Detection of dual infection of infectious myonecrosis virus (IMNV) and

Enterocytozoon hepatopenaei (EHP) in Penaeus vannamei farms

Infectious myonecrosis (IMN) is a viral disease of penaeid shrimp caused by infection

with infectious myonecrosis virus (IMNV) with symptoms of extensive white necrotic areas in
striated (skeletal) muscles, especially in the distal abdominal segments and tail fan, and redness
in some affected shrimp. Recently there have been some reports of IMNV from some farms in
the country and NSPAAD has alerted for targeted active surveillance in the country. A couple of
shrimp ponds (11 gm; DOC 53 days) in Nagapattinam with symptoms of redness in tail and pale
hepatopancreas (Fig. 1) was tested for IMNV by OIE primers and found positive by RT-PCR
(Fig. 2). Positive samples showed a single band on 2% agarose gel, estimated at 328 bp
representing the amplicon of IMNV, which upon sequencing gave 327 bp showing similarity
with other published sequences to the extent of 100%. All other OIE listed DNA (WSSV,
IHHNV) and RNA viruses (YHV, TSV) were not present; but positive for microsporidian
infection of Enterocytozoon hepatopenaei (EHP). Detailed history and clinical course was
monitored during the culture. The number of shrimp with white patches increased after a week
on check tray examination and shrimp started dying and floating in the centre of the pond. The
growth was not affected (1 g increment per week) and no mortality was reported until 76 DOC of
harvest. A re-visit of the affected area after a month showed similar symptoms in neighboring
ponds with IMNV at 60 DOC. Detailed histopathological study revealed necrosis and
myoliquifaction of muscles.

Fig. 1. The clinical signs of white patches, redness and pale hepatopancreas in shrimp showing IMNV
infection (Nagapattinam, TN).
 M S N P
st
1 step PCR

1000 bp

500 bp
328 bp

100 bp

M S N P

Nested PCR

1000 bp

500 bp

139 bp
100 bp

Lane M : 100bp DNA ladder

Lane S : Sample (P. vannamei, 53 DOC)
Lane N : (-) control
Lane P : (+) control

Fig. 2. Detection of infectious myonecrosis (IMNV) by RT-PCR using OIE primers (Farm 1,
Nagapattinam, TN). [Lanes S - Shrimp samples; N - Negative control and P - Positive control. Note that
the sample is positive by I step PCR]
 PCR result for the screening of IMNV

First step PCR Nested PCR

M 1 2 3 N P M 1 2 3 N P

1 kb
500 bp
328 bp
139 bp

Lanes M- DNA Marker

Lane 1-3shrimp samples (1-Nagapattinam, 2- Nellore 1, 3-Nellore 2)
Lane N-Negative control
Lane P- Positive control
Fig. 3. Electrophoresis in 2% agarose gel of products of IMNV by RT-PCR using OIE primers. Lanes 1-
3 Shrimp samples; N - Negative control and P - Positive control. Note that the sample 1 (Farm 2,
Nagapattinam) is positive for IMNV and 2 and 3 (Nellore) are negative for IMNV by nested PCR.
 st
1 step PCR

M 1 2 3 4 N P

1000 bp

500 bp
328 bp

100 bp

Nested PCR

M 1 2 3 4 N P

1000 bp

500 bp

139 bp
100 bp

Fig. 4. Electrophoresis in 2% agarose gel of products of IMNV by RT-PCR using OIE primers. Lanes 1-
4 Shrimp samples; N - Negative control and P - Positive control. Note that the sample number 1 and 4 are
positive by nested PCR.
Fig. 5. Histological section of shrimp muscle from abdominal segments showing coagulative necrosis and
haemocyte infiltration (40 x and 100 x, H&E stain)

K. P. Jithendran
February, 2017 CIBA, Chennai