title: Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.
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.
authors.affiliation: Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Garry O
authors.initials: GO
authors.lastname: Kelley
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Zagmutt-Vergara
authors.forename: Christian M
authors.initials: CM
authors.lastname: Leutenegger
authors.forename: Karin A
authors.initials: KA
authors.lastname: Myklebust
authors.forename: Mark A
authors.initials: MA
authors.lastname: Adkison
authors.forename: Terry S
authors.initials: TS
authors.lastname: McDowell
authors.forename: Gary D
authors.initials: GD
authors.lastname: Marty
authors.forename: Alex L
authors.initials: AL
authors.lastname: Kahler
authors.forename: Arla L
authors.initials: AL
authors.lastname: Bush
authors.forename: Ian A
authors.initials: IA
authors.lastname: Gardner
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Hedrick
mesh_terms: D000818:Animals; D001921:Brain / Q000469:parasitology; D020808:Central Nervous System Protozoal Infections / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D056890:Eukaryota / Q000235:genetics / Q000254:growth & development*; D005393:Fish Diseases / Q000175:diagnosis / Q000469:parasitology*; D006651:Histocytochemistry / Q000662:veterinary; D017686:Oncorhynchus mykiss; D010434:Pepsin A / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D014357:Trypsin / Q000378:metabolism
chemical_list: D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S; D014357:Trypsin; D010434:Pepsin A