Extraction method development and in vivo and in vitro toxicity studies of the etiologic agent of avian vacuolar myelinopathy.

摘要

Avian vacuolar myelinopathy (AVM) is a neurological disease affecting birds in the southeastern United States. The cause of the disease has not yet been determined but is believed to be a naturally produced toxin and is associated with aquatic vegetation. Current research on AVM is limited to in vivo studies utilizing whole tissue or vegetative samples. The objectives of this research were to develop extraction methods for isolating the putative AVM toxin from vegetative samples and to develop an in vitro bioassay for detection and study of the toxin. Samples of vegetation were collected from reservoirs known to be affected by AVM and confirmed to contain the toxin by mallard bioassay. The collected vegetation was then extracted with a series of solvents and the crude extracts were re-introduced to a mallard model to confirm the presence or absence of the AVM agent. All birds administered a methanol extract developed characteristic AVM lesions, indicating that methanol is a suitable solvent for AVM toxin extraction. Additional crude extracts were produced from vegetation collected at AVM-affected reservoirs as well as reservoirs with no known history of AVM and evaluated for their in vitro toxicity using established cell lines. Extracts produced from vegetation collected at AVM sites induced a significant cell cycle arrest in C6 glioma cells, while AVM-negative extracts induced only mild effects on this endpoint. To further evaluate this in vitro assay for its ability to detect the AVM toxin, as well as continue the development of toxin extraction methods, fractions were produced from a crude methanol extract and evaluated for toxicity by the cell cycle assay as well as an in vivo chicken bioassay. The fractionation methods were successful in isolating the in vitro toxicity, but the presence of the AVM toxin could not be confirmed in these fractions due to inconclusive in vivo results. Further testing will be necessary to determine if the observed cell cycle arrest is due to the etiologic agent of AVM as well as to advance the process of AVM toxin isolation and characterization, but this research has providing promising tools for future AVM investigations.