Study of amphibian immunotoxicology is a growing area of research, but very little information is available on how environmental contaminants affect disease resistance in urodele amphibians. Urodele amphibians lack the more highly evolved aspects of the specific immune system that are present in anurans, birds, and mammals. Instead, these animals rely more heavily on innate defense mechanisms than do anurans to provide rapid, nonspecific protection from pathogens. Thus, it is prudent that immunotoxicologic research with urodele amphibians includes an evaluation of effects of contaminant exposure on nonspecific immunity. The objectives of this study were to measure the phagocytic and oxidative-burst activity of peritoneal neutrophils collected from a urodele, the tiger salamander (Ambystoma tigrinum), and to evaluate the use of these assays in immunotoxicologic research using urodele amphibians. Using tiger salamanders collected in August 2000, phagocytosis and oxidative-burst assays modified from mammalian protocols were conducted through October 2001. Results indicated that large numbers of peritoneal neutrophils for use in immunotoxicologic tests can be collected from salamanders injected with thioglycollate. Moreover, these neutrophils readily engulfed foreign material (phagocytic activity) and produced measurable amounts of hydrogen peroxide (oxidative-burst activity). Phagocytosis was effectively inhibited by incubating cells with sodium azide (PϽ0.001), and quantification of phagocytosis using flow cytometry was well correlated with manual counts (rϭ0.84, PϽ0.001). Dexamethasone treatment reduced phagocytic activity as measured by manual counts (PϽ0.02), suggesting that this test is useful for detecting alteration by immunosuppressive agents. In contrast, oxidative function was unaffected by dexamethasone treatment, and results from the oxidative-burst assay were generally less consistent than those from the phagocytosis assay. Based on these results, phagocytic activity of peritoneal neutrophils may be a useful endpoint in immunotoxicologic studies to evaluate the impact of environmental contaminants on innate defense mechanisms in urodele amphibians.

One theory proposed to explain the global declines in amphibian populations involves contaminant-induced immune alteration and subsequent increased susceptibility to infectious disease. The goal of this study was twofold, to (1) study acute oral toxicity of deltamethrin (cyclopropanecarboxylic acid, 3-(2,2-dibromoethenyl)-2,2-dimethyl cyano(3-phenoxyphenyl)methyl ester) in tiger salamanders (Ambystoma tigrinum), and (2) evaluate whether the insecticide deltamethrin produces immunosuppression in these animals. In the acute toxicity study, tiger salamanders receiving single doses of deltamethrin ranging from 1 to 35 mg/kg displayed intention tremors, hypersalivation, ataxia, choreoathetosis (writhing), severe depression (immobility with minimal response to stimuli), and death. For acute effects, based on clinical signs, the median lethal dose (LD(50)) and lowest observed adverse effect level (LOAEL) were estimated to be 5 to 10 mg/kg and 1 mg/kg, respectively. The LOAEL in animals dosed 3 times per week for 4 wk was 400 microg/kg/d. The endpoints for the immunotoxicity study included lymphoid organ mass and histopathology, hematological variables, and functional assays of phagocytosis, oxidative burst, and lymphoblastic transformation. Tiger salamanders in 4 treatment groups (0, 4, 40, or 400 microg/kg/d) were dosed with deltamethrin via the diet 3 times per week for 4 wk. Deltamethrin exposure resulted in increased liver mass, packed cell volume, and total plasma protein concentration, but these effects were not dose dependent. The relative mass of kidney and spleen, plasma albumin and globulin concentrations, and circulating leukocyte numbers were not affected by deltamethrin exposure, nor were phagocytosis, oxidative burst, and lymphoblastic transformation. This study shows that at moderate levels of exposure, deltamethrin may be neurotoxic to tiger salamanders. However, based on the immune assays considered in this study there was no evidence of immunosuppression from dietary exposure to environmentally relevant concentrations of deltamethrin. In light of these findings, it is unlikely that exposure to environmental concentrations of deltamethrin has produced immunosuppression and contributed to the emergence of iridovirus outbreaks in tiger salamander populations.