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

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.

From the basics to emerging diagnostic technologies: What is on the horizon for tilapia disease diagnostics?

“…RT-qPCR or qPCR procedures have been developed and optimized for the detection and quantification of viral or bacterial loads in infected tilapia. Target pathogens include TiLV, 55,56,88 ISKNV, 89 TiPV, 74 S. agalactiae 64,90 and F. orientalis. [91][92][93] Several multiplex TaqMan qPCR assays have also been developed to detect and quantify three to four pathogen species in a single PCR test, such as F. orientalis, S. iniae and S. agalactiae 94 ; A. hydrophila, A. veronii and A. schubertii 95 ; and E. ictaluri, E. tarda, E. anguillarum and E. piscicida.…”

“…191,200 The application of eDNA/eRNA for tilapia disease diagnosis is still limited, however, a straightforward approach for TiLV detection and quantification from water that employed a simple iron flocculation method for viral concentration coupled with a probe-based RT-qPCR has been described. 55 TiLV nucleic acid was detected and quantified in water collected from affected ponds/cages as well as sewage, and a reservoir. This approach might be effective for noninvasive monitoring of TiLV in aquaculture environments, and allow suitable biosecurity interventions.…”

“…This approach might be effective for noninvasive monitoring of TiLV in aquaculture environments, and allow suitable biosecurity interventions. 201 Potential applications of eDNA/eRNA in disease diagnosis have also been described in other fish species and their pathogens, including the use of pathogen-specific detection approaches, metabarcoding strategies and a combination of both. For instance, the detection and quantification of red sea bream iridovirus (RSIV) in a challenge model with Japanese amberjack (Seriola quinqueradiata) and farmed red sea bream (Pagrus major) revealed high viral loads at least 5 days before fish mortality, suggesting potential application of eDNA assay for early forecast of disease.…”

“…For instance, the detection and quantification of red sea bream iridovirus (RSIV) in a challenge model with Japanese amberjack (Seriola quinqueradiata) and farmed red sea bream (Pagrus major) revealed high viral loads at least 5 days before fish mortality, suggesting potential application of eDNA assay for early forecast of disease. 55,202,203 Multiple target pathogens were detected using eDNA samples collected from Atlantic salmon (Salmo salar) farm sites to assess the potential of pathogen transmission from domesticated to wild fish populations sharing the same habitat. 204 The use of universal metabarcoding markers (e.g., mitochondrial genes, internal transcribed spacer (ITS) sequences and small-subunit ribosomal RNA gene) as potential monitoring tools for harmful parasites and microalgae in cultured fish have been described.…”

Detection of Fish Pathogens in Freshwater Aquaculture Using eDNA Methods: A Review

“…There are mainly two different forms of virus that infect fish species which are either DNA virus or the RNA virus. Several studies have shown that DNA and RNA from virus can be detected using eDNA or eRNA method (Minamoto et al 2009;Haramoto et al 2007;Kawato et al 2021;Taengphu et al 2021;Vilaca et al 2020;Miaud et al 2019;Weli et al 2021;Bernhardt et al 2020). Since DNA is more stable than RNA, detection of eDNA is more practical and easier than detecting RNA from an environmental source.…”