Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) vannamei in India

Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, has emerged as a serious pathogen reported to be associated with retarded growth in cultured shrimp in many of the shrimp growing countries in Asia. As a part of ongoing disease surveillance among the farmed shrimp, we have investigated Penaeus (Litopenaeus) vannamei cultured in the south-east coast of India for EHP infection using light and scanning electron microscopy, histopathology, polymerase chain reaction (PCR) and in situ hybridisation. Squash preparation of hepatopancreas and white faecal strings showed large number of microsporidian spores. Spores under scanning electron microscope appeared oval and measured 1.7×1.0μm. Histology of infected animals showed severe degeneration of hepatopancreatic tubules. Basophilic inclusions resembling the developmental stages of EHP were found in the epithelial cells and large number of spore aggregations was observed in the tubular lumen. Enlargement of haemal sinuses and encapsulation of hepatopancreatic tubules were also observed in some cases. DNA extracted from hepatopancreas was subjected to PCR amplification using primers targeting microsporidian ssu rRNA gene. The PCR yielded an expected product of ~951bp and the sequences showed 100% identity with the EHP reported from Vietnam, Thailand and China. Further screening of field samples was carried out using nested PCR employing EHP-specific primers. Of the 137 juvenile P. vannamei samples tested, 10 were found to be positive in the first step and 77 in the nested PCR. Overall prevalence of EHP was estimated to be 63.5%. However, only first step PCR-positive samples showed discernible number of spores in the hepatopancreas under a light microscope. Post-larvae of P. vannamei collected from a hatchery were found to be PCR negative for EHP. In situ hybridisation using EHP-specific DIG-labelled probe showed positive signals in infected hepatopancreatic tissue. Animals collected from white faeces syndrome (WFS)-affected pond showed higher prevalence of EHP (96.4%) compared to those from the unaffected pond (39.7%). On the contrary, slow growing animals showed low prevalence (58.5%) compared to normally growing animals (80.8%). Although EHP could be detected from slow-growing as well as WFS-affected animals, the present study could not conclusively elucidate the association of EHP with these clinical signs through experimental infection trials. This report forms the first record of the emergence of EHP infection in cultured SPF P. vannamei in India.