DAO 59:235-248 (2004)  -  doi:10.3354/dao059235

Identification of a serine protease gene expressed by Myxobolus cerebralis during development in rainbow trout Oncorhynchus mykiss

Garry O. Kelley1,*, Francisco J. Zagmutt-Vergara1, Christian M. Leutenegger2, Mark A. Adkison1, Dolores V. Baxa1, Ronald P. Hedrick1

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California 95616, USA
2Department of Medicine and Epidemiology, Lucy Whittier Molecular Core Facility, School of Veterinary Medicine, University of California, Davis, California 95616, USA

ABSTRACT: Serine proteases have been recognized as key factors in parasite physiology and disease development. We have identified a serine protease gene from Myxobolus cerebralis, MyxSP-1, the myxozoan parasite causing whirling disease in salmonid fishes. The amino acid sequence, as deduced from the cDNA sequence, included a catalytic residue arrangement similar to that of the chymotrypsin family of serine proteases. A real-time TaqMan polymerase chain reaction (PCR) analysis revealed differences in the transcription levels for the chymotrypsin-like protease as found in early, intermediate, and late developmental stages of the parasite in experimentally-infected rainbow trout Oncorhynchus mykiss. MyxSP-1 transcription differed between individual tissues at each sampling point and in the same tissues over time (p < 0.0001). A nonradioactive mRNA in situ hybridization (ISH) protocol was developed to detect MyxSP-1 transcripts. Using a mixture of 3 digoxigenin-labeled antisense mRNA probes, MyxSP-1 transcription was observed in developmental stages of the parasite during the acute and chronic phases of the disease over a 240 d time period in infected rainbow trout tissues. MyxSP-1 transcription observed by ISH in cartilage and as associated with cartilage destruction was consistent with our real-time TaqMan PCR findings that demonstrated high levels of MyxSP-1 transcription during lesion development. Identifying genes encoding these enzymes and characterization of their functions can lead to the development of new chemotherapeutic protocols and vaccine approaches to control parasitic diseases.

KEY WORDS: Salmonid whirling disease · Myxobolus cerebralis · Serine protease · Real-time TaqMan polymerase chain reaction · mRNA in situ hybridization

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