Shrimp injection with dsRNA targeting the microsporidian EHP polar tube protein reduces internal and external parasite amplification.

The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat to shrimp health worldwide. Severe EHP infections in shrimp cause growth retardation and increase susceptibility to opportunistic infections. EHP produces spores with a chitin wall that enables them to survive prolonged environmental exposure. Previous studies showed that polar tube extrusion is a prerequisite for EHP infection, such that inhibiting extrusion should prevent infection. Using a proteomic approach, polar tube protein 2 of EHP (EhPTP2) was found abundantly in protein extracts obtained from extruded spores. Using an immunofluorescent antibody against EhPTP2 for immunohistochemistry, extruded spores were found in the shrimp hepatopancreas (HP) and intestine, but not in the stomach. We hypothesized that presence of EhPTP2 might be required for successful EHP spore extrusion. To test this hypothesis, we injected EhPTP2-specific double-stranded RNA (dsRNA) and found that it significantly diminished EHP copy numbers in infected shrimp. This indicated reduced amplification of EHP-infected cells in the HP by spores released from previously infected cells. In addition, injection of the dsRNA into EHP-infected shrimp prior to their use in cohabitation with naïve shrimp significantly (p < 0.05) reduced the rate of EHP transmission to naïve shrimp. The results revealed that EhPTP2 plays a crucial role in the life cycle of EHP and that dsRNA targeting EHP mRNA can effectively reach the parasite developing in host cells. This approach is a model for future investigations to identify critical genes for EHP survival and spread as potential targets for preventative and therapeutic measures in shrimp.

A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.

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.