# Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies

Emira Cherif, Theo Deremarque, Fabienne Justy, Rodolphe Elie Gozlan, Marine Combe

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## To cite this version:

Emira Cherif, Theo Deremarque, Fabienne Justy, Rodolphe Elie Gozlan, Marine Combe. Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies. Journal of Fish Diseases, 2022, 46 (2), pp.177-180. 10.1111/jfd.13722. hal-04816016

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Submitted on 3 Dec 2024

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# Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies

Emira Cherif ID

Theo Deremarque

Fabienne Justy

Rodolphe Elie Gozlan

Marine Combe

ISEM, Univ Montpellier, CNRS, IRD, Montpellier, France

**Correspondence**

Emira Cherif, Rodolphe Elie Gozlan and Marine Combe, ISEM, Univ Montpellier, CNRS, IRD, Montpellier, France.

Emails: emira.cherif@ird.fr; marine.combe@ird.fr; rudy.gozlan@ird.fr

**Funding information**

ROSETTA project (AFB-IRD)

**Keywords:** disease surveillance, fish fungal parasite, intracellular parasite, nested-PCR, ONT sequencing, *Sphaerothecum destruens*

## 1 | INTRODUCTION

In 2005, a risk of global disease emergence in freshwater fish was identified in Europe and associated with the presence of the fungal-like pathogen *Sphaerothecum destruens* (Rosette Agent) (Combe & Gozlan, 2018; Gozlan et al., 2005). *Sphaerothecum destruens* is a unicellular, intracellular obligate and generalist parasite (Andreou et al., 2011; Arkush et al., 2003) that exhibits high levels of virulence (i.e. host mortality rates) in a wide variety of freshwater and marine fish species (including at least 18 cyprinids and salmonids species) in the wild and in aquaculture facilities and has been found in a wide range of climates such as the mountainous Tcheremoch River in Ukraine to the desert wadi Felrhir in Algeria (reviewed in Combe & Gozlan, 2018). For example, in 1984 S. destruens was responsible for high mortalities observed in Chinook salmon (*Oncorhynchus tshawytscha*) in seawater net pens in Washington state, USA (Harrell et al., 1986). Since then, it has been responsible for two mortality events of brown and rainbow trout in farms and experimental facilities in western France (Boitard et al., 2017) and the total extinction (up to 80%-90% of mortality) of native fish populations in a catchment area in southeast Turkey (Ercan et al., 2015). Importantly, the presence of this fungal-like parasite in such a variety of freshwater ecosystems is enhanced by the highly invasive nature of its healthy

(i.e. asymptomatic) carrier the topmouth gudgeon, *Pseudorasbora parva* (reviewed in Gozlan, 2011).

The intracellular and fungal-like nature of S. destruens combined with low parasite loads in healthy carriers makes it extremely difficult to detect in infected hosts and therefore introduces a bias when we estimate the disease's true prevalence in a population. Furthermore, the use of slightly different primer sets to target and amplify its DNA from complex environmental matrices (e.g. water and fish tissues) results in an underestimation of its prevalence among invasive fish populations (Combe et al., 2022) and significant variations in prevalence levels between different studies (see Combe et al., 2022; Ercan et al., 2015; Gozlan et al., 2005; Spikmans et al., 2013). After 15 years of research resulting in 28 publications and despite the high risk of disease emergence worldwide associated with irreversible impacts on fish biodiversity and the economy of the aquaculture sector, the scientific community still questions the reliability, sensitivity and efficiency of 3 different PCR primer sets commonly used to detect its DNA in healthy carriers and infected native fish species. However, the first step for effective control of the emergence and spread of the rosette agent in natural ecosystems and aquaculture facilities is a highly specific and sensitive DNA detection method that allows us to estimate the true extent of infection. Here, our aim was to provide decision-makers and
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stakeholders with a more reliable detection tool. We suggest that this optimized protocol becomes the one commonly used worldwide to obtain comparable results between studies.

## 2 | MATERIAL AND METHODS

First, the DNA of five fish (P12, B127, Abl76, G100, G138) previously found positive by qPCR for S. destruens (Combe et al., 2022) was used to test the detection efficiency of previously published primer sets. In parallel DNA from 40 fishes collected from endemic areas in France, such as Corsica, the Camargue and the Indre regions, was used to test several primer combinations and validate the best one. In total, forty fish DNA samples corresponding to nine species were analysed (Table S1). A pure S. destruens DNA (RA-1 isolate, ATCC® 50643™) provided by the Laboratoire des Pyrénées et des Landes (LPL) was used as a positive control and DNA from parasite-free common carp brain cells (CCB) available at the ISEM laboratory was used as a negative control. The specificity of 10 primer combinations (Figure 1; Table S2) corresponds to pre-existing nested-PCR assays from Gozlan et al. (2005), Mendonca and Arkush (2004) and Spikmans et al. (2020) and targeting an 18S rRNA gene sequence was tested following the author's amplification protocols. Next, the nested-PCR products (PCR amplification products) were validated using a low-cost amplicon-based nanopore sequencing approach. Sequencing was performed on an Oxford Nanopore Technologies (Oxford, UK) MinION device with a runtime of 12h. Then, for each nested-PCR result, the bases of the raw FAST5 files with a run sequencing tag were recalled using the super-accurate model implemented in the ONT Guppy v5.0.7 basecaller. The subsequent bioinformatic analysis included only reads between 100 and 700bp. The size filtering step was performed by the *guppyplex* command implemented in the ARTIC pipeline (https://

github.com/artic-network/fieldbioinformatics). The filtered ampli-con reads were mapped to the 18S sequence (GenBank accession: AY267345.1) of *Sphaerothecum destruens* isolate BML using minimap2 v2.17 (Li, 2018). BAM files were indexed, sorted and filtered with SAMtools v1.10 (Li et al., 2009). BAM files were filtered using the option '-F' of Samtools view with the flag "4" to remove unmapped reads (Li et al., 2009). The resulting filtered BAM files were used as input to Medaka v1.0.3 (https://github.com/nanoporetech/medaka) to build a consensus for each sample. A multi-sequence alignment step was performed between the samples and the reference sequence using MAFFT (Katoh et al., 2019). After analysing the results, an optimized PCR assay was sought. The most conserved region was identified and a new primer set was designed with Primer3Plus (Untergasser et al., 2007). The performance of the new set was evaluated by running an in silico PCR on the last release of the SILVA databases (Quast et al., 2012) using TestPrime 1.0 (Klindworth et al., 2013). Thus, a new nested-PCR assay (Figure 1; Table S2) was developed and tested (for the detailed protocol, see https://www.protocols.io/private/D04B5 C6A909711EC84BC0A58A9FEAC02).

## 3 | RESULTS

All the published primer combinations tested in this study generated multiple band profiles indicating possible nonspecific co-amplifications (Figure S1). Sequencing analyses of the PCR amplicons confirmed the hypothesis of the lack of specificity of the primers. For each combination tested, only 0.00069% to 5.45% (Table 1) of the reads mapped to the 18S ribosomal sequence of S. destruens. In comparison, 1.57%, 76%, and 99.72% of the positive control (pure RA-1 isolate) reads were mapped to the targeted 18S sequence using the published primer combinations.

<figure><img src="image_4.png" /><figcaption>FIGURE 1 Primer combinations tested in this study. The sequence corresponds to *Sphaerothecum destruens* 18S rRNA gene. The origin of each PCR assay is indicated by the author's name: Goz = primers from Gozlan et al. (2005); Ark = primers from Mendonca and Arkush (2004); Spik = primers from Spikmans et al. (2020). Ch = newly designed primer (this study). The location on the 18S rRNA gene of the targeted region is represented by coloured lines and arrows: F, forward primer; R, reverse primer; 1: first PCR; 2: nested-PCR. Dark colours correspond to published primer combinations. The light colours correspond to the published primers and to the new combinations tested in this study. The pink star marks the selected combination for optimized detection of S. destruens DNA.</figcaption></figure>
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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Cherif, E., Deremarque, T., Justy, F., Gozlan, R. E., & Combe, M. (2023). Assessing the specificity of the Rosette agent DNA amplification: An optimized protocol for the detection of standard DNA among studies. *Journal of Fish Diseases*, 46, 177-180. https://doi.org/10.1111/jfd.13722