Naslov
DEVELOPMENT OF DROPLET DIGITAL PCR ASSAY FOR MONITORING OF Saprolegnia parasitica, AND DEMONSTRATION OF ITS APPLICABILITY IN AQUACULTURE AND NATURAL FRESHWATER ENVIRONMENTS

Autori
Pavić, Dora ; Miljanović, Anđela ; Prosnec Zmrzljak, Uršula ; Košir, Rok ; Grbin, Dorotea ; Diéguez-Uribeondo, Javier, Bielen, Ana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Europe Aquaculture 2020 / - , 2021, 424-642

Skup
Aquaculture Europe 2020

Mjesto i datum
Online conference, 12.04.2021. - 15.04.2021

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Saprolegnia parasitica, ddPCR assay, specific primers

Sažetak
Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections in freshwater ecosystems and aquaculture facilities. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop droplet digital PCR (ddPCR) assay for detection and quantification of S. parasitica DNA in environmental samples. Material and methods Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. Specificity of the designed primer pair was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA. Assay performance was further assessed with water and swab samples from aquaculture (trout farms) and natural environments. Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin (30 samples) and eggs (15) of rainbow (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle (20) of signal (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Results Designed primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (like Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of reaction mixture. The assay performance was further assessed using environmental DNA samples (water and swab samples). Saprolegnia parasitica was detected in 16 out of 21 water samples (76 %) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Furthermore, S. parasitica was detected in swab samples collected from the host surface (i.e. adult trout, trout eggs and crayfish). Saprolegnia parasitica load was significantly higher in diseased trout samples then in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (median 8, agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had sings of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load then swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (median 63 agent level A5 vs. A4, respectively). Conclusion Our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environment. This would help in better understanding of S. parasitica ecology and its effects on the host populations.

Izvorni jezik
Engleski

Znanstvena područja
Interdisciplinarne prirodne znanosti, Biotehnologija

POVEZANOST RADA