Biology Ph.D. Dissertations

Molecular Detection and Quantification of the Fish Pathogen Saprolegnia spp. Using qPCR and Loop Mediated Isothermal Amplification

Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Vipaporn Phuntumart (Advisor)

Second Advisor

Lubomir Popov (Other)

Third Advisor

Raymond Larsen (Committee Member)

Fourth Advisor

Paul Morris (Committee Member)

Fifth Advisor

Scott Rogers (Committee Member)

Sixth Advisor

David Straus (Committee Member)

Abstract

Saprolegniasis, caused by oomycete pathogens of the Saprolegnia genus is a serious emergent disease of fish causing losses worth approximately $40 million annually in USA. This study aims to analyze the efficacy of copper sulfate and peracetic acid as chemical agents to combat saprolegniasis; and to develop a molecular strategy for the rapid, sensitive and specific detection of Saprolegnia spp., suitable for on-site applications. The study indicates that copper sulfate and peracetic acid effectively reduced the various parameters of Saprolegnia spp. growth. Peracetic acid was effective against other oomycete pathogens to varying degrees, and its persistence in environmental water samples depends on the organic matter content of the water samples. Peracetic acid has been proposed as an effective, non-toxic, and eco-friendly approach to combat saprolegniasis.

This study reports the isolation Saprolegnia spp. from various sources. Using cytochrome c oxidase subunit I (COI) and Internal Transcribed Spacer of rDNA (ITS) as molecular markers, these have been identified phylogenetically. Based on these markers, qPCR primers have been developed specific to the Saprolegnia genus and could detect as low as 2pg of Saprolegnia spp. genomic DNA. Also, qPCR based absolute quantification could be used as an approach to quantify the Saprolegnia spp. levels in environmental samples. Additionally, a LAMP assay was developed using the ITS marker. The established LAMP assay was specific to the Saprolegnia genus and could detect as low as 10 fg of Saprolegnia spp. genomic DNA within 30 min, thus making it significantly more sensitive compared to qPCR. Both qPCR and LAMP could also detect as low as 1 zoospore directly. The LAMP reactions could be performed using a simple equipment such as heat block, and results could be detected visually. Further, LAMP has the potential for direct on-field applications for detecting Saprolegniaspp. from water samples collected from Recirculating Aquaculture Systems (RAS).

Based on the results, we propose the application of LAMP in conjunction with qPCR, as a rapid and cheap diagnostic technique for Saprolegnia spp. pathogens directly from water samples, which would enable fish farmers to make informed decisions regarding the timing and extent of chemical treatment.

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