Final Report: Endocrine Disruptors and Host Resistance in Lake Apopka AlligatorsEPA Grant Number: R826127
Title: Endocrine Disruptors and Host Resistance in Lake Apopka Alligators
Investigators: Schoeb, Trenton R. , Brown, Mary B. , Gross, Timothy S. , Klein, Paul A.
Institution: University of Florida , United States Geological Survey [USGS]
EPA Project Officer: Hahn, Intaek
Project Period: January 3, 1998 through January 2, 2001
Project Amount: $598,253
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals
Objective:In the early 1980s, numbers of juvenile alligators in Lake Apopka, FL, declined by 90 percent in association with decreased egg viability, increased neonatal mortality, and organochlorine pesticide contamination. Hypoplastic lymphoid organs and bone marrow and weakened antibody responses in young Lake Apopka alligators were observed, suggesting that weakened host defenses could be a contributing factor. The objective of this project was to study effects on neonatal alligators of endocrine disrupting contaminants (EDCs) on measures of specific and nonspecific immunity as well as lymphoid organ and bone marrow development. The approach is to study alligators hatched from eggs from Lake Apopka, from reference lake eggs treated with endocrine disrupting contaminants found in tissues and eggs of Lake Apopka alligators, and from untreated reference lake eggs (controls) for ability to generate T-dependent humoral immune responses and for hematologic parameters, peripheral blood antibacterial activity, lymphoid organ and bone marrow morphology, and susceptibility to a pathogenic Mycoplasma sp. of alligators. Additional specific aims were developed this year based on the results of the first 2 years of study. These aims were to determine whether embryos naturally exposed to EDCs had increased numbers of bacterial and fungal isolates in yolk samples and to determine the causes of morbidity and mortality in pesticide-exposed perinatal alligators.
Summary/Accomplishments (Outputs/Outcomes):For studies begun in the second year and completed during this reporting period, egg treatments were done by injection. These studies were focused on an environmentally relevant range of doses of DDE, the predominant contaminant found in tissues and eggs of Lake Apopka alligators, and one mixture based on recent data. Treatments were up to 25 ppm p,p'-DDE; a mixture of DDE, chlordane, dieldrin, methoxychlor, and toxaphene; or DMSO vehicle control. Eggs also were collected from Lake Orange (reference site), Lake Griffin, Emeralda Marsh (adjacent to Lake Griffin), and both north and south shores of Lake Apopka, sites having a range of contaminant levels. Analysis of liver samples verified absorption and presence in tissues of DDE, chlordane, dieldrin, and toxaphene.
T-dependent antibody responses were assessed by immunization with keyhole limpet hemocyanin (KLH) esterified with dinitrophenol (DNP) (DNP-KLH) and measurement of DNP hapten-specific responses by enzyme-linked immunosorbent assay (ELISA). In Year 2 experiments, no significant differences were found among pesticide treatment or lake study sites. There were no statistically significant differences among the pesticide treatments or lake groups in white or red blood cell counts, packed cell volumes (hematocrits), plasma protein, or hemoglobin, except a higher mean heterophil count in the Emeralda group and a lower mean heterophil count in the Griffin group, neither of which was considered clinically significant. No statistically significant effects of pesticide treatments or lake groups were found on peripheral blood antibacterial activity against Staphylococcus aureus; histology of the spleen, thymus, or bone marrow; spleen weight/body weight ratios; or in vivo lymphocyte response to phytohemagglutinin.
In Year 2, alligators from control and DDE treatment groups were inoculated intravenously with the alligator pathogen, Mycoplasma alligatoris. Blood, brain, and pericardium were cultured, and joints, brain, and any tissues with gross lesions were prepared for histologic examination. There were no statistically significant differences in quantitative culture results among experimental treatments or lake sites. The histology for the Year 2 experiments was completed in Year 3. Joint or brain lesion scores determined by histologic examination were not significantly different among the pesticide groups or among the lake groups. However, there was a nonsignificant trend toward decreased joint lesion severity in the higher dose DDE groups. This decreased lesion severity could indicate a reduced host immune response in the higher DDE dose groups. Additional studies with more animals are necessary to evaluate that hypothesis.
In Year 3, egg microbiology was studied to determine if alligators exposed to EDCs had increased numbers of bacterial or fungal species in yolk compared to alligators from a relatively uncontaminated lake. Subsets of eggs from Lake Griffin, Lake Apopka, Emeralda Marsh Conservation Area, and Lake Woodruff (reference site) clutches were collected for a total of 151 eggs. The remaining eggs in each clutch were incubated at approximately 32?C until hatching, and clutch viabilities were determined. Both gram-positive and gram-negative aerobic bacteria (68 species) were isolated from yolk. Fungal isolates were rare. Organisms for which there was a statistically significant increase in their occurrence in dead eggs compared to viable eggs were Pseudomonas aeruginosa, Seratia marcescens, Proteus vulgaris, Brevundimonas diminuta, and Chromobacterium violaceum. There was no evidence of a relationship between the total number of aerobic bacteria in yolk and lake site or clutch.
Also, there was no evidence of a relationship between clutch viability and exposure to any or all of the pesticides in organochlorine analyses. This is a significant observation indicating that factors other than pesticide exposure are likely contributing to embryonic mortality and clutch viability.
Alligators naturally exposed to EDCs in Florida lakes were examined by gross and/or microscopic examination to determine whether morbidity and mortality were associated with infections that could provide evidence of compromised immune systems. Embryos and neonates were examined from Lakes Griffin, Apopka, Orange, Woodruff, Jessup, Okeechobee, Hancock, and Monroe. Of the more than 4,300 eggs incubated, 47 late-term embryos and neonates were necropsied. Lesions indicating in ovo disease included yolk sacculitis (with associated bacteria), inflammation of the albumen remnant with associated fungal hyphae and bacteria, and failure of the heart to migrate into the body cavity with associated severe chronic-active fibrinopurulent pericarditis.
The lesions in neonatal alligators included suppurative or pyogranulomatous and ulcerative inflammation of the gastrointestinal tract. Many of these lesions were severe and could have contributed to mortality. Other lesions included multifocal bacterial hepatitis, suppurative pyelonephritis, suppurative adrenalitis, suppurative interstitial pneumonia, and pyogranulomatous bacterial choroiditis and ventriculitis. These lesions were likely related to bacterial infection. From the embryos and neonates sampled for aerobic bacteria, 39 bacterial species, often opportunistic pathogens, were isolated with no consistent isolates relating to mortality.
A significant finding of unknown cause was the presence of focal areas of necrosis in the brains of 6 out of 31 animals. Additional observations included intracytoplasmic vacuolation in cortical neurons, vacuolation of the granule cell layer in the cerebellum, and pyknosis of granule cells. Affected animals were either observed with neurologic signs, including ataxia and loss of righting ability, or were found moribund or dead. The affected animals were from Lakes Apopka and Griffin and came from clutches with low clutch viability. Because more than 20 percent of the animals examined had this lesion, and because animals from these lakes were observed in past years with similar clinical signs, this lesion may play a significant role in the mortality of neonates, and possibly embryos, from these lakes. Possible causes of the lesions include toxicants; toxins; vascular disease (thromboembolism, infarcts); and nutritional disease (thiamine deficiency). Although possibly not related to the brain lesions in the neonatal animals, similar lesions were observed in the brains of adult Lake Griffin alligators associated with mortality. Although organochlorine contaminants would not be expected to cause such lesions in other animal species, additional studies are necessary to determine whether there is an association with EDC exposure and these brain lesions.