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 Sheathers sugar solution or sedimentation by formalin/diethyl ether method, followed
by a microsporidia-specific staining using modified trichome stain (Chromotrpe 2R) by Ryanblue 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. Jithendran
P. Ezhil Praveena
T. Bhuvaneswari
J. Joseph Sahaya Rajan
V. Jagadeesan
A. Navaneeth Krishnan
31-12.2015