Abstract
The Wildlife Conservation Society and the St Catherines Island Foundation have partnered with the Georgia and South Carolina Departments of Natural Resources, the U.S. Fish and Wildlife Service, and other organizations to establish a new conservation health initiative on and around St. Catherines Island, Georgia. This study is a product of these efforts. There are eleven species of seabirds and shorebirds that nest on the beaches of Georgia and South Carolina. All obligate beach-nesting birds are under pressure from human expansion and are a high conservation priority for governmental agencies and nonprofit organizations. The American oystercatcher (AMOY), Haematopus palliatus, is considered to be rare in both states. It is one of the few birds to be specialized feeders on bivalve mollusks living in saltwater.10 Due to these unique dietary preferences, this species has the potential to act as a bioindicator of the marine ecosystem in the southeastern United States. Human development of coastal and inland Georgia as well as South Carolina is increasing at a rapid rate with increased water contamination coming from industry, agriculture, recreation, and nonpoint-source runoff. The purpose of this presentation is to provide some preliminary information on the health assessment of the AMOY.
Over the past year, our research team has caught 31 (18 adult males, seven adult females, two subadult males, two subadult females, one unknown, and one pending results) AMOY for banding and collection of biomaterials with the objective of establishing baseline values in clinical pathology, toxicology, reproductive physiology, microbiology, parasitology, morphometrics, as well as confirmation of sex with DNA technology.
A cannon netting technique to capture the birds has been developed by some of the investigators (TMN, BW, RCG) and has proven to be safe and effective. This method of capture is labor intensive, but has been found to be the most successful technique for this species. This is the first time that this method has been used to capture AMOY in North America. Twenty-six birds were captured on Little St. Simons Island, Georgia, and 5 birds were captured at the Cape Romain Wildlife Refuge in South Carolina.
Once in hand, the birds are manually restrained and 5–6 ml of blood is obtained via the right jugular vein in a sodium heparinized syringe. This volume drawn has not resulted in any obvious negative effects. Blood smears are made immediately and stained with Wrights-Giemsa. A differential white blood cell (WBC) count is performed. Red blood cell and WBC morphology are evaluated. White blood cell counts are performed on a hemocytometer using the eosinophil Unopette method. Packed cell volume is manually read from centrifuged microhematocrit tubes. A plasma biochemical profile is performed and includes uric acid, glucose, blood urea nitrogen, creatinine, triglyceride, cholesterol, amylase, lipase, calcium, phosphorus, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, gamma-glutamyltransferase, creatinine phosphokinase, sodium, potassium, chloride, total protein, albumin, and globulin. This is performed using standard dry-slide determinations with a Johnson & Johnson (Kodak) 700XR analyzer. Bile acid determinations are performed on plasma samples by radioimmunoassay (RIA). Total protein is determined by a handheld refractometer, and protein fractions are analyzed by plasma protein electrophoresis.2 Clinical pathology data obtained on the 31 birds captured have been similar to those reported for other avian species.
Aspergillosis antibody (IgG) and circulating antigen plasma titers are determined by ELISA.6 Current data indicates that 34% of birds sampled have a weak to moderate antibody titer and 38% of the birds are antigen positive, which is indicative of either infection or more likely exposure to the organism. Chlamydia antibody titers are evaluated by an indirect immunofluorescence technique, which quantitates IgG. Positive antibody titers were found in 38% of the birds, indicating infection, previous infection, or exposure to the organism. West Nile virus antibody titers were negative on 15 birds sampled in January 2001. We will continue to perform the above infectious disease serology on birds captured in the future.
One milliliter of whole blood is used for heavy metal analysis, (chromium, mercury, copper, lead, zinc, tin, strontium, and vanadium), and 1 ml of whole blood is used for a pesticide screen (PCBs, DDT and metabolites, toxaphene, chlordane, benzene hexachloride). Feathers are plucked from the same site on the breast of each bird and placed in aluminum foil and in whirlpack plastic bags and stored at –70°C for potential heavy metal analysis if any parameters are elevated in the blood or yolk. Blood samples have been evaluated for heavy metals and pesticides from 25 birds caught on Little St. Simons Island, GA and only five birds from Cape Romain, SC. The heavy metal and pesticide screens are performed at the STL Savannah Toxicology Laboratory in Savannah, GA utilizing standard operating procedures.3 Heavy metal analyses are performed by utilizing a graphite furnace and cold vapor atomic absorption measurements. The organochlorine analytic methods, including preparation, Soxhlet extraction, and lipid removal have been previously described.3 Pesticides in each fraction are quantified with a gas-liquid chromatograph (GLC), equipped with a 63Ni electron capture detector. Residues in 10% of the samples will be confirmed by gas chromatography/mass spectrometry. Preliminary results revealed elevations in blood mercury and toxaphene in some of the birds. More samples are needed to determine the significance of these findings.
American oystercatchers will generally lay one to four eggs per clutch; however, they only rarely raise even one chick successfully (C. George, unpublished data). Predation and inundation are the most common causes of mortality in eggs and chicks, so it is not uncommon for no offspring to be produced. Four eggs were collected during the summer of 2001 to obtain some preliminary contaminant data and to assess whether egg yolk would be a more useful biomaterial than whole blood for obtaining this information. Results are pending. A high percentage of birds caught to date and sampled were most likely winter migrants (based on banded bird returns from the January 2001 capture). Results from evaluating egg yolk for contaminants should be more representative of the local (Georgia and South Carolina) environment. One egg from three or four nests will be collected from six river watersheds in Georgia and an additional three or four eggs will be pulled from nests at the Cape Romain Wildlife Refuge. Eggs will only be taken if there are more than two eggs in the nest, the nest is in an area with high predation, or if the egg is known to be infertile. Egg collection will take place during the estimated peak period of egg laying in late April 2002. Each egg is weighed and measured. Egg shell thickness is measured with a micrometer and pore density is measured with a phase contrast microscope. Yolk is collected from the egg, weighed, and then frozen until analysis. A similar heavy metal and pesticide panel and methodology described for the whole blood will be performed on each egg yolk.
One of the authors (ND) has developed specific monoclonal antibodies that react with vitellogenin from other bird species, including tern, quail, and barn swallow. Initial experiments were performed to find out whether the existing monoclonal antibody would react with AMOY vitellogenin in plasma samples taken on January 2001. There was no detectable vitellogenin. This indicated that no cross-reaction occurred or that vitellogenin was nondetectable at this time of year in AMOY. Egg yolk was collected from an infertile AMOY egg and the main egg yolk protein fraction was purified by centrifugation using standard procedures. The egg yolk protein was separated by SDS-PAGE gel electrophoresis and transferred to a membrane and probed by western blot. The existing monoclonal antibody was found to cross-react with vitellogenin in the egg yolk. The western blot test is not very sensitive, requiring at least 50 µg vitellogenin/ml for a positive reaction. We plan on developing a more sensitive ELISA (detection limit 1 µg/ml) in order to measure circulating levels of plasma vitellogenin. The presence of vitellogenin in male birds would indicate their exposure to estrogen-like contaminants. Vitellogenin expression in males is being recognized as an excellent biomarker of exposure to estrogen mimicking environmental contaminants. Plasma samples collected from birds captured from October through December of 2001are currently being analyzed. We will also use the vitellogenin assay to characterize the normal seasonal reproductive cycle for females. Vitellogenin is a protein that is made in the liver under estrogen control. It is secreted into the blood stream and then is taken up by the developing oocytes. Its main function is to be a nutritional source for the developing embryo. Females will produce vitellogenin in relatively high concentrations as the eggs develop. By mapping the appearance of vitellogenin in the plasma of reproductively active females, we will be able to map their normal cycle. Vitellogenin levels will be compared to plasma steroid hormone levels to help characterize the full cycle. Details of the experimental design being used in this portion of the study can be found in the references provided.4,5,7-9,11
Plasma samples from 25 (14 adult male, two subadult male, seven adult female, and two subadult female) AMOY were analyzed for sex hormones (testosterone and 17β-estradiol) using a radioimmunoassay (RIA) technique. Fifty microliters of plasma were extracted twice with 5 ml of diethyl ether before RIA analysis. Samples were then analyzed in duplicate for both hormones and corrected for extraction efficiencies. Standard curves were buffered with known concentrations of radioinert 17β-estradiol (ICN Biomedicals, Costa Mesa, CA) or testosterone (Sigma Chemical, St. Louis, MO) (1, 5, 10, 25, 50, 100, 250, 500, and 1,000 pg per ml). The minimum concentration detectable for both hormones is approximately 7 pg/ml. Cross-reactivities of 17β-estradiol and testosterone antiserum (produced and characterized by T. S. Gross, University of Florida) with other steroids (e.g., estrone, estriol, 17α-estradiol, α-dihydrotestosterone, and androstenedione) are expected to be less than 10%. The mean and standard deviation for hormones measured thus far are males: mean 17β-estradiol 213.88 pg/ml ±66.42, testosterone 110.56 pg/ml ±70.32; females: 17β-estradiol 200.89 pg/ml ±76.82, testosterone 190.56 pg/ml ±97.50. These data were obtained during the nonbreeding season and are highly variable and represent background hormonal levels. Seasonal trends still need to be established for this information to be useful in monitoring reproductive success in AMOY populations.
Fecal samples are collected opportunistically and placed in polyvinyl alcohol and 5% formalin to evaluate the incidence of protozoan and nematode parasites. To date, group fecal samples have been collected due to the small volume of feces available. An Eimeria sp. of coccidia and Acanthocephalan sp. have been identified. Feces are also placed in enrichment media and submitted within 24 hours for enteric bacterial pathogen culture. No enteric bacterial pathogens have been cultured to date. Cotton-tip swabs are placed into the cloaca and subsequently placed in viral transport media and then cultured for avian influenza virus in a collaborative study with the Southeastern Wildlife Disease Cooperative (SCWDS).12 All samples have been negative. Feather lice have been documented to occur on a high percentage of the birds captured. The lice load is estimated (none, mild, moderate, severe) and recorded. Lice samples have been submitted to a parasitologist for identification.
A complete physical examination, body weight, bill length, tarsus length, and wing chord measurements are obtained from each bird. Adults and juveniles are identified by bill and ocular color. Digital images are taken of the entire bird, the head and eyes, and the back of each bird. Gender is determined by DNA sexing technology and compared to body measurements. These birds appear to be sexually monomorphic based on data analyzed to date, which conflicts with the existing literature.10 Methods for determining sex currently used by field biologists may not be 100% accurate. More data is needed to confirm this; thus, morphometric data and DNA sexing will be performed on every bird captured. A ventral pupillary defect in the eye is apparent in some birds. The presence or absence and the severity of the defect is noted in each bird. Feather quality and molting condition is noted.
Future objectives for continuation of this study will be to expand the existing data and add new objectives in nutrition and toxicology. They will include: 1) Evaluate seasonal and age-related changes of baseline health, contaminant, and reproductive parameters. To date, we have caught most birds during late fall and winter. Most birds sampled have been adults, and to a lesser extent, subadults. No juvenile birds have been sampled, although safe capture techniques for 30-day-old chicks were developed by some of the authors (BW and CG). 2) Establish normal nutritional parameters for the various age classes. 3) Perform nutritional and toxicologic analysis on the three most commonly consumed prey items by the oystercatcher found at different locations along the Georgia and South Carolina coasts. 4) Perform toxicologic analysis on American oystercatcher eggs collected at several different drainage sites along both coasts. 5) Continue to investigate sexual dimorphism in this species. 6) Land use practices and industrial development within the watersheds of South Carolina and Georgia vary considerably; thus, a comparison of the data will be made between the two sites.
Acknowledgments
This work is being supported by a grant from the Disney Conservation Fund. Funding has also been provided by the U.S. Fish and Wildlife Service, the Wildlife Conservation Society’s Field Veterinary Program and St. Catherines Island Wildlife Survival Center, and the St. Catherines Island Foundation.
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