Bioassay for spore polar tube extrusion of shrimp Enterocytozoon hepatopenaei (EHP)

The microsporidian Enterocytozoon hepatopenaei (EHP) is an intracellular spore-forming parasite, which has recently become a serious threat of cultivated penaeid shrimp in Asia. These studies were aimed to generate comprehensive study on EHP infection as well as establishing innovative strategies to reduce its viability and potential infectivity in shrimp farms. The EHP spore purification method by using Percoll gradient separation with ultracentrifugation step at 15 °C has been developed. The viability of the purified spores has been evaluated by its polar tube extrusion with Phloxin B staining. The active EHP spores were exposed to different temperature (−20 °C, 4 °C and 33 °C) and chemical treatments including calcium hypochlorite, formalin, potassium permanganate (KMnO4) and ethanol to identify the conditions that can be used to inhibit the extrusion of the polar tube. Complete inhibition of activity was demonstrated either by freezing the spores at −20 °C for at least 2 h or by treating them with chemicals. The chemicals that yielded 100% inhibition were 15 ppm KMnO4 for 15 min, 40 ppm of 65% active chlorine for 15 min or 10 ppm of 65% active chlorine for 24 h and 20% ethanol for 15 min. However 200 ppm formalin resulted in a maximum reduction of 95.33%. Taken together, our protocol demonstrates for the first time that living EHP spores can be isolated and purified, providing a potential platform for future testing and development of EHP's control strategies.