Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Suwimon Taengphu; Pattanapon Kayansamruaj; Yasuhiko Kawato; Jerome Delamare-Deboutteville; Chadag Vishnumurthy Mohan; Ha Thanh Dong; Saengchan Senapin

doi:10.7717/peerj.13157

Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

PeerJ. 2022 Apr 18:10:e13157. doi: 10.7717/peerj.13157. eCollection 2022.

Authors

Suwimon Taengphu¹, Pattanapon Kayansamruaj², Yasuhiko Kawato³, Jerome Delamare-Deboutteville⁴, Chadag Vishnumurthy Mohan⁴, Ha Thanh Dong⁵, Saengchan Senapin^{1

6}

Affiliations

¹ Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Mahidol University, Phayathai, Bangkok, Thailand.
² Center of Excellence in Aquatic Animal Health Management, Faculty of Fisheries, Kasetsart University, Chatuchak, Bangkok, Thailand.
³ Pathology Division, Nansei Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Minami-Ise, Mie, Japan.
⁴ WorldFish, Bayan Lepas, Penang, Malaysia.
⁵ School of Environment, Resources and Development, Asian Institute of Technology, Klong Luang, Pathum Thani, Thailand.
⁶ National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong Luang, Pathum Thani, Thailand.

Abstract

Background: Tilapia tilapinevirus, also known as tilapia lake virus (TiLV), is a significant virus that is responsible for the die-off of farmed tilapia across the globe. The detection and quantification of the virus using environmental RNA (eRNA) from pond water samples represents a potentially non-invasive and routine strategy for monitoring pathogens and early disease forecasting in aquaculture systems.

Methods: Here, we report a simple iron flocculation method for concentrating viruses in water, together with a newly-developed hydrolysis probe quantitative RT-qPCR method for the detection and quantification of TiLV.

Results: The RT-qPCR method designed to target a conserved region of the TiLV genome segment 9 has a detection limit of 10 viral copies per µL of template. The method had a 100% analytical specificity and sensitivity for TiLV. The optimized iron flocculation method was able to recover 16.11 ± 3.3% of the virus from water samples spiked with viral cultures. Tilapia and water samples were collected for use in the detection and quantification of TiLV disease during outbreaks in an open-caged river farming system and two earthen fish farms. TiLV was detected from both clinically sick and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms (i.e., river water samples from affected cages (8.50 × 10³ to 2.79 × 10⁵ copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 × 10³ to 3.53 × 10⁴ copies/L)). By contrast, TiLV was not detected in fish or water samples collected from two farms that had previously experienced TiLV outbreaks and from one farm that had never experienced a TiLV outbreak. In summary, this study suggests that the eRNA detection system using iron flocculation, coupled with probe based-RT-qPCR, is feasible for use in the concentration and quantification of TiLV from water. This approach may be useful for the non-invasive monitoring of TiLV in tilapia aquaculture systems and may support evidence-based decisions on biosecurity interventions needed.

Keywords: Environmental RNA; Iron flocculation; Quantification; RT-qPCR; TiLV; Tilapia lake virus; Viral concentration; eRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Fish Diseases* / diagnosis
Flocculation
RNA Viruses*
Tilapia*
Viruses*
Water

Substances

Water

Grants and funding

This research was conducted under the CGIAR Research Program on Fish Agri-Food Systems (FISH) led by WorldFish and as part of the CGIAR Initiative “Protecting human health through a One Health approach”, which are both supported by contributors to the CGIAR Trust Fund (https://www.cgiar.org/funders/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.