Collaborative experiments with Clemson University (Dr. William Bowerman and Anna Birrenkott) produced the disease experimentally by feeding farm-raised mallards hydrilla from Lake Thurmond, GA/SC. This demonstrated that the primary disease agent is within the hydrilla mats. Also, a sentinel bird study using naturally occurring blooms of cyanobacteria on hydrilla leaves and farm-raised mallard ducks (Anas platyrhynchos) induced the disease experimentally (Wilde et al. 2005). AVM brain lesions were formed in 6 of 9 hydrilla-feed birds, and no lesions were present in the control mallards. The hydrilla used in the positive feeding trial was then evaluated for toxicity in cell lines bioassays by the NOS Biotoxin Group, Charleston (Faith Wiley and Fran Van Dolah). They found that the methanol fraction from the AVM lake hydrilla sample was toxic relative to hydrilla collected from a lake where the bird disease has never been documented. Field collections, feeding trials and cell line bioassays are ongoing to discover if the hydrilla and associated epiphytes is the source of the AVM neurotoxin.
Searching for the common factor in all AVM sites
A survey of reservoirs by SCHABP from 2001-2005 investigated the potential involvement of plant-associated (epiphytic) cyanobacteria as the causative agent of AVM associated with bald eagle and other bird deaths in SC, NC, AR and GA lakes. In these reservoirs, there is very little water-column (phytoplankton) production, but aquatic plants such as hydrilla are abundant. Plant samples were collected from all sites where AVM positive birds have been recovered. Epiphytic cyanobacteria covered 20-90% of the leaf and stem surface of the aquatic plants (primarily hydrilla).
Among the many algal species surveyed from the water, plants and sediment, eleven species/genera were capable of producing toxins (Table 1). No specific cyanotoxin screening was conducted on samples during this monitoring study. The species listed in Table 1, however, have been documented with the potential to produce a range of hepatotoxins (liver toxins), LPS (lipopolysaccharides — mostly dermal toxins) and neurotoxins (brain, nervous system toxins). The primary known cyanotoxins that have neurotoxic effects are anatoxins and saxitoxins. There are many other potential neurotoxins produced by cyanobacteria that have yet to be characterized. While there were native aquatic species with cyanobacterial epiphytes, native aquatic plants never dominate the entire water column with dense biomass in the manner of hydrilla, egeria and eurasian watermilfoil.
One unknown Stigonematalan species of epiphytic cyanobacteria has been noted from all the AVM sites sampled during 2001-2005. Additionally, this species was not or rarely present on plant samples collected from sites where AVM has not been documented. American coots are known to feed primarily on aquatic plants and algae, and thus may be at the greatest risk (among waterbirds) of contracting AVM by ingesting cyanobacteria. Bald eagles, in turn, are thought to contract AVM by preying on sick diseased coots. A conceptual model has been developed to show transmission from aquatic vegetation and associated epiphytic cyanobacteria to waterfowl to bald eagles.
Describing a novel algal species
In an effort to formally describe the epiphytic cyanobacteria responsible for AVM, investigators are working to describe the target species, based on morphological and molecular characteristics. Morphological and genetic data revealed this target species is a previously unidentified species in the Stigonematales order. Morphologic images are shown in Figure 1. 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). Additional morphological and genetic information allowed for formal description of this new genus/species, Aetokthonos hydrillicola (Wilde, et al 2014).
Summary of research findings
AVM is considered the most significant cause of unknown eagle mortality in US history. The AVM disease is caused by a neurotoxin that affects coots and waterfowl by ingesting plant material and associated epiphytes. Eagles are affected through ingested diseased prey (e.g. coots). The working hypothesis is that the suspect Stigonematales species is producing a novel neurotoxin. This coupled with the fact that the AVM-suspect cyanobacteria, a stigonematalan species, grows more densely on the hydrilla leaves than on any other aquatic plant may be the reason why hydrilla is the common denominator at all sites with the highest incidence of the AVM disease.