Genetic diversity and distribution of Tilapia lake virus in fish polyculture system in Bangladesh

Abstract

Tilapia is the world's second most important farmed fish species, following carp. Bangladesh is the world's fourth-largest tilapia producer and has largely adopted fish polyculture. Few research on factors associated with mortality and economic losses following such deaths have been undertaken in Bangladesh due of its perceived hardiness. 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 could be widespread, we surveyed 565 tilapia farms in 15 of Bangladesh's most important tilapia-producing districts using online tilapia epidemiology and health economics survey tool, followed by a surveillance program on tilapia grow-out farms and hatcheries in 10 districts of Bangladesh in 2017 and 2019. Furthermore, several unusual mortalities observed in species co-cultivated with TiLV-infected tilapia prompted us to examine whether any of the co-cultivated species would test positive for TiLV and whether they were susceptible to TiLV infection in controlled laboratory experiments. The survey examined a range of factors, including geographical factors, farmer characteristics, farm characteristics, stocking factors, biosecurity measures, and baseline – and unusual mortality levels and characteristics. A total of 18.2 % of farms reported having experienced unusual mortality, with an average mortality level of 23.2 %. Farm size, baseline mortality level, farmer concern about baseline mortality, and antibiotic treatment were all significantly associated with increased odds of reporting unusual mortality in a multivariable model. Similarly, in basic statistics, farming region, water source, dead fish removal frequency, and antibiotic treatment were all found to be significantly associated with the level of unusual mortality, where water source and dead fish removal frequency remained significant in the multivariable model. Based on the baseline and unusual mortality in tilapia, a total hidden loss of 875.7 million USD was estimated. Considering this unusual mortality, biological sampling was done through surveillance study where 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. Test result was confirmed through histopathology as an alternate method. 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. Additionally, using 183 samples obtained from 15 polyculture 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) were 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. To reduce the nationwide spread and severity of TiLV infection, we propose that TiLV-targeted surveillance activities continue to detect contaminated sources. 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 the new host(s).