Identification, quantification, and localization of protease genes expressed by Myxobolus cerebralis and their potential role in the pathogenesis of whirling disease

Myxobolus cerebralis, the agent causing whirling disease, is considered a significant pathogen in salmonid aquaculture and has recently been implicated in population losses in wild salmonid fish. The microscopic pathology associated with the development of M. cerebralis in the fish host provides empirical evidence that tissue destruction may be the result of proteolytic enzymes produced by the parasite. The identification and initial characterization of protease gene(s) from M. cerebralis and their expression associated with lesion formation were therefore examined.; A serine protease gene (MyxSP-1) and a cysteine protease gene ( MyxCP-1) from M. cerebralis were identified. Real-time TagMan polymerase chain reaction (PCR) analysis demonstrated differences in the transcription levels for both MyxSP-1 and Myx CP-1 in early, intermediate, and late developmental stages of the parasite. A mRNA in situ hybridization (ISH) protocol was developed to detect MyxSP-1 gene transcription during the acute and chronic phases of the disease. Phylogenetic comparisons of the M. cerebralis MyxCP-1 to other representative eukaryotic full-length cathepsin-like genes indicated MyxCP-1 is the earliest lineage in the cathepsin Z group and clearly differs from cathepsin L, B, and C-like proteases. Protease transcription at key stages of parasite development indicated that MyxSP-1 and MyxCP-1 potentially facilitate initial penetration of host tissue, provide the means to traverse host epithelial and nervous tissues, and eventually cause cartilage lysis at the site of sporulation.; A TagMan PCR for the 18S rDNA of M, cerebralis was developed as a standard for protease gene expression and the test was evaluated with 4 traditional assays for parasite detection in fish tissues: (1) pepsin-trypsin digest (PTD); (2) two different histopathology grading scales; (3) a conventional single round polymerase chain reaction (PCR); and (4) a nested PCR assay. There were no significant differences (P > 0.05) between the 5 diagnostic assays in distinguishing between experimentally-infected and uninfected control fish. Quantification of parasite levels in cranial tissues using PTD and real-time TagMan PCR were significantly correlated r = 0.540 (P < 0.001). These results confirm that currently used procedures and a newly developed TagMan PCR are effective tools in detecting and evaluating the severity of M. cerebralis infections in moderately to heavily infected rainbow trout.