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Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia

Senapin S, Dong HT, Meemetta W, Gangnonngiw W, Sangsuriya P, Vanichviriyakit R, et al.

J Fish Dis, 2019 Jan;42(1):119-127.
PMID: 30397913 DOI: 10.1111/jfd.12915

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2-1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density-gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral-like particles (~100-180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%-99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow-out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.
Fulltext Concentration and quantification of Tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR

Taengphu S, Kayansamruaj P, Kawato Y, Delamare-Deboutteville J, Mohan CV, Dong HT, et al.

PeerJ, 2022;10:e13157.
PMID: 35462762 DOI: 10.7717/peerj.13157

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.
Two-year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh

Debnath PP, Delamare-Deboutteville J, Jansen MD, Phiwsaiya K, Dalia A, Hasan MA, et al.

J Fish Dis, 2020 Nov;43(11):1381-1389.
PMID: 32851674 DOI: 10.1111/jfd.13235

Tilapia lake virus (TiLV) is an emerging pathogen in aquaculture, reportedly affecting farmed tilapia in 16 countries across multiple continents. Following an early warning in 2017 that TiLV might be widespread, we executed a surveillance programme on tilapia grow-out farms and hatcheries from 10 districts of Bangladesh in 2017 and 2019. Among farms experiencing unusual mortality, eight out of 11 farms tested positive for TiLV in 2017, and two out of seven tested positive in 2019. Investigation of asymptomatic broodstock collected from 16 tilapia hatcheries revealed that six hatcheries tested positive for TiLV. Representative samples subjected to histopathology confirmed pathognomonic lesions of syncytial hepatitis. We recovered three complete genomes of TiLV from infected fish, one from 2017 and two from 2019. Phylogenetic analyses based on both the concatenated coding sequences of 10 segments and only segment 1 consistently revealed that Bangladeshi TiLV isolates formed a unique cluster within Thai clade, suggesting a close genetic relation. In summary, this study revealed the circulation of TiLV in 10 farms and six hatcheries located in eight districts of Bangladesh. We recommend continuing TiLV-targeted surveillance efforts to identify contaminated sources to minimize the countrywide spread and severity of TiLV infection.
Fulltext Tilapia Lake Virus was not detected in non-tilapine species within tilapia polyculture systems of Bangladesh

Debnath PP, Dinh-Hung N, Taengphu S, Nguyen VV, Delamare-Deboutteville J, Senapin S, et al.

J Fish Dis, 2022 Jan;45(1):77-87.
PMID: 34580880 DOI: 10.1111/jfd.13537

Sixteen countries, including Bangladesh, have reported the presence of tilapia lake virus (TiLV), an emerging tilapia pathogen. Fish polyculture is a common farming practice in Bangladesh. Some unusual mortalities reported in species co-cultivated with TiLV-infected tilapia led us to investigate whether any of the co-cultivated species would also test positive for TiLV and whether they were susceptible to TiLV infection under controlled laboratory experiments. Using 183 samples obtained from 15 farms in six districts across Bangladesh, we determined that 20% of the farms tested positive for TiLV in tilapia, while 15 co-cultivated fish species and seven other invertebrates (e.g. insects and crustaceans) considered potential carriers all tested negative. Of the six representative fish species experimentally infected with TiLV, only Nile tilapia showed the typical clinical signs of the disease, with 70% mortality within 12 days. By contrast, four carp species and one catfish species challenged with TiLV showed no signs of TiLV infection. Challenged tilapia were confirmed as TiLV-positive by RT-qPCR, while challenged carp and walking catfish all tested negative. Overall, our field and laboratory findings indicate that species used in polycultures are not susceptible to TiLV. Although current evidence suggests that TiLV is likely host-specific to tilapia, targeted surveillance for TiLV in other fish species in polyculture systems should continue, in order to prepare for a possible future scenario where TiLV mutates and/or adapts to new host(s).
Fulltext A multiplexed RT-PCR assay for nanopore whole genome sequencing of Tilapia lake virus (TiLV)

Delamare-Deboutteville J, Meemetta W, Pimsannil K, Sangpo P, Gan HM, Mohan CV, et al.

Sci Rep, 2023 Nov 20;13(1):20276.
PMID: 37985860 DOI: 10.1038/s41598-023-47425-w

Tilapia lake virus (TiLV) is a highly contagious viral pathogen that affects tilapia, a globally significant and affordable source of fish protein. To prevent the introduction and spread of TiLV and its impact, there is an urgent need for increased surveillance, improved biosecurity measures, and continuous development of effective diagnostic and rapid sequencing methods. In this study, we have developed a multiplexed RT-PCR assay that can amplify all ten complete genomic segments of TiLV from various sources of isolation. The amplicons generated using this approach were immediately subjected to real-time sequencing on the Nanopore system. By using this approach, we have recovered and assembled 10 TiLV genomes from total RNA extracted from naturally TiLV-infected tilapia fish, concentrated tilapia rearing water, and cell culture. Our phylogenetic analysis, consisting of more than 36 TiLV genomes from both newly sequenced and publicly available TiLV genomes, provides new insights into the high genetic diversity of TiLV. This work is an essential steppingstone towards integrating rapid and real-time Nanopore-based amplicon sequencing into routine genomic surveillance of TiLV, as well as future vaccine development.
Rapid genotyping of tilapia lake virus (TiLV) using Nanopore sequencing

Delamare-Deboutteville J, Taengphu S, Gan HM, Kayansamruaj P, Debnath PP, Barnes A, et al.

J Fish Dis, 2021 Oct;44(10):1491-1502.
PMID: 34101853 DOI: 10.1111/jfd.13467

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid, accurate diagnosis and genotyping of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions, occasionally combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the lengthy turnaround time. Here, we report an innovative approach using a previously developed specific PCR assay for pathogen diagnosis combined with a new Oxford Nanopore Technologies (ONT)-based amplicon sequencing method for pathogen genotyping. Using fish clinical samples, we applied this approach for the rapid confirmation of PCR amplicon sequences identity and genotyping of tilapia lake virus (TiLV), a disease-causing virus affecting tilapia aquaculture globally. The consensus sequences obtained after polishing exhibit strikingly high identity to references derived by Illumina and Sanger methods (99.83%-100%). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low- and medium-income countries, for fast identification and genotyping of emerging infectious pathogens from field samples within a single day.