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The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a diverse range of chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Although no endogenous physiological ligand for the AhR has yet been described, numerous studies support the existence of such a ligand(s). Here we have examined the ability of prostaglandins and related chemicals to activate the AhR signaling system. Using two AhR-based bioassay systems we report that relatively high concentrations of several prostaglandins (namely, PGB3, PGD3, PGF3α, PGG2, PGH1, and PGH2) can not only stimulate AhR transformation and DNA binding in vitro, but also induce AhR-dependent reporter gene expression in mouse hepatoma cells in culture. PGG2 also induced AhR-dependent reporter gene expression to a level three-to four fold greater than that observed with a maximal inducing dose of TCDD. Sucrose gradient ligand binding analysis revealed that PGG2 could competitively displace [3H]TCDD from the AhR. Overall, our results demonstrate that selected prostaglandins are weak agonists for the AhR and they represent a structurally distinct and novel class of activator of the AhR signal transduction pathway. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:187–196, 2001

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.

Free-ranging marine birds are severely impacted by petroleum released into the environment. Although petroleum can affect many organ systems, oxidative damage to red blood cells (RBC) and development of Heinz body anaemia is the only known mechanism of RBC damage in oil-exposed marine birds. Rhinoceros auklets (Cerrorhinca monocerata) were orally exposed to 0, 2.5 or 10 ml of Prudhoe Bay crude oil/kg body weight for five consecutive days by gavage tube. No statistically significant differences between treatment groups were evident for the following blood parameters: packed cell volume (PCV); haemoglobin concentration (Hb); mean corpuscular haemoglobin concentration (MCHC); reticulocyte percentage: fibrinogen concentration; white blood cell count (WBC); and cell counts of heterophils, lymphocytes, monocytes, eosinophils and basophils. After petroleum exposure, blood samples from auklets did not have any evidence of haemolysis, oxidative RBC damage or Heinz body formation as determined by new methylene blue staining of blood smears. Anaemia developed in both oil-exposed and unexposed auklets within 3 weeks of being captured and placed in captivity. Anaemia persisted throughout the duration of the study (77 days). Statistically significant differences over time were identified for PCV, Hb concentration, MCHC, reticulocyte percentage, fibrinogen concentration and lymphocyte, eosinophil and basophil numbers in auklets, without regard to petroleum exposure status. A mild transient regenerative response was noted after gavaging birds with either petroleum or sterile saline, however, the PCV never returned to levels measured immediately after capture. It is likely that anaemia associated with petroleum exposure in seabirds is multifactorial and may be associated with the sedentary nature of captivity, a variety of captivity associated stressors and possibly, the age of affected birds. Furthermore, neither inflammatory nor acute phase responses were consistent indicators of petroleum ingestion.

We assessed temporal and spatial patterns of chronic oiling of seabirds in California during 2005–2010, using data on: (1) live oiled birds reported to the Oiled Wildlife Care Network (OWCN) from throughout the state, and (2) dead oiled birds found during systematic monthly beached-bird surveys in central California. A mean of 245 (±141 SD) live miscellaneous oiled birds (not associated with known oil spills) were reported to the OWCN per year, and 0.1 oiled dead birds km−1 per month were found on beach surveys in central California. Chemical fingerprinting of oiled feathers from a subset of these birds (n = 101) indicated that 89% of samples tested were likely from natural petroleum seeps off southern and central California. There was a pronounced peak during late winter in the number of oiled birds reported in southern California, which we theorize may be related to large storm waves disturbing underwater seeps.

Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting a range of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1--3)-beta-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commercially available BG assay (Fungitell, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, and aspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds with confirmed aspergillosis had the highest mean BG concentrations (M = 3098.7 pg/dl, SD = 5022.6, n = 22) followed by companion avian species and raptors with confirmed aspergillosis (M = 1033.8 pg/dl, SD = 1531.6, n = 19) and experimentally infected Japanese quail (Coturnix japonica; M = 1066.5 pg/dl, SD = 1348.2, n = 17). Variation in severity of disease, differences among species of birds with and without disease, and also different levels in environmental exposure likely contribute to the differences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0 and 92.7%, respectively, with an overall optimized avian cut-offvalue ofor = 461 pg/dl for positive disease. Our findings suggest that, although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctive diagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor of infection.

Blood and cloacal swabs were collected from 100 (66 female, 34 male) wild Tundra Swans (Cygnus columbianus) molting in northwestern Alaska, USA, 25–28 July 2008, to establish hematologic and serum chemistry reference values and to isolate enteric Salmonella spp. and Escherichia coli O157:H7. Plasma biochemistry and hematology values did not vary significantly by sex or age. Tundra swans had high levels of creatine kinase, lactate dehydrogenase, amylase, and alkaline phosphatase compared with some other avian species (values were up to 7 times greater), possibly indicating capture myopathy. However, concentrations were much lower (up to 8 times lower) than in other waterfowl exposed to similar or more intensive capture methods. White blood cell count and hematocrit values were similar to other waterfowl species, and enteric Salmonella spp. and E. coli O157:H7 were not present among birds sampled. Our data provide the first biochemical, hematologic, and bacteriologic reference values for wild Tundra Swans.

We reviewed medical records from select wildlife rehabilitation facilities in California to determine the prevalence of injury in California Brown Pelicans (Pelecanus occidentalis), gulls (Larus spp.), and pinniped species (Zalophus californianus, Mirounga angustirostris, and Phoca vitulina) due to fishing gear entanglement and ingestion from 2001 to 2006. Of 9,668 Brown Pelican, gull, and pinniped cases described during the 6-yr study period (2001-06), 1,090 (11.3%) were fishing gear-related. Pelican injuries caused by fishing gear were most common in the Monterey Bay region, where 59.6% of the pelicans rescued in this area and admitted to a rehabilitation center were injured by fishing gear over the 6-yr period. The highest prevalence of fishing gear-related injury in gulls was documented in the Los Angeles/Orange County region (16.1%), whereas the highest prevalences in pinnipeds were seen in the San Diego region (3.7%). Despite these higher prevalences of gull and pinniped fishing gear-related injuries in these specific regions, there was no statistical significance in these trends. Juvenile gulls and pinnipeds were more commonly injured by fishing gear than adults (gulls: P = 0.03, odds ratio = 1.29; pinnipeds: P = 0.01, odds ratio = 2.07). Male pinnipeds were twice as likely to be injured by fishing gear as females (P0.01, odds ratio = 2.19). The proportion of fishing gear-related injury cases that were successfully rehabilitated and released (percentage of cases successfully rehabilitated to the point of release out of the total number of fishing gear-related injury cases) was high in all three species groups (pelicans: 63%; gulls: 54%; pinnipeds: 70%). Fishing gear-related injuries in Brown Pelicans and gulls were highest in the fall, but there was only a significant difference between seasons for fishing gear-related injuries in pelicans. Fishing gear-related injuries in pinnipeds most commonly occurred in summer; however, a statistical difference was not detected between seasons for pinnipeds. Derelict fishing gear-lost, abandoned or discarded sport and commercial line, nets, traps, etc.-in the marine environment is a significant cause of injury in California coastal marine wildlife. We evaluated data for stranded animals only; our results may underestimate the true number of coastal marine animals injured by lost or discarded fishing gear in California.

Marine mammals can be infected with zoonotic pathogens and show clinical signs of disease, or be asymptomatic carriers of such disease agents. While isolated cases of human disease from contact with marine mammals have been reported, no evaluation of the risks associated with marine mammal work has been attempted. Therefore, we designed a survey to estimate the risk of work-related injuries and illnesses in marine mammal workers and volunteers. The 17-question survey asked respondents to describe their contact with marine mammals, injuries sustained, and/or illnesses acquired during their period of marine mammal exposure. Most respondents, 88% (423/483), were researchers and rehabilitators. Of all respondents, 50% (243/483) reported suffering an injury caused by a marine mammal, and 23% (110/483) reported having a skin rash or reaction. Marine mammal work-related illnesses commonly reported included: 'seal finger' (Mycoplasma spp. or Erysipelothrix rhusiopathiae), conjunctivitis, viral dermatitis, bacterial dermatitis, and non-specific contact dermatitis. Although specific diagnoses could not be confirmed by a physician through this study, severe illnesses were reported and included tuberculosis, leptospirosis, brucellosis, and serious sequelae to seal finger. Risk factors associated with increased odds of injury and illness included prolonged and frequent exposure to marine mammals; direct contact with live marine mammals; and contact with tissue, blood, and excretions. Diagnosis of zoonotic disease was often aided by veterinarians; therefore, workers at risk should be encouraged to consult with a marine mammal veterinarian as well as a physician, especially if obtaining a definitive diagnosis for an illness becomes problematic.

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P)or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/Por = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.

To reduce rehabilitation time and improve survival, a practical, objective test is needed to determine if marine birds are contaminated with oil before they enter captivity. The RaPID Assay ® and EnviroGard TM immunoassays for detection of polycyclic aromatic hydrocarbons (PAHs) in soil were evaluated for their ability to detect petroleum on feather samples from 30 intermediate fuel oil contaminated and 30 uncontaminated common murres ( Uria aalge ). Sensitivity, specificity, positive and negative predictive value, precision, and time required to run each assay were determined. The RaPID Assay ® was 96.7% sensitive and specific, while the EnviroGard TM assay was 93.3% sensitive, and 90.0% specific. Sensitivity decreased at higher dilutions for both assays. Intra-replicate variation was less than 20%. Our evaluation showed that these immunoassays are rapid and cost-effective methods for detecting oil-contamination on the plumage of seabirds, with the EnviroGard TM assay being more practical in most oil spill response situations due to ease of use and rapidity of results.

Objective—To validate a luciferase bioassay, which is based on a recombinant mouse hepatoma cell line, for the detection of exposure to petroleum in mustelid species. Animals—122 American mink (Mustela vison) and 15 sea otters (Enhydra lutris). Procedures—Mink were exposed to Bunker C fuel oil or Alaska North Slope crude oil externally as a single exposure or internally via low dose concentrations in their ration for 6 months. Serum samples were analyzed for cytochrome P450 1A1 induction by quantification of luciferase activity in the bioassay. Mink liver specimens were also evaluated for cytochrome P450 1A1 induction by quantification of ethoxyresorufin-o-deethylase activity. Serum collected from exposed and unexposed sea otters was also analyzed using the luciferase bioassay. Results—Serum samples from mink externally exposed to petroleum had significantly increased luciferase activities at 1 week after exposure. Serum samples taken at later time points or from mink exposed to either product in the ration did not cause significant luciferase induction. Samples from otters exposed to petroleum had significantly higher luciferase induction as compared with samples from otters not exposed to petroleum at 2 and 8 years after the spill. Cytochrome P450 1A1 activity in liver specimens collected from mink that were internally exposed through diet was significantly increased at the conclusion of our study. Conclusion and Clinical Relevance—The luciferase bioassay is a sensitive and specific method for determining recent exposure to petroleum in mink. The lack of luciferase activity in serum samples collected from mink greater than 1 week after experimental exposure was likely attributable to lower overall petroleum exposure in our trial, compared with natural exposures. (Am J Vet Res 2002;63:963–968)

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range,0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Induction of CYP1A1 gene expression is one such response that is known to be regulated by the AhR complex. It was recently reported (Ledirac et al., Toxicol. Appl. Pharmacol. 144, 177-182, 1997) that, although carbaryl, a carbamate insecticide, can induce AhR-dependent expression of CYP1A1, it was not an AhR ligand. Since this apparent ligand-independent activation of the AhR is difficult to reconcile given what is known about the mechanism of AhR action, we have examined the ability of carbaryl to stimulate the AhR signaling pathway. Not only was dioxin responsive element-driven luciferase gene expression induced by carbaryl in stably transfected mouse, rat, guinea pig, and human cells, gel retardation analysis revealed that carbaryl stimulated AhR transformation and DNA binding in vitro and in cells in culture. Dose-response experiments revealed that carbaryl was 300,000-fold less potent that the prototypical inducer, TCDD, in both inducing luciferase gene expression and stimulating AhR transformation and DNA binding in vitro, suggesting that carbaryl itself was the inducing agent. The identification of carbaryl as an AhR ligand was demonstrated by its ability to competitively inhibit [3H]-TCDD to the guinea pig hepatic cytosolic AhR. Our results confirm that carbaryl is both a weak AhR ligand and inducer of AhR-dependent gene expression and argue against its proposed ligand-independent mechanism of AhR activation.

Western blots and a kinetic enzyme-linked immunosorbent assay (ELISA) were used to characterize and quantify the prevalence of antibodies to Psoroptes sp. mites in elk (Cervus elaphus) from nine herds in North America. Sera from infested (n = 18) and non-infested (n = 22) elk were used to optimize test methodology and to define cut-off values for negative, suspect, and positive samples. Among 357 samples, 35 (9.8%) of the animals were seropositive, 259 (73%) were negative, and 63 (18%) were suspect. Six of nine herds (67%) contained positive animals and two additional herds (22%) had suspect animals. Sex was not associated with prevalence of antibodies, but adults greater than 2 yr old were approximately five times more likely (95% confidence interval = 2.6-15.4) to be seropositive than calves. Based on these results, we propose that exposure to Psoroptes sp. mites may be widespread in free-ranging elk of North America.

The use of chemical dispersants has often been discussed for larger spills due to the perceived advantage to the environment. Evaluation and development of dispersant use plans were undert...

Following the spill of bunker oil from the M/V Cosco Busan into San Francisco Bay, California on 7 November 2007, we evaluated the foraging characteristics, survival and movements of surf scoters (Melanitta perspicillata) that had been rehabilitated following formal rehabilitation protocols through the use of intracoelomic implants of radio transmitters and aerial/land based tracking. We compared these behavioral attributes of oiled surf scoters to surf scoters that were not oiled, but instead captured and released or put through the same rehabilitation procedure (thus separating the rehabilitation/captivity effects from the oiling). Initial behavioral observations have shown that mean home range size was greatest for unrehabilitated birds, and oiled birds were observed closer to shore than either the un-oiled rehabilitated or control group. Survival was also seen to differ among treatment groups and over encounter occasions, with cumulative winter survival estimates being lowest for oiled birds ...