An improved microscopic method for the diagnosis of

Enterocytozoon hepatopenaei in shrimp farms

Hepatopancreatic microsporidiosis caused by Enterocytozoon hepatopenaei
(abbreviated as EHP), is an emerging parasite of penaeid shrimp in several
shrimp farming countries in Asia. Light microscopic examination of the
stained clinical smear is an inexpensive method of diagnosing microsporidian
infections even though it does not allow species level identification. An
improved microscopic method for the diagnosis of EHP in shrimp was
standardized by employing conventional concentration techniques for
parasitic stages by sedimentation (formalin-diethyl ether) or flotation
(Sheathers sugar solution) followed by a microsporidia-specific staining
technique by modified trichrome stain (Ryan-Blue formulation). The method
was found ideal for biological samples such as faeces, hepatopancreas and
pond sediments. Ryan-Blue stains the microsporidian spore pinkish-red due
to dye content chromotrope 2R and provides a good differentiation from the
lightly stained bluish background debris materials by aniline blue, so that the
ovoid and refractile spores stand out for easy visualization by light
microscopy. The method showed varying sensitivity depending upon the
level of infection in shrimp.

Figure 1. Spores of Enterocytozoon hepatopenaei from shrimp faeces after sedimentation, stained with
Chromotrope 2R stain

K. P. Jithendran
Principal scientist,
Central Institute of Brackishwater Aquaculture, Chennai-600 028