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 including Vietnam,

Thailand, Malaysia, Indonesia, China, and recently in India also. The disease is reported to be associated with

growth retardation and/or white faeces symptoms in farmed shrimp. EHP infections have been found in both

black tiger prawns Penaeus monodon and Pacific white shrimp Penaeus vannamei causing considerable

economic loss to shrimp aquaculture. Difficulty in confirmatory diagnosis of microsporidian (human) by

conventional parasitological techniques has been reported in literature primarily due to very small size of the

spores and non-specific staining of sample debris originated from faeces or tissues by conventional stains

such as Giemsa, H&E etc. EHP diagnosis is further complicated due to tiny spores (1.1x 0.7mm) in relatively

large volume of tissue/faecal debris in diagnostic samples. The problem is compounded when dealing with

very low infection, which again may warrant specialized hand.

To address these issues, we attempted a concentration and staining techniques in conventional

parasitology using field samples of hepatopancreatic microsporidiosis in shrimps. This method involves a

concentration of microsporidian spores either by floatation using Sheather’s sugar solution or sedimentation

by formalin/diethyl ether method, followed by a microsporidia-specific staining using modified trichome

stain (Chromotrpe 2R) by Ryan-blue method. The Ryan-blue method could resolve pink coloured spores

against bluish (stained debris) background and could detect light and heavy infections with ease by light

microscopy. The method is considerably specific, but exhibited varying sensitivity depending on the degree of

infection in samples. The protocol is simple, fast and may be used for routine diagnosis and monitoring of

EHP in faecal strings or hepatopancreas of shrimp and even pond sediments.

K. P. JithendranP. Ezhil PraveenaT. Bhuvaneswari

J. Joseph Sahaya RajanV. Jagadeesan

A. Navaneeth Krishnan 31-12.2015

1