Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.

OBJECTIVE: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc. METHODS: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays. RESULT: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book. CONCLUSION: The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.

Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease II: Efficacy of Using Antibiotics in Diluents and During Water Hardening.

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, has lead to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid and can enter the egg during fertilization. The addition of antibiotics to diluents and water-hardening solutions could prevent the spread of the disease. In separate trials, a mixture of 0.197 mg/mL penicillin plus 0.313 mg/mL streptomycin was added to both a 0.5% sodium chloride fertilization diluent and hatchery well water during hardening. Tests showed that the addition of the antibiotics to the diluent and during up to 60 min of water hardening had no effect on the eye-up, hatch and deformity rates of Rainbow Trout Oncorhynchus mykiss eggs compared with the nonantibiotic-treated controls. Also, significant reductions in the prevalence of F. psychrophilum on the surface and inside eggs were observed when compared with controls. These results indicate that the addition of penicillin and streptomycin to diluents and during water hardening can prevent the vertical transmission of bacterial coldwater disease.

Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss.

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.