An Improved Microscopic Method for The Rapid Diagnosis of Emerging Microsporidian Parasite, Enterocytozoon hepatopenaei in Shrimp Farms

Penaeus (Litopenaeus) vannamei shrimp samples (hepatopancreas and faeces) collected from grow-out farms were evaluated for the presence of newly emerged microsporidian parasite Enterocytozoon hepatopenaei by concentration techniques using either water–ether sedimentation or Sheather’s sugar solution for spore recovery and subsequent microscopic detection by modified trichrome stain (Ryan-blue method). This improved selective staining protocol can easily resolve the identification of microsporidian spores in hepatopancreatic tubules compared to conventional stains. This method enables differential diagnosis of microsporidian spores by a characteristic staining pattern of pinkish-red, often with a belt-like diagonal stripe seen in the middle of the spore and a halo of unstained area at one end which could be easily distinguished from similarly staining particles or debris.