TitleDevelopment of a sensitive assay for the detection of Pseudoloma neurophilia in laboratory populations of the zebrafish Danio rerio.
Publication TypeJournal Article
Year of Publication2011
AuthorsSanders, JL, Kent, ML
JournalDis Aquat Organ
Date Published2011 Sep 09
KeywordsAnimals, Base Sequence, DNA, Fungal, Fish Diseases, Laboratory Animal Science, Microsporidia, Microsporidiosis, Polymerase Chain Reaction, Sensitivity and Specificity, Spores, Fungal, Zebrafish

The zebrafish Danio rerio is an increasingly important biological model in many areas of research. Due to the potential for non-protocol-induced variation, diseases of zebrafish, especially those resulting in chronic, sub-lethal infections, are of great concern. The microsporidium Pseudoloma neurophilia is a common parasite of laboratory zebrafish. Current methods for detection of this parasite require lethal sampling of fish, which is often undesirable with poorly spawning mutant lines and small populations. We present here an improved molecular-based diagnostic assay using real-time polymerase chain reaction (PCR), and including sonication treatment prior to DNA extraction. Comparisons of several DNA extraction methods were performed to determine the method providing the maximum sensitivity. Sonication was found to be the most effective method for disrupting spores. Compared to previously published data on PCR-based assay using a dilution experiment, sensitivity is increased. This shows that our assay, which includes sonication, is capable of detecting parasite DNA at 1 log higher dilution than the conventional PCR-based assay, which does not include sonication. Furthermore, we demonstrate the application of this method to testing of water, eggs, and sperm, providing a potential non-lethal method for detection of this parasite in zebrafish colonies with a sensitivity of 10 spores 1(-1) of water, 2 spores per spiked egg sample, and 10 spores microl(-1) of spiked sperm sample.

Alternate JournalDis Aquat Organ
PubMed ID22013754
PubMed Central IDPMC3358228
Grant ListP40 RR012546 / RR / NCRR NIH HHS / United States
R24 RR017386 / RR / NCRR NIH HHS / United States
5R24RR017386-02 / RR / NCRR NIH HHS / United States
R24 RR017386-02 / RR / NCRR NIH HHS / United States
R24 OD010998 / OD / NIH HHS / United States