Enterocytozoon hepatopenaei (EHP), the etiologic agent of Hepatopancreatic M icrosporidiosis (HPM) has emerged from an obscurity to the forefront of infectious diseases in shrimp aquaculture in just over ten years. As the disease spreads across Asia, and more recently in the western hemisphere, there is an urgent need to prevent its further spread and develop genetically resistant line of shrimp.
EHP is an obligate parasite and currently there is no in vitro culture method to propagate the parasite in a robust and reproducible manner . The iI nability to produce a large quantity of inoculum to carry out an experimental challenge is an impediment in screening for EHP resistance in shrimp, and in conducting many other laboratory experiments.
We describe here a simple yet robust method to generate a large quantity of EHP inoculum in approximately 30 days using live shrimp. This method involves directly injecting EHP inoculum into the hepatopancreas of Specific Pathogen Free shrimp (Penaeus vannamei) . At fifteen, thirty, and forty-five days post-injection, EHP-infected hepatopancreata were dissected and an aliquot of hepatopancreas tissue was taken to assess infection level by histopathology and quantitative PCR. The remaining tissues at each of the three time points were used to challenge SPF P. vannamei shrimp via feeding . Following feeding EHP-infected tissue, animals were sampled at 15, 30, and 45 days post-challenge with infection status determined by H&E histology and quantitative PCR. The data shows that feeding SPF P. vannamei with hepatopancreas tissue originally derived from EHP- injected P. vannamei will provide a large pool of infected tissue within 30 days. This tissue can then be prepared into inoculum and made available for experimental bioassay work. The method is robust enough and overcomes the bottleneck of EHP inoculum availability for screening resistance in shrimp. Until an in vitro culture method for EHP is developed , the method described can be used to generate a large quantity of EHP inoculum for an experimental bioassay.
Key words: Penaeus vannamei, EHP amplification