Defection of Myxobolus cerebralis in rainbow trout and oligochaete tissuesby using a nonradioactive in situ hybridization (ISH) protocol

A nonradioactive in situ hybridization (ISH) protocol was developed to dete ct Myxobolus cerebralis, the causative organism of whirling disease, in its primary host, rainbow trout Oncorhynchus mykiss; and in its alternate olig ochaete host, Tubifex tubifex. A cocktail of three oligonucleotide primers (derived from the small subunit ribosomal DNA sequence) directed at target sequences of the parasite DNA was tailed at the 3' end with digoxigenin-lab eled deoxyuridine triphosphate (DIG-dUTP). Labeled probes were hybridized t o parasite DNA present in deparaffinized tissue sections from infected trou t and oligochaetes. The bound probes were visualized after modifications of existing ISH protocols. By using the new ISH procedure, the parasite was f ound in target tissues of subclinically and clinically infected fish and tu bificid oligochaetes after exposures of these hosts to triactinomyxons and mature spores, respectively. The probe did not bind with salmonid tissues i nfected with two other myxosporean parasites, Ceratomyxa shasta or the PKX organism, or to a Myxobolus sp. infecting the cartilage of plain sculpin My oxocephalus jaok. These initial results indicate that ISH is an effective a nd specific test for detecting Myxobolus cerebralis in its fish and oligoch aete hosts.