Describing a novel algal species
The most abundant cyanobacteria present during disease outbreaks at all the AVM sites was an epiphytic colony forming species. Morphological and genetic data revealed this target species is a previously unidentified species in the Stigonematales order. Morphologic images are shown below. 16S rRNA sequence identity was determined from environmental isolates of this unknown Stigonematalan species using DGGE (density gradient gel electrophoresis). The entire 16S gene sequence has been submitted to the international database GenBank and received a unique accession number # AY785313. 16S rRNA sequence data has been aligned with additional cyanobacteria sequences to determine designations for Real-time PCR assays in order to rapidly genetically detect the Stigonematales species from environmental samples (Williams, et al. 2007). The morphological and genetic information will also be used in a pending manuscript to determine phylogeny and formally describe this species.
Mallard feeding trials
Feeding trials were conducted in the laboratory with farm-raised mallards and hydrilla collected from AVM positive sites during a disease outbreak. Hydrilla with dense colonies of Stigonematales colonies was used for the treatment groups. Hydrilla was also collected from control sites with no birds deaths, no birds with AVM lesions, and no Stigonematales colonies on the leaves. The birds were given as much hydrilla as they could consume each day for a three week trial. Some of the ducks fed hydrilla with the Stigonematales colonies became neurologically imparied before the end of the trial. Only the birds that were fed the treatment hydrilla had brain lesions at the conclusion of the trial (Wiley, et al 2007).
Mammal feeding trials
Five six-week old swine were fed hydrilla with the epiphytic suspect cyanobacterium for a 6-week period The hydrilla material was collected from an AVM reservoir during a time period when birds were nuerologically impaired. Pigs were euthanized at end of treatment period, brains removed, fixed in formalin, and analyzed for AVM lesions. None of the pigs developed AVM lesions at the end of the trial (Haynie 2008). Another study of mammalian susceptibility used AVM affected coot tissues collected from an AVM outbreak and fed them to swine (Lewis-Weis et al. 2004). None of these pigs became positive for AVM.
Cell line bioassays
Previous to the development of the cell line assay, the only means of detecting and studying the AVM toxin was by live animal studies. These studies have used several birds and mammal species, typically last one to six weeks, and involve feeding large quantities of vegetation, tissues, or other test materials. It is through these studies that researchers have been able to locate the source of exposure to the AVM toxin (aquatic vegetation and associated epiphytes), have demonstrated the conveyance of toxicity from prey items to predators (Fischer et al. 2003), and have explored mammalian susceptibility (Lewis-Weis et al. 2004).
But these studies they have numerous disadvantages. The trials are labor intensive, generally require large quantities of test material, and take extensive time to plan and conduct. The endpoint of these studies – the determination of AVM characteristic lesions in the brain – also takes time to analyze. So researchers worked to develop a cell based bioassay for AVM toxin detection that could be used as an alternative to the large scale animal assays. In addition to reducing the need for animal subjects, an cell line assay would allow for small-scale testing, using minimal amounts of the test solution/substance.
The hydrilla used in the positive feeding trial with mallards was then evaluated for toxicity in cell lines bioassays (Wiley, et al 2009). Specifically, the methanol fraction from the AVM lake hydrilla sample was toxic relative to hydrilla collected from a control lake where the bird disease has never been documented.