Enterocytozoon hepatopeneaei (EHP) is a microsporidial pathogen of farmed shrimp including Penaeus vannamei, P. monodon, and P. stylirostris. The fungus causes the disease Hepatopancreatic Microsporidiosis (HPM) which is listed as an emerg ing disease by the World Organization for Animal Health (WOAH, Paris, France) . Currently, methods for detecting or confirming EHP infection are primarily based on Hematoxylin and Eosin (H&E) histology, PCR -based diagnosis targeting the 18S rRNA gene and the spore wall protein gene, as well as in-situ hybridization (ISH) using a DIG-labelled 18S rRNA gene probe . We report the use of fluorescence in-situ hybridization (FISH) as a new diagnostic method for EHP detection . Due to the use of direct FISH procedures, this protocol is approximately 30% faster than the traditional ISH method.
To determine the feasibility of the newly developed FISH method for EHP detection, sections of Davidson’s-fixed, paraffin-embedded shrimp tissue from a n experimental EHP challenge study of P. vannamei was used. Specific-Pathogen-Free (SPF) tissue was used as a negative control , and a known EHP-infected tissue with at least Grade 3 infection level was used as a positive control . EHP detection was carried out using H&E histology , ISH using the 18S rRNA gene probe , and the newly developed FISH protocol. The FISH probes (EHP510 F/R) w ere labeled at the 5’ end with an Alexa Fluor 594 fluorophore (IDT) . EverBrite TrueBlack ® Hardset Mounting Medium with DAPI (Biotium) was used to provide a nuclear counterstain and reduce autofluorescence in tissue sections.
The EHP positive block was confirmed for the presence of EHP infection by traditional pathological analysis and ISH . The new FISH protocol was successful in detecting EHP in the hepatopancreas of the shrimp (see Figure 1). This new method will not only reduce turnaround times in EHP diagnosis, but also allow for detection of multiple pathogens in a single tissue section and the visualization of spatial relationships between pathogens in coinfections.