DNA-based diagnostic test for detecting myxobolus, the cause of salmonid whirling disease

#The present invention is directed to compositions and methods for detecting the presence of the myxozoan parasite Myxobolus spp. in aquatic samples.

#Fish of the family Salmonidae are amongst the most commercially important of game fish. This taxonomic family includes such well known species as Atlantic salmon, brown trout, rainbow trout, coho salmon, and arctic char. Several species of salmonid fish are parasitized by the myxozoan parasite Myxobolus spp. (e.g., M. cerebralis, M. insidiosus, M. squamalis) which is responsible for the chronic inflammatory condition called whirling disease. The radical tail chasing behavior of infected fish, which characterizes the disease, results from pressure on the auditory capsule due to developmental stages of the parasite and the accompanying host's inflammatory response. The origins of the pathogen are suspected to be central Europe and Asia but movements of frozen and live trout have spread the agent to a total of 21 countries and 20 of the states in the USA. Recent increases in outbreaks of whirling disease have highlighted the vulnerability of stocks of salmonid fish in state and private hatcheries. The costs associated with control programs can be staggering. From 1965 to the present in California and Michigan alone these costs are estimated to exceed $30,000,000. This has brought to the forefront the concerns of transmission of pathogens between cultured and wild fish populations and the need to readily identify infected fish populations.

#The phylum Myxozoa comprises a group of multicellular organisms that principally infect fish (Lom, J., Parasitol. Today 3:327 (1982)). Myxozoans found in freshwater have complex life cycles with alternating forms found in fish and aquatic oligochaete worms (Kent, M. L., et al., Can. J. Zool. 72:932 (1994); Wolf, K., et al., Science 255:1449 (1984); Wolf, K., et al., J. of Fish Dis. 9:83 (1986)). These stages are designated as myxosporean and actinosporean in their respective fish and worm hosts. The parasite stages designated as spores and triactinomyxons, respectively, emerge from the fish and worm hosts and they are infectious only for the other host (Wolf, K., et al., Science 255:1449 (1984)).

#Example 1 describes the isolation and sequencing of the 18S rRNA gene of Myxobolus cerebralis.

#Both myxosporean and actinosporean spores were generated in the laboratory at the Fish Health Research Center, National Biological Survey, Leetown, W. Va. The alternating stages represented a single source of spores used to initiate the infections in oligochaetes and then fish. Myxobolus cerebralis spores were obtained directly from infected rainbow trout (Oncorhynchus mykiss) tissues using the pepsin trypsin digest as described by Markiw and Wolf (J. of the Fisheries Res. Board of Canada 31:15 (1974)). Partially purified spores were stored at -20.degree. C. until used. Actinosporean stages were obtained as described by Markiw (J. of Fish Dis. 12:137 (1989)) following experimental infections of the oligochaete (Tubifex tubifex). The actinosporean stages were partially purified using PERCOLL (modified-sucrose) gradient centrifugation. These stages were also stored at -20.degree. C. prior to use.

#A. DNA Extractions