Evaluation of five diagnostic methods for the ...

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Title Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis
Author(s) G. O. Kelley, F. Zagmutt-Vergara, C. M. Leutenegger, K. A. Myklebust, M. A. Adkison, T. S. McDowell, G. D. Marty, A. L. Kahler, A. L. Bush, I. A. Gardner, R. P. Hedrick
Journal Journal of Veterinary Diagnostic Investigation
Date 2004
Volume 16
Issue 3
Start page 202
End page 211
Abstract Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: (1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, (2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, (3) a conventional single-round polymerase chain reaction (PCR), (4) a nested PCR assay, and (5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P>0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P<0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r=0.540 (P<0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.
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