose tissues contained desmethylbromethalin. Desmethylbromethalin is the active metabolite of bromethalin, uncouples oxidative phosphorylation, and results in cerebral edema. Bromethalin is a rodenticide that is visually indistinguishable from many other rodenticides, making identification of poisonings by appearance alone nearly impossible. Based on the pathological and toxicological findings, a diagnosis of bromethalin toxicosis was established. In cases of wildlife species with unknown deaths or inconsistent clinical signs with normal or minimal histological findings, bromethalin toxicosis should be considered as a differential. Adipose tissue is the tissue of choice and can be easily harvested from a live or deceased animal to help confirm or rule out bromethalin exposure or intoxication.

Oral rabies vaccination variation in tetracycline biomarking among Ohio raccoons.

Oral rabies vaccination (ORV) programs have traditionally relied on tetracycline marking as an index to bait uptake. Whether tetracycline serves well in this capacity depends on its deposition affinity and ability to be detected consistently among tissues selected for analysis from target species. We evaluated samples from 760 hunter-harvested raccoons (Procyon lotor) from areas in Ohio where ORV had been conducted during 1998, 1999, and 2001. Tetracycline marking was evaluated within and among first premolar (PM1), second premolar (PM2), and canine (CN) teeth, and mandibular bone (MB) by side (left versus right); and by tissue type. Tetracycline detection ranged from 6.5% in PM1 in 1998 to 56.3% in right-side MB in 2001. PM1 teeth were less frequently marked (21.7%) than PM2 (27.7%), CN (33.0%), or MB (42.0%). Tetracycline detection was similar in left and right PM1, PM2, and CN teeth, but differed in MB. Tetracycline marking was significantly different among all tissue types.

Monitoring perchlorate exposure and thyroid hormone status among raccoons inhabiting a perchlorate-contaminated site.

Perchlorate is a water soluble anion that is readily accumulated in vegetation. It inhibits uptake of iodide into thyroid gland tissue, thereby reducing production of thyroid hormones. Potential raccoon food items including berries, fish, and vegetation collected at a contaminated site contained quantifiable concentrations of perchlorate as determined by ion chromatography. Therefore, we monitored resident raccoons for exposure to perchlorate by examining plasma perchlorate and thyroid hormone concentrations. Resulting analytical data failed to demonstrate perchlorate exposure among raccoons that likely consumed food items collected along perchlorate-contaminated water bodies. There were no correlations between triiodothyronine or thyroxine and thyroid stimulating hormone concentrations, but triiodothyronine concentrations in raccoon plasma were significantly higher in 2000 than in 2001 (p = 0.0081). These data suggest that natural attenuation and remedial efforts initiated in January of 2001 may have reduced perchlorate exposure among raccoons inhabiting this site from 2000 to 2001. Temporal, spatial, and analytical factors limited our ability to quantify exposure among raccoons, however, our data do not indicate that raccoons currently inhabiting this site are at risk for significant exposure to perchlorate and subsequent effects.

The development of a spatially explicit model to estimate radiocaesium body burdens in raccoons (Procyon lotor) for ecological risk assessment.

A spatially explicit model of raccoon (Procyon lotor) distribution for the U.S. Department of Energy's (DOE) Savannah River Site (SRS) in west-central South Carolina was developed using data from a raccoon radio-telemetry study and visualized within a Geographic Information System (GIS). An inductive approach was employed to develop three sub-models using the ecological requirements of raccoons studied in the following habitats: (1) man-made reservoirs, (2) bottomland hardwood/riverine systems, and (3) isolated wetland systems. Logistic regression was used to derive probabilistic resource selection functions using habitat compositional data and landscape metrics. The final distribution model provides a spatially explicit probability (likelihood of being in an area) surface for male raccoons. The model is a stand-alone tool consisting of algorithms independent of the specific GIS data layers to which they were derived. The model was then used to predict contaminant burdens in raccoons inhabiting a riverine system contaminated with radiocaesium (137Cs). The predicted 137Cs burdens were less than if one would assume homogeneous use of the contaminated areas. This modelling effort provides a template for DOE managed lands and other large government facilities to establish a framework for site-specific ecological assessments that use wildlife species as endpoints.

Localization of epidermal hyaluronan in the foot pads of the North American raccoon (Procyon lotor).

The localization of the epidermal hyaluronan in the digital foot pads of the North American raccoon (Procyon lotor) was examined by light and electron microscopic histochemical methods. The thick epidermis contained five epidermal layers composed of typical keratinocytes. In the stratum basale and stratum spinosum, hyaluronic acid was clearly detectable at cytoplasmic processes, in close vicinity to the surface coat of the plasma membrane, and/or in the intercellular space between the basal and spinous cells. The results suggest that epidermal hyaluronan that fills the intercellular space may particularly control water maintenance in the epidermis of the raccoon digital pads to guarantee the optimal mechanoreceptive function of the very specific and sensitive sensoric subepidermal equipment in this mammalian species.

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads.

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.

Environmental polychlorinated biphenyl exposure and cytochromes P450 in raccoons (Procyon lotor).

An investigation involving raccoons as a sentinel species at the Paducah Gaseous Diffusion Plant (PGDP) and Ballard Wildlife Management Area in western Kentucky (USA) delineated the extent of exposure to polychlorinated biphenyls (PCBs). Three separate measures of hepatic cytochrome P450 (CYP) induction were used to evaluate raccoon physiological responses to PCB exposure. Hepatic CYP induction was estimated via determination of total CYP, dealkylase activities, and immunoreactive proteins. There were no differences in raccoon biomarker responses between study sites. Significant relationships between and among PCB residues and biomarkers indicated that hepatic CYP induction had occurred in response to PCB exposure. Pentoxyresorufin O-deethylase (PROD) activity, CYP1A1, and CYP1A2 were biomarkers most closely associated with PCB exposure. The rank order of responses was CYP1A1 > CYP1A2 > PROD > ethoxyresorufin O-deethylase (EROD) as related to raccoon liver PCB concentrations, whereas the order was CYP1A1 > PROD > EROD > CYP1A2 when regressed with total PCB concentrations in abdominal fat.

Environmental exposure to polychlorinated biphenyls among raccoons (Procyon lotor) at the paducah gaseous diffusion plant, Western Kentucky, USA.

An investigation involving raccoons (Procyon lotor) as a sentinel species at the Paducah Gaseous Diffusion Plant (PGDP) in Western Kentucky (USA) delineated the extent of exposure to polychlorinated biphenyls (PCBs) and PCB spatial distribution. Raccoon exposure to PCBs was demonstrated through analysis of subcutaneous fat, abdominal fat, liver, and brain tissues from raccoons collected at the PGDP but also was clearly evident in raccoons from a reference area situated along the Ohio River (USA). Raccoons with the highest tissue PCB concentrations appeared to be those inhabiting areas nearest the plant itself and most likely those that ventured into the plants interior. Male raccoons at the PGDP had similar concentrations of total PCBs in subcutaneous fat (1.86 +/- 0.64 microg/g) as males from the reference site (1.41 +/- 0.35 microg/g), but females had higher PCB body burdens than those at the reference site (9.90 +/- 6.13 microg/g vs 0.75 +/- 0.40 microg/g). Gross measurements of exposure to radiation-producing materials revealed that counts per minute exceeded background in 61% of PGDP raccoons compared with 27% at the reference site and five raccoons at the PGDP had beta counts that were more than twice the background. Differences among trapping success, growth rates, and serum chemistry parameters were noted but may have been related to habitat and other environmental and population density factors.

Metal levels in raccoon tissues: differences on and off the Department of Energy's Savannah River Site in South Carolina.

Levels of arsenic, cadmium, chromium, copper, lead, manganese, selenium, and strontium88 were examined in heart, kidney, muscle, spleen and liver of raccoons (Procyon lotor) from four areas on the Department of Energy's Savannah River Site (SRS), including near a former reactor cooling reservoir and a coal ash basin, and from public hunting areas within 15 km of the site. Mercury is mentioned briefly because it is discussed more fully in another paper. We test the hypotheses that there are no differences in metal levels between raccoons on SRS and off the SRS (off-site), and among different locations on the SRS. Although raccoons collected off-site had significantly lower levels of mercury and selenium in both the liver and kidney, there were few consistencies otherwise. There were significantly higher levels of cadmium in liver of on-site compared to off-site raccoons, and significantly higher levels of chromium and strontium88 in kidney of on-site compared to off-site raccoons. Copper and manganese were highest in the liver; cadmium, lead, mercury and selenium were highest in the liver and kidney; chromium was highest in the spleen and muscle; arsenic was highest in the heart, and strontium88 was slightly higher in the kidney than other organs. Where there were significant differences on site, chromium, manganese were highest in raccoon tissues from Steel Creek; arsenic, lead and selenium were highest in the Ash Basin; cadmium was highest at Upper Three Runs; and strontium88 was highest at Upper Three Runs and Steel Creek. The patterns were far from consistent.

Effect of dietary glycine and benzoate level on benzoate metabolism in mink (Mustela vision), blue fox (Alopex lagopus), and raccoon dog (Nyctereutes procyonoides).

Three 2 x 4 factorial experiments were carried out from August to September with 30 juvenile male mink, 24 raccoon dogs, and 24 blue foxes to investigate the effect of dietary glycine supply (low or high) on the efficiency of these species to excrete hippuric acid with incremental benzoate intake (0, 1, 2, or 4 mmol/kg BW). For mink, two additional treatments with 1 or 2 mmol/kg BW of ethyl benzoate were included. A basal low-glycine diet was formulated to meet the minimum protein requirements of fur animals (30% of ME). This diet was supplemented with 0 or 3 g/kg of glycine, or with 0, 1.0, 2.07, or 4.15 g/kg of sodium benzoate for mink and blue foxes, and with 0 or 4.5 g/kg of glycine and 0, 1.58, 3.17, or 6.34 g/kg of sodium benzoate for raccoon dogs, respectively. Two additional diets with .76 or 1.53 g/kg of ethyl benzoate were made for mink. Fecal and urinary benzoic and hippuric acid excretion were measured for 3 d. The 24-h recovery of [14C]benzoic acid injected intraperitoneally was measured from urine, the liver, and the kidneys. All animals appeared healthy and no clinical signs of benzoate overdose were observed. Dietary benzoate level did not affect ADFI or ADG in any species. Glycine supplementation lowered ADFI in mink. The majority of ingested benzoates were absorbed from the gut (over 95%), except in blue foxes, which excreted 6 to 15% of ingested benzoates in feces with incremental increases in benzoate intake. Urinary free benzoic acid excretion accounted for 10% of the ingested benzoates in blue foxes but less than 5% in mink and raccoon dogs. When benzoate intake was 1 mmol/kg BW, mink, blue foxes, and raccoon dogs excreted 71, 77, and 34% of ingested benzoates as hippuric acid in urine, respectively. With higher benzoate intakes, urinary hippuric acid excretion decreased quadratically with mink to 20%, and linearly with blue foxes and raccoon dogs to 45 and 16%, respectively. The hippuric acid pathway appears to be the principal route of benzoate elimination in the mink and blue fox, whereas, in the raccoon dog, other pathways appear to be more important. In mink, the elimination of ethyl benzoate did not differ from that of sodium benzoate. Because glycine conjugation is the primary route of benzoate elimination, it is recommended that benzoate content in fur animal feeds should not exceed 1 g/kg feed on an as-fed basis.

Effects of contaminants in dredge material from the lower Savannah River.

Contaminants entering aquatic systems from agricultural, industrial, and municipal activities are generally sequestered in bottom sediments. The environmental significance of contaminants associated with sediments dredged from Savannah Harbor, Georgia, USA, are unknown. To evaluate potential effects of contaminants in river sediments and sediments dredged and stored in upland disposal areas on fish and wildlife species, solid-phase sediment and sediment pore water from Front River, Back River, an unnamed Tidal Creek on Back River, and Middle River of the distributary system of the lower Savannah River were tested for toxicity using the freshwater amphipod Hyalella azteca. In addition, bioaccumulation of metals from sediments collected from two dredge-disposal areas was determined using the freshwater oligochaete Lumbriculus variegatus. Livers from green-winged teals (Anas crecca) and lesser yellowlegs (Tringa flavipes) foraging in the dredge-spoil areas and raccoons (Procyon lotor) from the dredge-disposal/river area and an upland site were collected for metal analyses. Survival of H. azteca was not reduced in solid-phase sediment exposures, but was reduced in pore water from several locations receiving drainage from dredge-disposal areas. Basic water chemistry (ammonia, alkalinity, salinity) was responsible for the reduced survival at several sites, but PAHs, metals, and other unidentified factors were responsible at other sites. Metal residues in sediments from the Tidal Creek and Middle River reflected drainage or seepage from adjacent dredge-disposal areas, which could potentially reduce habitat quality in these areas. Trace metals increased in L. variegatus exposed in the laboratory to dredge-disposal sediments; As, Cu, Hg, Se, and Zn bioaccumulated to concentrations higher than those in the sediments. Certain metals (Cd, Hg, Mo, Se) were higher in livers of birds and raccoons than those in dredge-spoil sediments suggesting bioavailability. Cadmium, Cr, Hg, Pb, and Se in livers from raccoons collected near the river and dredge-disposal areas were significantly higher than those of raccoons from the upland control site. Evidence of bioaccumulation from laboratory and field evaluations and concentrations in sediments from dredge-disposal areas and river channels demonstrated that some metals in the dredge-disposal areas are mobile and biologically available. Drainage from dredge-disposal areas may be impacting habitat quality in the river, and fish and wildlife that feed and nest in the disposal areas on the lower Savannah River may be at risk from metal contamination.

Calcitonin gene-related peptide and neuropeptide Y in the raccoon cuneate nucleus.

Immunocytochemical methods were used to determine the distribution of calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in the middle region of the adult raccoon cuneate nucleus. Extensive CGRP-immunoreactive fibers and darkly stained punctate structures, thought to be terminals, were concentrated in the dorsal cap and basal region of the middle cuneate nucleus. These regions receive input from the claws and the hairy skin of the paw and forearm. The NPY-immunoreactive fibers and terminals were also found within the dorsal cap of the cuneate nucleus, but were less abundant than CGRP. However, most of the NPY-immunostained fibers and terminals were found in the cluster region of the cuneate nucleus, which receives input from glabrous skin. No CGRP- or NPY-immunoreactive cell bodies were found in the raccoon middle cuneate nucleus. This description of the distribution of CGRP and NPY in the normal animal provides a baseline for future investigations into injury-induced neuropeptide plasticity in the raccoon middle cuneate nucleus.

Distribution of heat-stable enterotoxin/guanylin receptors in the intestinal tract of man and other mammals.

The human intestinal tract, as well as that of several eutherian and metatherian mammals, was examined for the distribution of heat-stable enterotoxin (ST)/guanylin receptors. These receptors were confined to the intestinal epithelium lining the lumen and forming the intestinal glands throughout the length of both the small intestine and colon of all species examined. In man and most other mammalian species, there appeared to be a decrease in receptor density distally along the longitudinal axis of the small intestine. ST/guanylin receptors were not observed in other strata forming the gut wall. Along the vertical axis of the human small intestine (villus/crypt unit), as well as that of most other mammals, receptor density was greatest in enterocytes located near the base of villi and in those forming the proximal portion of the intestinal glands. ST/guanylin receptors were for the most part confined to the region of the plasmalemma forming the microvillus border. In the colon of man and the other species examined, receptor density was greatest in enterocytes forming the proximal region of the intestinal glands. Receptors were present in the intestinal epithelium lining the lumen of the colon, but generally were fewer in number. The distribution of cellular cGMP accumulation responses to E. coli ST and guanylin in the opossum (Didelphis virginiana) and raccoon (Procyon lotor) revealed that proximal small intestine had greater magnitudes of cGMP responses than did the distal small intestine. Proximal colon had greater cGMP responses than distal colon, which had no significant cGMP responses to either ST or guanylin.

Background prevalence of tetracycline-like fluorescence in teeth of free ranging red foxes (Vulpes vulpes), striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) in Ontario, Canada.

We collected and examined teeth from 3406 red foxes (Vulpes vulpes) collected in Ontario, Canada, from 1978 to 1986, prior to large scale rabies vaccine baiting. We found tetracycline-like fluorescence in five (0.2%) of the samples. Also, we observed similar fluorescences in five (0.4%) of 1103 striped skunks (Mephitis mephitis) and in six (0.8%) of 744 raccoons (Procyon lotor). The low prevalence of such marks would not appear to invalidate the use of tetracycline as a marking agent in vaccine baiting trials.