Assessing the effect of probiotics on tilapia lake virus-infected tilapia: Transmission and immune response.

Probiotics have been used to alleviate disease transmission in aquaculture. However, there are limited studies on probiotic use in modulating tilapia lake virus (TiLV). We assessed commercially available probiotic supplements used in TiLV-infected tilapia and performed mortality and cohabitation assays. We developed a mechanistic approach to predict dose-response interactions of probiotic effects on mortality and immune gene response. We used a susceptible-infected-mortality disease model to assess key epidemiological parameters such as transmission rate and basic reproduction number (R0 ) based on our viral load dynamic data. We found that the most marked benefits of probiotics are significantly associated with immune system enhancements (~30%) and reductions in disease transmission (~80%) and R0 (~70%) in tilapia populations, resulting in a higher tolerance of farming densities (~400 fold) in aquaculture. These findings provide early insights as to how probiotic use-related factors may influence TiLV transmission and the immune responses in TiLV-infected tilapia. Our study facilitates understanding the mode of action of probiotics in disease containment and predicting better probiotic dosages in diet and supplements to achieve the optimal culturing conditions. Overall, our analysis assures that further study of rationally designed and targeted probiotics, or mechanistic modelling is warranted on the basis of promising early data of this approach.

Persistence of Tilapia tilapinevirus in fish rearing and environmental water and its ability to infect cell line.

Tilapia tilapinevirus, or Tilapia Lake Virus (TiLV), is a RNA virus associated with mass morbidity and mortality in tilapia, leading to severe economic losses for global tilapia aquaculture. In this study, we investigated the persistence of TiLV in water by spiking sterile distilled water (SDW), freshwater collected from rearing fish tanks (FW) and natural pond water (PW) at 27°C as a representative of environmental water conditions with 0.6 ml of stock virus (3.18 × 107 viral copies/ml of water). The water samples were filtered through an electronegative charge membrane and quantified using reverse transcriptase quantitative PCR at 0, 3, 5, 7, 10 and 14 days post-inoculation. The results revealed that TiLV RNA in SDW was reduced by 1.34 log10 in 14 days. A similar approximately 4 log10 removal of the virus in FW and PW was observed at 3 and 7 days, respectively. Moreover, the infectivity of TiLV was further studied; the virus lost its infectivity in E-11 cells after 1 day in SDW, FW and PW water samples, even though the virus was spiked 10 more times than in the viral persistence study. Taken together, the results could be applied to improving biosecurity practices in tilapia farms by disinfecting or resting reservoir water for at least three to five days prior to stocking tilapia, to limit the spread of TiLV.

Development and application of TaqMan probe-based quantitative PCR assays for the detection of tilapia parvovirus.

Tilapia parvovirus (TiPV) is a novel parvovirus associated with high mortality in Nile tilapia and red hybrid tilapia, leading to severe economic losses for tilapia aquaculture. It is critical to develop a sensitive and accurate assay to detect TiPV in fish tissues. In this study, new TaqMan probe-based quantitative PCR (qPCR) assays targeting the non-structural (NS) and viral protein (VP) genes of TiPV were developed. The standard curves of the assays were 95.64%-98.96% over a wide linear range of 109 -101 copies of the corresponding standard DNA per reaction. The intra- and inter-assay coefficients of variation were in the ranges 0.54%-2.50% and 0.13%-1.17%, respectively, which suggests good repeatability and reproducibility. The detection limit of the TaqMan TiPV assays was 10 copies/µl. The application of the TaqMan qPCR assays to field samples revealed that they had comparable sensitivity to a previously developed SYBR Green qPCR, but more sensitive than the conventional PCR. No cross-reactivity of the TaqMan TiPV assays was found with the samples infected with other viruses and bacteria. Overall, the assays offered high sensitivity and specificity in the detection of low concentrations of TiPV DNA in infected tilapia samples. These new TaqMan qPCR assays could provide a valuable diagnostic tool for the reliable and specific detection of TiPV in experimental and field samples.

Weight-dependent susceptibility of tilapia to tilapia lake virus infection.

The emergence of tilapia lake virus (TiLV) has had a severely negative impact on global tilapia aquaculture. TiLV infection has been reported at different life stages of tilapia, with more emphasis on fry and fingerlings; however, the virulence and pathology of TiLV at different tilapia size remains unexplored. In this study, tilapias from a single spawning were raised to 5 g, 25 g, and 65 g, and subsequently challenged by the intraperitoneal injection and cohabitation of a virulent strain of TiLV. The cumulative mortality, viral load, and histopathology of the fish were determined until 22 days post-infection (dpi). The cumulative mortality of the 5 g, 25 g, and 65 g fish was 85% (±1.67), 55% (±2.89), and 51.67% (±7.49), respectively. At 14 dpi, the mean TiLV load in the liver of the 5 g fish was significantly higher than in the 25 g and 65 g fish. All the weight groups showed severe pathological changes in the liver, spleen, and intestine after TiLV infection, but no particular difference was otherwise noted during the study with the exception of higher pathological scores in the liver of the small fish at 14 dpi. Overall, this study indicated that small fish are more susceptible to TiLV infection than large fish. Although multiple factors, including environmental factors, farm management practices, strains of virus could contribute to different susceptibility of fish to viral infection, the present study provides the evidence to support that fish weight affects the mortality and clinical outcome during TiLV infection. More intensive measures such as strict biosecurity and disease surveillance during the susceptible weight should therefore be emphasized to reduce the impact of this virus.

Uncovering the first occurrence of Tilapia parvovirus in Thailand in tilapia during co-infection with Tilapia tilapinevirus.

The recently discovered Tilapia parvovirus (TiPV) was the first Parvovirus confirmed to infect fish, causing mortality outbreaks in farmed adult Nile tilapia in China. Severe mortality outbreaks caused by Tilapia tilapinevirus (TiLV) to farmed tilapia in Thailand revealed the concomitant occurrence of TiPV. Out of ten fish farms screened, TiPV was detected in one site rearing juvenile red hybrid tilapia. Clinical signs included abnormal swimming, scale protrusion, skin and muscle haemorrhaging, exophthalmia and generalized anaemia. Histological findings showed extensive infiltration of lymphocytes, with increased melanomacrophage centres in the anterior kidney and spleen, erythrocyte depletion in the spleen and hepatic syncytial cells. Both TiLV and TiPV were systemically distributed in the body of moribund fish. The analysis of the near-complete TiPV genome isolated from Thailand revealed 98.74% sequence identity to the formerly isolated from China, together with a highly conserved and comparable genomic organization and with a 3 nucleotides deletion in the 5-UTR. The viral genome structure was highly conserved for each of its components, with nucleotide and amino acid identity ranging from 100% for ORF1 to 97% for ORF2, and with conserved HuH and Walker loop motifs within NS1. Taken together, our results document the first detection of TiPV outside China, thus for the first time in Thailand. Moreover, TiPV was detected for the first time during a natural occurrence in farmed red hybrid tilapia and involved in co-infection pattern with TiLV. Diagnostic investigations during tilapia disease outbreaks should include the screening for TiPV. Further studies are needed to elucidate TiPV genomic variance, pathobiology, including focussing on the outcomes of TiLV-TiPV co-infection patterns, necessary to enable risk assessment for the worldwide spreading of TiPV and to design adequate control measures against these emerging viruses in tilapia.

Diagnostic sensitivity of pooled samples for the detection of tilapia lake virus and application to the estimation of within-farm prevalence.

Tilapia lake virus (TiLV) is a highly contagious novel orthomyxo-like RNA virus that is negatively impacting tilapia production worldwide. To prevent TiLV from spreading globally, the infection status of source farms needs to be established prior to the movement of live tilapia to minimize the risk of horizontal transmission. However, testing individual fish for TiLV requires large sample sizes, when within-farm prevalence is low and is costly, time-consuming, and labour-intensive. The objective of the present study was to evaluate the use of pool testing for TiLV detection and to estimate within-farm prevalence based on the percentage of positive pooled samples. Pooled samples of liver and spleen were prepared by diluting different numbers of positive tissue samples with negative homogenate tissue samples. A tissue pool from 5 or 10 individual fish containing at least one TiLV-positive sample was sufficient to yield a positive result except when cycle threshold (Ct) values were between 31 and the cut-off value of 34. Additionally, our study characterized viral load in two farms after TiLV outbreaks. Bayesian modelling showed that within-farm prevalence could be estimated from the percentage of positive pools of size 5 using prior information about pool sensitivity and specificity, and prevalence, and assuming random sampling of tilapia from infected ponds. Ninety-five percent posterior intervals for prevalence were slightly wider than those obtained based on the results of individual samples. Findings in the present study corroborate the use of a pooling strategy for post-outbreak surveillance of TiLV.

Evidence of potential vertical transmission of tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease in tilapia with worldwide distribution. Although the horizontal transmission of TiLV has been demonstrated through the cohabitation of infected fish with susceptible fish, no direct experiment showed the potential of vertical transmission from broodstock to progeny. In this study, natural outbreaks of TiLV in broodstock and fry in two tilapia hatcheries were confirmed. The TiLV genomic RNA was detected in liver and reproductive organs of infected broodstock, while infective virus was isolated in susceptible cell line. In situ hybridization assay confirmed the presence of TiLV in the ovary and testis of naturally infected fish and experimentally challenged fish. Moreover, early detection of TiLV in 2-day-old fry and the presence of TiLV genomic RNA and viable virus in the testis and ovary suggested the possible transfer of this virus from infected broodstock to progenies. As infective virus was present in gonads and fry in natural outbreak and experimental fish, the importance of biosecurity and prevention of the virus to establish in the hatchery should be emphasized. Hence, the development of TiLV-free broodstock and the maintenance of high biosecurity standards in the hatcheries are essential for any attempt of virus eradication.