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

Abstract

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is an important virus responsible for die-off of farmed tilapia globally. Detection and quantification of the virus from environmental DNA/RNA (eDNA/eRNA) using pond water represents a potential, noninvasive routine approach for pathogen monitoring and early disease forecasting in aquaculture systems. Here, we report a simple iron flocculation method for viral concentration from water combined with a newly developed hydrolysis probe quantitative RT-qPCR method for detection and quantification of TiLV. The RT-qPCR method targeting a conserved region of TiLV genome segment 9 has a detection limit of 10 viral copies per uL 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 virus from water samples spiked with viral cultures. During disease outbreak cases from an open-caged system and a closed hatchery system, both tilapia and water samples were collected for detection and quantification of TiLV. The results revealed that TiLV was detected from both clinically sick fish and asymptomatic fish. Most importantly, the virus was successfully detected from water samples collected from different locations in the affected farms e.g. river water samples from affected cages (8.50 x 102 to 2.79 x 104 copies/L) and fish-rearing water samples, sewage, and reservoir (4.29 x 102 to 3.53 x 103 copies/L) from affected and unaffected ponds of the hatchery. In summary, this study suggests that the eRNA detection system using iron flocculation coupled with probe based-RT-qPCR is feasible for concentration and quantification of TiLV from water. This approach might be useful for noninvasive monitoring of TiLV in tilapia aquaculture systems and facilitating appropriate decisions on biosecurity interventions needed.

Efficacy of heat‐killed and formalin‐killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia (Oreochromis niloticus)

“…Representative dead fish from each group were subjected for TiLV diagnosis using an in-house RT-qPCR (Taengphu et al, 2021).…”

Immunization of Nile Tilapia (Oreochromis niloticus) Broodstock with Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer

“…Fish were cultured in dechlorinated tap water and half of the water volume was renewed weekly. Prior to the experiment, blood samples were taken from ten randomly selected tilapia and tested for the presence of TiLV using RT -qPCR [29], and their TiLV-free status at the point of sampling was confirmed. All the animal experiments and procedures used in this study were ethically approved by the Kasetsart University Institutional Animal Care and Use Committee (ACKU62-FIS-008).…”

“…Clinically healthy tilapia juveniles (body weight 20.3 ± 6.7 g; length 10.9 ± 0.7 cm) were acclimated in dechlorinated tap water using 100-L tanks, with a density of 20 fish per tank. Prior to the experiment, five fish were randomly tested for the presence of TiLV using a RT-qPCR assay [29] and confirmed as negative. Prior to immunization, fish were anaesthetized using clove oil (100 ppm).…”

“…House-keeping gene elongation factor-1α (EF1α) was used as an internal control for the RT-qPCR. To quantify TiLV copy number, a standard curve was produced using ten-fold dilution of plasmid pSeg9-351 containing 351 bp of TiLV segment 9 open reading frame [29] (Figure S1).…”

Tilapia Lake Virus was not detected in non‐tilapine species within tilapia polyculture systems of Bangladesh

“…In addition to TiLV detection from field samples by semi-nested RT-PCR, TiLV in tissue samples from experimentally TiLV-challenged animals was detected using a newly developed quantitative one-step RT-qPCR protocol targeting TiLV genome segment 9 (Taengphu et al 2021).…”