Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.
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.
Kelley
Garry O
GO
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Zagmutt-Vergara
Francisco J
FJ
Leutenegger
Christian M
CM
Myklebust
Karin A
KA
Adkison
Mark A
MA
McDowell
Terry S
TS
Marty
Gary D
GD
Kahler
Alex L
AL
Bush
Arla L
AL
Gardner
Ian A
IA
Hedrick
Ronald P
RP
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
D016054:DNA, Protozoan; D012337:RNA, Ribosomal, 18S; D014357:Trypsin; D010434:Pepsin A