SHORT COMMUNICATION D.B. Antonio á M. El-Matbouli á R.P. Hedrick Detection of early developmental stages of Myxobolus cerebralis in ®sh and tubi®cid oligochaete hosts by in situ hybridization Received: 19 April 1999 / Accepted: 16 June 1999 Abstract The myxosporean and actinosporean spores of Myxobolus cerebralis develop through many stages in their respective hosts, salmonid ®shes and a tubi®cid oligochaete. Using a modi®ed, non-radioactive in situ hybridization protocol, the parasite, which exhibits radically dierent structural forms during its develop- ment in each host, could be speci®cally detected in paran-embedded tissues of both ®sh and oligochaetes. Our study aims to demonstrate the application of the technique for detection of early stages of M. cerebralis in both hosts. Myxobolus cerebralis, the causative organism of whirling disease, is a major pathogen among farmed salmonids worldwide (Homan 1990). The parasite has been as- sociated recently with the decline of wild trout popula- tions in Colorado and Montana (Nehring and Walker 1996; Vincent 1996) and river systems with a history of the disease (Hedrick et al. 1998). The need to identify speci®c stages of the parasite is critical in controlling the spread of whirling disease (Markiw 1992). Current techniques used to detect M. cerebralis in its ®sh host include cartilage digestion (Markiw and Wolf 1974) and centrifugation (O'Grodnick 1975), light microscopy of stained tissue sections (Thoesen 1994) and direct ¯uo- rescent antibody tests (Markiw 1989). These procedures have been useful for detecting the parasite in its ®sh host after the beginning of sporogonesis (2±3 months post- infection depending on water temperature). However, they cannot be used to diagnose ®sh exposed to whirling disease for less than 3 months. DNA-based approaches such as the polymerase chain reaction (PCR, Andree et al. 1998) and in situ hybridization (ISH, Antonio et al. 1998) tests recently developed in our laboratory have augmented existing techniques for detecting M. cerebralis in both the ®sh and oligochaete hosts. Although PCR can detect the parasite in both hosts at lower thresholds than currently used diagnostic proce- dures (Andree et al. 1998), it does not have the ability to visualize and localize stages of the parasite as with ISH. Extraction and concentration techniques destroy early developmental forms in the ®sh and no current proce- dures identify directly the parasite stages as seen in the oligochaete host. We aim to demonstrate the application of the ISH technique in detecting very early stages of the parasite in its exact location in both ®sh and tubi®cid oligochaete hosts. Experimental infection of ®sh and oligochaete hosts was accomplished by initially exposing a sample of oligochaetes, Tubifex tubifex, to mature spores of M. cerebralis to produce the infective stage for ®sh (triactinomyxon). Production of mature spores and triactinomyxons has been previously described (Antonio et al. 1998). For the experiments described in this study, the hosts were experimentally infected in the following manner. Brie¯y, ca. 300 actinosporean-free T. tubifex were exposed to a dose of 500 M. cerebralis spores/worm prepared from heads of infected adult rainbow trout Oncorhynchus mykiss, following extraction and centrif- ugation procedures (Markiw and Wolf 1974; O'Grod- nick 1975). The inoculum was added to oligochaetes placed in a 1-l container with 2.5 cm sterile sand sub- strate and water of up to 5.0 cm above the substrate. Following an overnight incubation with the inoculum at 15 °C, the annelids were separated, rinsed in dechlori- nated tap water and transferred to a new container with Parasitol Res (1999) 85: 942±944 Ó Springer-Verlag 1999 D.B. Antonio á R.P. Hedrick (&) School of Veterinary Medicine, Department of Medicine and Epidemiology, University of California, Davis, CA 95616, USA e-mail: rphedrick@ucdavis.edu; Fax: +1-530-7520414 M. El-Matbouli University of Munich, Institute of Zoology, Fish Biology and Fish Diseases, D-80539 Munich, Germany