Pre-fired microsporidian spores lose their infectivity in shrimp

Shrimp industry has been negatively impacted by growth retardation and size variation, which are caused by an infectious disease called hepatopancreatic microsporidiosis (HPM). The causative agent of HPM is a microsporidian Ecytonucleospora (Enterocytozoon) hepatopenaei or EHP. EHP invades shrimp hepatopancreatic cells by a spore stage, which can germinate when the spore encounters suitable conditions. During the spore germination, a specialized organelle called the polar tube extrudes from the spore and pierces the host cell membrane. Recently, our lab has identified that potassium hydrogen phthalate (KHP) and sodium hydrogen phthalate (NaHP) are able to induce the EHP spore germination, in vitro. We hypothesized that if the spores are germinated prior to encountering shrimp, the spores would lose their infectivity. To test this hypothesis, we pre-germinated spores with optimal concentrations of KHP, NaHP, and phloxine B, which are 50 mM, 100 mM, and 2 %, respectively. Pre-germinated spores were immersed into the aquarium containing naive Penaeus vannamei (PL12 stage). The EHP spores in 1X PBS were used as a control. After 14 days of immersion, the EHP infections were tested by a nested PCR using a spore wall protein gene as a target. The results showed that all pre-germinated spore groups had no infection, while the infection rate was 60 % in the control group. These results were confirmed by histology. Staining the hepatopancreatic tissues using a nuclear staining dye showed positive EHP infection in the control group, but not in the pre-germinated spore groups. Our results suggest that pre-germinated EHP spores lose their infectivity in the laboratory setting. The use of KHP and NaHP as anti-EHP agents need to be further tested in the farm setting. The in vivo infection assay developed here could also be used as a pipeline to investigate new compounds that potentially decrease EHP spore infectivity.