Master of Science / Department of Diagnostic Medicine/ Pathobiology / Lisa M. Pohlman / Background: Iron storage disease has proven to be a serious health concern for captive toucans. Physiologic mechanisms to efficiently extract iron from naturally iron-deficient diets appear the likely cause of iron overload when fed iron-sufficient diets in captivity. Iron overload can result in diabetes, heart failure, and even death. Serum ferritin concentrations are considered the most reliable screening tool to predict total body iron stores in many species, but an assay has not been available to measure serum ferritin in toucans. Objective: The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum ferritin in toucans using a polyclonal antibody in a sandwich arrangement. Methods: Ferritin was isolated from toucan liver and used as a standard. A rabbit polyclonal anti-toucan antibody was used as the capture antibody and as a detection antibody conjugated to horseradish peroxidase. Linearity of toucan ferritin standards, effect of serum dilution, recovery of added ferritin standards, and intra- and inter-assay variability were determined. Results: Ferritin standards were linear from 0 to 50 ng/ml. The relationship between serum dilution and serum ferritin concentration was also linear. When 10, 20, 30, 40, or 50 ng/ml of purified toucan ferritin were added to diluted serum, the recoveries varied from 69% to 104%. The intra-assay variability for four test serum samples averaged 11% and the inter-assay variability for the same four samples averaged 11%. Conclusions: Although the results from the linearity and recovery studies are promising for assay development when viewed independently, preliminary ferritin concentrations from all toucans studied are much higher than expected. Upon further evaluation including Dot blot assays, Western blot assays, SDS-PAGE, and protein determination of the ferritin stock solution, it was determined that the ferritin stock solution did not contain a pure protein and therefore likely renders the assay invalid. Further testing is needed to confirm these findings.

Iron storage disease is a significant cause of liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The nature of the susceptibility in this and other captive exotic species to iron storage disease is not clear. Hepcidin, a key iron regulatory hormone, is involved in the regulation of iron absorption in humans and other mammalian species and a deficiency in hepcidin has been associated with a number of genetic mutations resulting in hemochromatosis in humans. The objectives of this thesis were to identify whether there is a functional mutation in the hepcidin gene in the Egyptian fruit bat that may increase the susceptibility of this species to iron storage disease, and whether there is a functional deficiency in hepcidin gene expression in the Egyptian fruit bat in response to iron challenge. We compared the coding region of the hepcidin gene amongst several species of bats and investigated hepcidin response to intramuscular injection of iron dextran amongst three species of bats with variable susceptibility to iron storage disease; the Egyptian fruit bat, the straw-colored fruit bat (Eidolon helvum), and the common vampire bat (Desmodus rotundus). While a number of genetic differences were identified amongst species, a functional mutation that could result in decreased hepcidin activity was not identified in the Egyptian fruit bat. Bats exhibited marked variation in hepcidin gene expression, with the highest level of hepcidin response to iron challenge in the common vampire bat. While the Egyptian fruit bat exhibited significant hepcidin response to iron challenge, the magnitude of response was lower than that in the common vampire bat and lower than expected based on findings in healthy humans. The straw-colored fruit bat did not exhibit any hepcidin response despite a significant increase in iron stores, which suggests this species may have evolved an alternate mechanism for coping with excessive iron or may be more susceptible to iron overload than previously recognized. / Toronto Zoo Scholarship Fund