Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis.
Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. gokelley@ucdavis.edu
Garry O
GO
Kelley
Mark A
MA
Adkison
Francisco J
FJ
Zagmutt-Vergara
Christian M
CM
Leutenegger
Jeffery W
JW
Bethel
Karin A
KA
Myklebust
Terry S
TS
McDowell
Ronald P
RP
Hedrick
D000818:Animals; D020808:Central Nervous System Protozoal Infections / Q000175:diagnosis / Q000235:genetics / Q000662:veterinary*; D016054:DNA, Protozoan / Q000032:analysis; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification*; D005393:Fish Diseases / Q000175:diagnosis* / Q000469:parasitology; D017686:Oncorhynchus mykiss / Q000469:parasitology*; D011529:Protozoan Infections, Animal / Q000175:diagnosis* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D018571:Sentinel Surveillance / Q000662:veterinary; D013047:Specific Pathogen-Free Organisms
D016054:DNA, Protozoan