Development of a sensitive assay for the detection of Pseudoloma neurophilia in laboratory populations of the zebrafish Danio rerio

Abstract

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.

Fig. 1

Partial ssrDNA sequence alignment of Pseudloma neurophilia (Genbank AF322654), Loma embiotocia (Genbank AF320310) , Ovipleistophora mirandellae (Genbank AF356223), Loma salmonae (Genbank U78736), Glugea sp. (Genbank AY090038), Loma morhua (Genbank GQ121037), Loma psittaca (Genbank FJ843104), Pleistophora hyphessobryconis (Genbank GU126672), Heterosporis anguillarum (Genbank AF387331), Ichthyosporidium giganteum (Genbank L13430), Pleistophora mulleri (Genbank FN434084), Dasyatispora levantinae (Genbank GU183263), Glugea stephani (Genbank AF056015), Loma salmonae (Genbank HM626215), Loma sp. (Genbank HM626217), Glugea anomala (Genbank AF044391), Loma sp.(Genbank AF104081), Glugea atherinae (Genbank U15987), Loma acerinae (Genbank AJ252951), Pleistophora typicalis (Genbank AJ252956), Pleistophora sp (PA) (Genbank AJ252958), and Ichthyosporidium sp.(Ganbank L39110) Pn10F and Pn10R primer locations are underlined and PN10 probe location is shaded. Asterisks denote regions of nucleotide similarity.

Fig. 2

Pseudoloma neurophilia spore sonication time study. Quantification cycle thresholds (Cq) of triplicate suspensions of 1000 spores of P. neurophilia sonicated at 55W for different time points and tested by real-time PCR. Bars represent mean Cq; Points represent individual sample Cqs.

Fig. 3

Pseudoloma neurophilia spore extraction method comparison by quantification cycle (Cq). NT: no treatment, M: MoBio, Q: QIAgen, P: peroxide, P+M: peroxide + Mobio, P+Q: peroxide + QIAgen, S: sonication, S+M: sonication + Mobio, S+Q: sonication + QIAgen, B+Q: bead beating + QIAgen, C+Q: chitinase + QIAgen. Bars represent mean Cq; points represent actual Cq for individual samples. Sonication treatment alone showed the lowest mean Cq in Trial A, 26.74 followed by sonication with DNA extraction by QIAgen silica gel membrane method (mean Cq = 27.73). Trial B results showed the lowest mean Cq for sonication alone, 28.74, followed by sonication and QIAgen DNA extraction combined, 29.93.