PCR DETECTION, HISTOLOGICAL AND IN SITU HYBRIDIZATION OF Enterocytozoon hepatopenaei (EHP), A MICROSPORIDIAN PARATIES INFECTING PENAEID SHRIMP, IN INDONESIA  

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an emerging pathogen for penaeid shrimp. The economic losses attributed to EHP infection have been rapidly growing, and EHP is now considered to be a critical threat to shrimp aquaculture.  EHP has been found in shrimp farms throughout Asia including Thailand, Malaysia, Vietnam, China, and India. Here we describe the presence of EHP in farmed shrimp in Indonesia. EHP-infected shrimp can not be determined by simple visual inspection; there are no obvious clinical signs of infection. Diagnosis of this pathogen is accomplished through histological examination, in situ hybridization (ISH), and PCR analyses. Histological examination with H&E staining reveals the presence of basophilic inclusions within the cytoplasm of the hepatopancreas tubule epithelial cells (Fig 1A) of infected shrimp. These inclusions appear to be a plasmodium stage of the parasite, but mature, basophilic, spores are also present. To detect EHP in infected Penaeus vannamei tissue sections through ISH, an 18S rRNA gene fragment labeled with digoxigenin is used as in situ hybridization probe, with the probe reacting intensely to the basophilic inclusions within the cytoplasm (Fig 1B). The hepatopancreas is the only tissue that reacts to the EHP probe, with the associated lesions corresponding to those detected by H&E histology. For PCR diagnosis, we apply a PCR assay from the 18S rRNA gene region and detected EHP in hepatopancreatic tissues of infected shrimp. The PCR amplified products were sequenced to confirm that they are from EHP. The ability to rapidly and correctly diagnose EHP-infection can aid in preventing the rapid spread of this parasite.