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United States Department of Agriculture

Agricultural Research Service

Title: Real-Time Quantitative Polymerase Chain Reaction (Qpcr) to Identify Myxobolus Cerebralis in Rainbow Trout Oncorhynchus Mykiss

Authors
item Overturf, Kenneth
item Cain, K. - UNIV OF IDAHO, MOSCOW
item Cavender, W. - UNIV OF IDAHO, MOSCOW
item Powell, M. - UNIV OF ID, HAGERMAN
item Wood, J. - PISCES MOLECULAR, BOULDER

Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 28, 2004
Publication Date: September 5, 2004
Citation: Overturf, K.E., Cain, K.D., Cavender, W.P., Powell, M.S., Wood, J.S. 2004. Real-time quantitative polymerase chain reaction (qpcr) to identify myxobolus cerebralis in rainbow trout oncorhynchus mykiss. Diseases of Aquatic Organisms. v.60.p.205-213.

Interpretive Summary: This study describes the development of a real-time quantitative polymerase chain reaction (QPCR) technique for a heat-shock protein (Hsp ) and the ribosomal DNA (rDNA) sequences specific to the disease organism for whirling disease in trout. The assay also attempt to quantify infection severity levels within rainbow trout fry Oncorhychus mykiss. Rainbow trout for this study were exposed to the causative disease agent of whirling disease (Myxobolus cerebralis) under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than detection using 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 at very low levels for the Hsp and rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp probe when parasite copy number and histology scores were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe and the 70 probe. 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.

Technical Abstract: This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 0 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhychus 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 ul-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.

Last Modified: 9/3/2014
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