CIBA-TM Series-2017-No.

Field guide for

diagnosis prevention and control of diseases of
shrimp and finfish
in brackishwater aquaculture

Aquatic Animal Health and Environment Division

Central Institute of Brackishwater Aquaculture,
75 Santhome High Road, R. A. Puram, Chennai 600 028
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Brackishwater Aquaculture for food, employment and prosperity

Published by
Dr. K.K. Vijayan
Director

Prepared and edited by

Subendu Kumar Otta, S.V. Alavandi and K.K. Vijayan

Cite as: Otta SK, Alavandi SV and Vijayan KK. Field guide for diagnosis, prevention and
control of diseases of shrimp and finfish in brackishwater aquaculture,
Central Institute of Brackishwater Aquaculture, 2017, 38 pp

DISCLAIMER
All rights reserved. No part of this publication may be reproduced, stored in a
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mechanical, recording or otherwise, without the prior written permission of
Director, CIBA.

The author and publisher are providing this book and its contents on an “as is”
basis and make no representations or warranties of any kind with respect to this
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current.

Except as specifically stated in this book, neither the author or publisher, nor any
authors, contributors, or other representatives will be liable for damages arising
out of or in connection with the use of this book.

2
Preface

I t is 20 years since CIBA published a bulletin on diagnosis and prevention and

control of disease shrimp in brackishwater aquaculture in India. Since then the
aquaculture scenario in the country has undergone paradigm shift with regard to
culture practices and issues faced in the farming sector. The finfish culture sector
still is under nascent phase despite breeding technologies, and indigenous feed
available. India’s shrimp production recorded a phenomenal rise from less than
1.0 lakh metric tonnes in 1995-96 to over 3.80 lakh tonnes during 2014-15 with the
total export value from marine and brackishwater sector reaching an all-time high
of over US $ 5000 million. Availability of imported specific pathogen free (SPF)
broodstock provided the much needed growth rate to India’s brackishwater
aquaculture sector. However, the intensification has exacerbated the epizootics and
disease issues are becoming a major constraint affecting productions. Starting from
1994, the annual loss to shrimp culture industry of India has been estimated to be
300 crores and this is due to a single causative agent, white spot syndrome virus
(WSSV). An assessment in 2009 by CIBA indicated a disease related loss to the tune
of over 48717 metric tons of production valued at 1022 crores.

During the initial phase of brackishwater aquaculture development, which was

traditional low stocking density culture, it was only the non-infectious (nutritional/tox-
in) or less dangerously endemic pathogen (bacteria, parasites) related diseases were
prevalent. During during late 80’s, diseases such as soft shell syndrome of shrimp
in traditional aquaculture ponds of Kerala and West Bengal and epizootic ulcerative
syndrome (EUS) of finish were plaguing our brackish water sector. In the initial years
of semi-intensive brackishwater aquaculture development, bacterial diseases such
as vibriosis caused limited problems in grow-out aquaculture. Since 1994 white spot
disease (WSD) has been devastating our shrimp farming sector even today. Many
brackishwater shrimp farmers shifted to scampi farming in brackishwater ponds,
which also suffered setbacks due to white tail disease (WTD). Later on since 1998, the
black tiger shrimp farming continued to face challenges such as loose shell syndrome
(LSS), followed by the monodon slow growth syndrome (MSGS).

After introduction of the exotic Pacific white shrimp, Penaeus vannamei,

following import risk analysis (IRA), the aquaculture sector in India has been booming.
However, in addition to the already widespread WSD, the sector is being challenged
with several uncharacterized disease syndromes. During the last five years, issues of
concern in shrimp farming include stunted growth or growth variation, white faeces
syndrome (WFS) low level daily mortalities (popularly called by shrimp farmers as
running mortality syndrome or RMS) and white muscle syndrome (WMS). Stunted
growth and WFS are often associated with the hepatopancratic microsporidiosis caused
by Enterocytozooan hepatopenaei (EHP).In this context the present publication
has been brought out now and is largely intended as a field guide for presumptive
identification of diseases.

K.K. Vijayan

3
 Content
1. Introduction 05
2. OIE Listed diseases of Shrimp 07
i. White spot disease 07
ii. Infectious hypodermal and haematopoietic necrosis 09
iii. Taura syndrome 11
iv. Yellow head disease 13
v. Infectious myonecrosis 14
vi. Acute hepatopancreatic necrosis disease 16
vii. Necrotising hepatopancreatitis 17

3. Diseases prevalent in brackishwater aquaculture in India 19

3.1 Shrimp diseases 19
i.
Hepatopancreatic microsporidiosis 19
ii.
White faeces syndrome 20
iii.
Running (chronic) mortality syndrome 21
iv.
Vibriosis 22
v.
Black gill disease 25
vi.
Protozoan fouling 26

3.2 F
infish diseases 27
i.
Iridovirus infection 27
ii.
Viral nervous necrosis 28

4. Level 1 Diagnosis of diseases of shrimp and finfish 30

5. Disease Prevention and Control in Brackishwater aquaculture 32

6. Anatomy of shrimp and fish and sampling for disease investigation 34

7. CIBA’s Referral laboratory services 38

4
 1. INTRODUCTION

Brackishwater aquaculture has been playing a very crores to the Indian aquaculture sector. Besides,
significant role in terms of its contribution to the global other important infectious agents including viruses,
seafood supply. From the ancient traditional status, bacteria and parasites have been responsible for
the industry has evolved to the present developed issues related to slow growth, mortalities and crop
form through several scientific interventions and losses. In the recent past, emerging diseases such
thereby has been able to constantly increase its as early mortality syndrome, running mortality
share in terms of both production and value. During syndrome, white faces syndrome and zoea
2014-15, India’s seafood export reached all-time high syndrome etc. are added problems to the already
of about US $5000 million. More than 70% of this existing misery of farmers.
share was from farmed shrimp alone and thereby
Many of these problems can be prevented using
signifying the importance of shrimp aquaculture.
better management practices. Good pond prepara-
Brackishwater aquaculture is predominated by tion, maintenance of biosecurity, stocking ponds
shrimp. Several coastal states of India are involved with healthy disease-free seed, regular health and
in the culture practice of penaeid shrimps. Until water quality monitoring and judicious input appli-
2009, it was totally dominated by the indigenous cation including feed are some of the key factors
species, tiger shrimp, Penaeus monodon. Pacific that decide successful harvest.
white shrimp, Penaeus vannamei was introduced
In this booklet, a brief outline on important diseas-
into India for culture in 2009, and presently, more
es in brackishwater aquaculture, with special refer-
than 80% of farmed shrimp constitutes this exotic
ence to shrimp and finfish, the causative agents,
shrimp.
symptoms, mode of transmission, possible meth-
India also has a great potential for the culture of ods of prevention and control are provided for the
brackishwater fin fishes. Biology and breeding information of the shrimp farmers. This publica-
of some of the species such as sea bass, Cobia tion is also intended to be useful to beginners who
and Milk fish etc has been taken up with success wish to understand disease issues in brackishwater
and culture of these finfish species has also been aquaculture.
going on in certain areas. The potential areas for
Considering the expertise, resource and infrastruc-
brackishwater aquaculture in India has not yet been
ture required for disease diagnostics, FAO/NACA
exploited to full extent and thereby further scope for
(2000) has recommended the promotion of three
expansion of this industry is expected.
levels of diagnostics according to existing resourc-
A number of biotic and abiotic factors influence es (see Table 1). The different levels provide a broad-
aquaculture productivity. White spot disease (WSD), scale application to disease detection and diagnos-
caused by white spot syndrome virus (WSSV) has tics where countries can move from one level to the
often been the single most dreaded threat to the next as capacities are improved and as resources
aquaculture industry, with global losses exceeding become available.
US $6 billion and an estimated annual loss over `300

5
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Table 1. Three levels of disease detection and diagnostics

Level Site Activity Requirements

I Field Observation of ani- Little basic equipment or no equipment required. Training of field
mal and the environ- personnel on the basic biology and clinical examination and co-op-
ment Clinical exam- eration of culture-site managers/employees to access to infor-
ination mation would help in achieving preliminary understanding and to
some extent, diagnose the problem.
II Lab Parasitology, Bacte- Significant investment in skill development / training of technical
riology, Mycology, personnel, equipment and recurring costs. Access to current scien-
Histopathology tific information would enable technicians to achieve diagnosis of
the problem.
III Lab Virology, Electron mi- Considerable investment in equipment and in skill development /
croscopy, Molecular training of manpower required to effectively utilise equipment with
biology, Immunology considerable recurring costs. Access to current information in all
etc the activities required to achieve accurate diagnosis.

6
 2. OIE LISTED DISEASES OF SHRIMP

More than 20 viruses have been identified that are White Spot Syndrome (WSD)
known to infect penaeid shrimp. The OIE now lists
White spot disease (WSD) is the most serious threat
two bacterial diseases, the acute hepatopancreatic
faced by the shrimp farming industry worldwide.
necrosis disease (AHPND), necrotizing hepatopa-
WSD was first reported in farmed P. japonicus from
ncreatitis (NHP) and five viral diseases, white spot
Japan in 1992-93, but was thought to have been im-
disease (WSD), yellow head disease (YHD), Taura
ported with live infected post-larvae from mainland
syndrome (TS), infectious myonecrosis (IMN) and
China. WSD has been identified from crustaceans in
infectious hypodermal and haematopoietic necro-
China, Japan, Korea, South-East Asia, South Asia,
sis (IHHN) in the Aquatic Animal Health Code (OIE,
the Indian sub -continent, the Mediterranean, the
2015), which are considered to be transmissible and
Middle East, and the Americas. WSSV can infect a
of significant socio-economic importance. All OIE
wide range of aquatic crustaceans including marine,
member countries are obliged to report these dis-
brackish and freshwater penaeids, crabs and cray-
eases so that disease spread can be monitored and
fish. All decapod crustaceans including crabs, cray-
legislation instituted to prevent disease spread. IMN
fish, freshwater prawns, spiny lobsters and clawed
and TS have not affected shrimp aquaculture in In-
lobsters in marine, brackish and freshwater sourc-
dia. Although YHV has been reported from India in
es are susceptible, but morbidity and mortality as a
one instance in farmed black tiger shrimp (using his-
consequence of infection is highly variable. Penaeid
topathological techniques), its economic impact was
shrimp species are highly susceptible to infection,
negligible. All these diseases are known to cause
often resulting in high mortality. Prevalence of WSSV
considerable loss to farmed P. vannamei.
is reported as highly variable, from <1% in infected
wild populations to up to 100% in captive populations.

7
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Fig 1a. Shrimp affected with white spot disease showing typical white spot on the carapace of black tiger shrimp and
vannamei shrimp

What is the causative agent of WSD? and rapid mortalities. White spots may not be always
seen in the early stages of infection in shrimp. WSSV
WSD is caused by a double-stranded DNA virus of can be detected using polymerase chain reaction
120-150 x 270-290 nm size, assigned to a new virus (PCR), or with molecular tools such as dot-blot and in
family, whispoviridae. situ hybridisation (ISH) tests. WSD can be also con-
firmed histologically (particularly in asymptomatic
What are the symptoms of WSD?
carriers) by the presence of large numbers of Cow-
The virus severely damages the stomach, gills, an- dry type-A nuclear inclusions and hypertrophied nu-
tennal gland, heart and eyes. WSSV affects organs clei in haematoxylin and eosine (H&E)-stained tissue
of ectodermal and mesodermal origin and in later sections, or simply by rapid fixation and H&E stain-
stages of infection, many cells get lysed and the ing of gill tissue.
organs are destroyed. Affected shrimp are lethargic
How is WSD transmitted?
show reddish body discolouration and during the
advanced stage of disease, characteristic 1-2 mm di- WSD can be transmitted vertically and horizontally
ameter white spots could be seen on carapace, ap- by cannibalism, predation, etc. and by water-borne
pendages and inside surfaces. Cumulative mortality routes. Dead and moribund animals can be a source
typically reaches 100 percent within three to seven of disease transmission. Outbreaks are usually trig-
days of infection. gered by latent carriers due to environmental chang-
es, such as osmotic stress induced through changes
How WSD is Diagnosed?
in salinity or hardness or rapid fluctuations in tem-
WSD may be diagnosed based on gross signs such perature.
as the presence of the characteristic white spots,

8
How WSD can be prevented / controlled? tion of eggs and nauplii may prevent vertical trans-
mission of WSSV from infected broodstock to larval
So far, no measures are known for controlling WSD stages. Live feed including polychates, should be
in aquaculture. Pathogen exclusion or biosecurity ensured to be free from WSD before using in the
and adoption of best management practices (BMPs) hatchery. Aquafarms should provide reservoirs and
are the only means of prevention of WSD. Specific disinfect water by chlorination prior to use in the
pathogen free (SPF) broodstock only need be used farms. All effluent from farms or processing plants
in the hatcheries. Hatcheries should quarantine should be disinfected with formalin or chlorine pri-
broodstock and screen for WSSV by PCR before or to discharge to avoid transmission of disease to
breeding. PL should also be screened for WSD by other farms.
PCR before stocking ponds. Washing and disinfec-

Fig.1 b Histopathological demonstration of basophilic hypertrophied nuclei in gill and diagnosis of WSD by PCR

Infectious Hypodermal and Haematopoietic Necrosis

Infectious hypodermal and haematopoietic necro- P. vannamei culture in the Americas during the
sis (IHHN) was first discovered in P. vannamei and 1990s.
P. stylirostris in the Americas in the year 1981, pos-
What is the Causative agent of IHHN?
sibly introduced along with live P. monodon from
Asia. IHHN probably existed for some time in Asia IHHN is caused by a small (20-22 nm) sin-
without detection due to its insignificant effects on gle-stranded DNA-containing parvovirus.
P. monodon, the major cultured species in Asia. Re- What are the Symptoms of IHHN?
cent studies have revealed geographic variations in
Gross signs of disease are not specific to IHHN, but
infectious hypodermal and haematopoietic necro-
may include reduced feeding, elevated morbidity
sis virus (IHHNV) isolates, and suggested that the
and mortality rates, fouling by epicommensals and
Philippines was the source of the original infection
bluish body coloration. Larvae, post-larvae (PL) and
in Hawaii, and subsequently in most shrimp farm-
broodstock rarely show symptoms. In P. vannamei,
ing areas of Latin America. Large-scale epizootics
IHHNV can cause runt deformity syndrome (RDS),
were responsible for multi-million dollar losses in

9
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Fig 2. IHHN infection in shrimp showing rostrum (source: http://bioaqua.vn/en/the-infectious-hypodermal-and-haema-

topoietic-necrosis-virus-a-brief-review-of-what-we-do-and-do-not-know/) and abdominal deformity characteristic of
runt deformity syndrome (RDS)

10
which typically results in cuticular deformities How IHHN is transmitted?
(particularly bent rostrums), slow growth, poor food
Transmission of IHHN is known to occur rapidly
conversion ratio (FCR) and a greater size variation
by cannibalism of weak or moribund shrimp,
at harvest, contributing substantially to reduction in
cohabitation and waterborne route. Vertical
profits. These effects are typically more pronounced
transmission from broodstock to larvae is common
when the shrimp are infected at larval stages. IHHN
and has been shown to originate from the ovaries
typically causes no problems for P. monodon since
of infected females. Insects and birds have been
they have developed a tolerance to it over a long
shown to act as mechanical carriers of IHHN and
period of time, but they may occasionally suffer with
may also transmit the disease.
RDS. P. merguiensis and P. indicus appear refractory
to the IHHNV. How IHHN can be prevented / controlled?

How is IHHN Diagnosed? Strict hatchery biosecurity including screening out

broodstock by PCR, or the use of SPF broodstock,
IHHN can be diagnosed by histological
washing and disinfecting of eggs and nauplii are
demonstration of intracellular Cowdry type A
useful in combating this disease. IHHNV is reported
inclusion bodies in ectodermal and mesodermal
to be highly resistant to all the common methods of
tissues such as cuticular epithelium, gills, foregut,
disinfection including chlorine, lime and formalin.
hind gut, lymphoid organ and connective tissues
One of the big problems with IHHNV is its eradication
of H&E-stained sections. Sensitive specific and
in infected facilities. Complete eradication of all
rapid methods such as PCR, dot blot, ISH are also
stocks, complete disinfection of the culture facility
available for IHHN diagnosis.
and avoidance of restocking with IHHNV-positive
animals may bring down incidences of IHHNV
infections.

Taura Syndrome
Taura Syndrome (TS) was first identified in shrimp prone to mutations causing more concern.
farms around the Taura River in Ecuador in 1992 and
What are the symptoms of TS?
the disease spread rapidly to the whole of Latin and
North America within three years. Subsequently, TSV infections occur in juvenile shrimp (0.1-1.5 g body
TS was also reported from Asia including Mainland weight) within two to four weeks of stocking ponds
China and Taiwan (from 1999), and in late 2003 and occur largely within the period of a single moult
in Thailand, probably through the regional and cycle. In the acute phase of the disease, during pre-
international transfer of live PL and broodstock of moult stage, the shrimp are weak, soft-shelled, have
P. vannamei. TS is so far not reported from India. empty gut and diffuse expanded chromatophores
that appear red, particularly in the tail (hence the
What is the causative agent of TS?
common name - red tail disease). Such animals
Initial work suggested that TS was caused by a toxic will usually die during moulting (5-95 percent).
pesticide. However, it is now known that a single or Adult shrimp are known to be more resistant than
perhaps several very closely related mutant strains juveniles. Shrimp that survive infection show signs
of the Taura syndrome virus (TSV) are responsible of recovery and enter the chronic phase of the
for the TS. TSV is a single stranded RNA virus of 32 disease. Such shrimp show multiple, randomly
nm size, non-enveloped icosahedrons and more distributed, irregular, pitted, melanised lesions of

11
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Fig 3. Juvenile, pond-reared P.vannamei showing melanized foci mark sites of resolving cuticular epithelium necrosis (left)
and tail fan showing reddish discoloration and rough edges of the cuticular epithelium in the uropods suggestive of focal
necrosis (right) due to TSV infection (Pictures from Bondad-Reantaso et al, 2001)

the cuticle. These gross lesions will persist, but may Recently it has been shown that mechanical transfer
be lost during moulting, and the shrimp thereafter through insect and avian vectors is likely route
appear normal. of infection. Shrimp-eating seagulls can transmit
TSV through their faeces. Hence birds are likely to
How is TS Diagnosed?
transmit TSV.
TS can be diagnosed using standard histological
How to prevent / control TS?
and molecular methods of detection. Reverse-
transcriptase PCR (RT-PCR) assay is commonly used. The disease can be prevented by use of SPF
Specific DNA probes applied to ISH assays with broodstock in hatcheries and stocking ponds with
paraffin sections and histological demonstration seed tested to be free of TS. Precaution should be
of enlarged lymphoid organs (LO) with multiple LO taken to prevent reintroduction of the virus from
spheroids and multifocal areas of necrosis in the nearby facilities, wild shrimp and carriers. Other
cuticular epithelium of the general body surface, methods suggested for controlling the virus include
appendages, gills, hindgut, and foregut (the BMPs and maintenance of optimal environmental
oesophagus, anterior and posterior chambers of the conditions, weekly applications of hydrated lime
stomach) provide the confirmatory diagnosis (CaOH) at 50 kg/ha, polyculture with fish (to
consume dying and dead carriers). Infected stocks
How is TS transmitted?
must be totally destroyed and the culture facility
TSV is horizontally transmitted through infected must be disinfected.
animals, cohabitation and water borne routes.

12
Yellow head disease

Fig 4. Gross sign showing yellow cephalothorax of YHD in Penaeus monodon

(Picture from Bondad-Reantaso et al, 2001)

Yellow head disease (YHD) was the first major viral days of the first appearance of clinical signs. GAV
disease that caused extensive losses to black tiger has been reported to be associated with mortalities
shrimp farms in Thailand during 1990-91. YHD has of up to 80% in P. monodon ponds in Australia.
been reported in China, Taipei, Indonesia, Malaysia, YHV infections can cause swollen and light yellow
Philippines, Sri Lanka, Thailand and Vietnam. colored hepatopancreas in infected shrimp, and
a general pale appearance, before dying within
What is the causative agent of YHD?
a few hours. YHV affects tissues of ectodermal
YHD is caused by a rod-shaped enveloped virus of and mesodermal origin such as lymphoid organ,
40-60 nm by 150-200 nm size, containing single haemocytes, haematopoietic tissue, gill lamellae
stranded RNA, the yellow head virus (YHV). Less and spongy connective tissue of the subcutis, gut,
virulent related genotypes known as gill-associated antennal gland, gonads, nerve tracts and ganglia.
virus (GAV) are reported to be highly prevalent in
How is YHV Diagnosed?
farmed and wild populations in Australia, Asia, East
Africa and Mexico. YHD can be diagnosed using RT-PCR or with a probe
designed for dot-blot and ISH tests. It can also be
What are the symptoms of YHV?
diagnosed histologically in moribund shrimp by
YHD principally affects pond reared juvenile microscopic demonstration of intensely basophilic
stages of 5 -15 g. Affected shrimp typically feed inclusions in H&E stained sections of stomach or gill.
voraciously for two to three days and then stop
How is YHD transmitted?
feeding abruptly and are seen swimming near the
periphery of the pond. YHD can cause up to 100% The primary mechanism of spread of YHD is through
mortality in infected P. monodon ponds within 3-5 horizontal route, either water-borne or carrier

13
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

organisms, cohabitation and mechanical means. How to prevent / control YHD?

YHV is reported to remains viable in aerated
YHD eradication in ponds is similar to that for other
seawater for up to three days. Other shrimp such
viruses and involves practicing BMPs that include
as P. merguiensis, P. indicus, Metapenaeus ensis,
pond preparation by disinfection and elimination
Palaemon styliferus and Acetes spp. may become
of carriers, chlorination of reservoir water, filtering
infected and act as carriers. Other crustaceans,
inlet water with fine screens, avoidance of live feeds,
such as Macrobrachium rosenbergii and many crab
maintenance of stable environmental conditions,
species and Artemia appear to be refractive to YHD.
disinfection of infected ponds before discharge, and
Infected broodstock can pass on the virus to larvae
routine monitoring.
in the maturation/hatchery facilities if thorough
disinfection protocols are not strictly adhered to.

Infectious Myonecrosis (IMN)

Infectious myonecrosis was first detected in Brazil become necrotic and reddened similar to the colour
during 2004 in P. vannamei and then in Indonesia in of cooked shrimp. Juveniles and sub-adults of P.
2006. To date, IMN has been detected in East Java, vannamei, farmed in marine or low salinity brackish
Bali, and West Nusa Tenggara provinces. water, appear to be the most severely affected by
IMN disease. The principal target tissues for IMNV
What is the causative agent of IMN?
include the striated muscles, connective tissues,
IMN is caused by a putative totivirus. IMNV particles haemocytes and the lymphoid organ parenchymal
are icosahedral in shape and 40 nm in diameter. cells. Severely affected shrimp become moribund
What are the symptoms of IMN? and mortalities can be instantaneously high and
continue for several days. Mortalities from IMN
IMN disease causes significant mortality in grow out
range from 40 to 70% in cultivated P. vannamei, and
ponds and is characterised by acute onset of gross
FCR of infected populations increase from normal
signs including focal to extensive whitish necrotic
values of ~ 1.5 to 4.0 or higher.
areas in the striated muscle, especially of the distal
abdominal segments and the tail fan, which may

14
Fig 5. P. vannamei affected with IMNV showing whitish necrotic areas of the distal abdominal segments,
diagnosis of IMNV by RT-PCR

How is IMN Diagnosed? eggs) to progeny is also likely to occur. IMNV may
also be transmitted among farms by feces of sea-
IMN can be confirmed by histopathology using rou-
birds or shrimp carcasses. Outbreaks of IMN with
tine H&E stained paraffin sections and demonstrat-
sudden high mortalities may follow stressful events
ing characteristic coagulative necrosis of striated
such as capture by cast-net, feeding, sudden chang-
skeletal muscle fibers, often with marked oedema
es in salinity or temperature, etc., in early juvenile,
among affected muscle fibers. IMN may be also rap-
juvenile, or adult P. vannamei in regions where IMNV
idly diagnosed using a nested RT-PCR. Published
is enzootic.
methods available are ISH, nested RT-PCR and re-
al-time RT-PCR for the molecular detection of IMNV. How IMN can be prevented /controlled?

How is IMN transmitted? IMN can be prevented using SPF broodstock and
practicing BMPs. No effective therapeutants have
IMNV has been demonstrated to be trans-
been reported for IMN.
mitted through cannibalism. Transmission via wa-
ter and vertical transmission from broodstock
(trans-ovarian or by contamination of the spawn

15
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND) rence of EMS in 2009 was initially ignored by most
earlier known as early mortality syndrome (EMS) or farmers. But in 2011, outbreaks became more serious
acute hepatopancreatic necrosis syndrome (AH- especially in farms with culture history of more than
PNS) has been causing significant losses in shrimp five years and those closer to the sea using high sa-
farms in China, Vietnam, Malaysia and Thailand line water. Shrimp farming in Hainan, Guangdong,
since 2009. The disease affects both black tiger Fujian and Guangxi suffered during the first half of
shrimp and Pacific white shrimp and is character- 2011 with almost 80% losses. In Malaysia, EMS was
ised by mass mortalities during the first 20-30 days first reported in mid-2010 in the east coast of penin-
of stocking. In Vietnam, the disease was observed sular states of Pahang and Johor. Thailand also suf-
since 2010, but the most widespread devastation fered serious setback due to AHPND.
due to EMS was reported since March 2011 in the
Mekong Delta (South Vietnam). In China, the occur-

Fig 6. Gross clinical signs of Penaeus vannamei affected by acute hepatopancreatic necrosis disease (left) compared
to normal shrimp (right). Source: AHPND disease card (http://www.enaca.org/publications/health/disease-cards/ah-
pnd-disease-card-2014.pdf) and Loc Tran (2014)

What is the causative agent of AHPND? hepatopancres (HP), which may be often pale to
white due to pigment loss, sometimes with visible
The disease is caused by a unique strain of Vibrio
black spots or streaks, which does not squash
parahaemolyticus, that can produce toxins respon-
easily between the thumb and forefinger could be
sible for the primary pathology in affected shrimp.
used for presumptive diagnosis in cases of shrimp
What are the symptoms of AHPND? mortality starting as early as 10 days post-stocking.
The clinical signs such as significant atrophy of the Progressive degeneration and dysfunction of the

16
HP tubule epithelial cells progressing from proximal and Penaeus vannamei are known to be susceptible.
to distal ends of HP tubules and its degenerative Infected live shrimp and fresh (never frozen) shrimp
pathology suggested of a toxic etiology. tissues can effectively transmit the disease to
“clean” shrimp
How to Diagnose AHPND?
How to prevent / control AHPND?
AHPND can be diagnosed based on gross signs along
with histopathological examination and culture and AHPND can be prevented by use of SPF broodstock
isolation and of V. parahaemolyticus, supplemented and strict adoption of BMPs in hatcheries and grow-
by bioassay studies following the challenge tests. out farms. The practice of nursing post larvae to a
AHPND can be confirmed using PCR method (AP3 larger size before stocking into ponds has been
or AP 4 primers). Commercial diagnostic kits are also strongly encouraged. Nurseries have to be physically
available (e.g., IQ Plus POCKIT by M/s GeneReach separated from the grow-out area and very strict
Technologies, Taiwan) biosecurity measures have to be in place. Applying
disinfectant during pond preparation reduces the risk
How is AHPND transmitted?
of horizontal transfer. Pond sludge management is
Vibrios are ubiquitous organisms in marine another important strategy, as V. parahaemolyticus
ecosystem and transmission of AHPND through oral can persist in the organic matter that accumulates
route is most likely. AHPND has been transmitted in pond bottom. Affected ponds must be disinfected
experimentally by immersion. Transmission by before release of pond water into wild.
cohabitation is expected. P. monodon, P. chinensis

Necrotizing Hepatopancreatitis (NHP)

This disease is also known as Texas necrotizing intracellular Rickettsia-like bacterium, a member
hepatopancreatitis (TNHP), Texas pond of the order α-Proteobacteria (Gram-negative,
mortality syndrome (TPMS), Peru necrotizing pleomorphic, rod-shaped or helical-shaped
hepatopancreatitis (PNHP). NHP has been reported bacterium).
as an important disease since its first diagnosis in
What are the symptoms of NHP?
1985. It has been reported to cause mass mortalities
to the tune of 20-90 percent of P. vannamei in Affected shrimp are lethargic, anorexic with empty
highly saline commercial grow-out ponds nearly gut and show epibiotc fouling. Exoskeleton becomes
every year since then. By 1993, NHP spread to soft and show abdominal muscle atrophy. Affected
Ecuador, Guatemala, Honduras, Mexico, and Peru ponds have increased FCR and growth of affected
and by 1995, coincided with warm waters with high shrimp is retarded. The hepatopancreas becomes
salinity associated with El Nino, and caused severe watery with white or black streaks. Mortality rates
mortalities (60-80 percent mortality) of P. vannamei reach up to 90% within 30 days of the appearance
and P. stylirostris throughout Ecuador. NHP has not of clinical signs.
yet been reported in Asia, but could cause significant How NHP is diagnosed?
damage were it to be transferred here with untested
NHP can be diagnosed by microscopic demonstration
shrimp introduction.
of lipid droplets and melanisation of hepatopancreas
What is the causative agent of NHP? in wet mount of preparations. It may be confirmed by
Necrotizing hepatopancreatitis is caused by obligate histopathological examination showing atrophy and

17
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

the presence of granulomata in the hepatopancreas, How NHP can be prevented /controlled?
and haemocyte aggregations around the
Strict adoption of BMPs, use of SPF broodstock
hepatopancreatic tubules. Intracytoplasmic
and stocking NHP free seed in the farms is the best
Rickettsia-like bacteria may be prominently seen in
way to prevent NHP. Adhering to strict biosecurity
the cytoplasm. Molecular diagnostic tools such as in
protocols and practicing BMPs will be useful in
situ hybridization, dot blot hybridisation, and PCR for
preventing NHP.
specific α-Proteobacterial DNA are also available.

How is NHP transmitted?

NHP could be transmitted horizontally with

infected PL.

Fig 7. Juvenile P. vannamei affected with NHP showing markedly atrophied hepatopancreas (Picture from Bondad-
Reantaso et al, 2001)

18
 3. Diseases prevalent in brackishwater aquaculture in India

Among the diseases explained in the earlier section, It has been found that EHP can be transmitted
only two diseases, viz., WSD and IHHNV have been directly from shrimp to shrimp by cannibalism and
reported from Indian aquaculture sector. Other cohabitation. EHP is confined to tubule epithelial
OIE listed diseases have so far not been reported cells of the shrimp HP and shows no gross signs
in Indian aquaculture so far. However, during the of disease except retarded growth. It is urgent that
recent times, several shrimp diseases have emerged these possibilities be explored in order to improve
in Indian brackishwater aquaculture the cause of control measures. Although EHP does not appear
which are yet to be determined. Many of the farms to cause mortality in P. monodon and P. vannamei,
suffer serious morbidity and mortality of stock, information from shrimp farmers indicates that it
resulting in economic losses due to these diseases. is associated with severe growth retardation in P.
vannamei. The best approach for maturation and
3.1. Shrimp diseases
hatchery facilities to avoid EHP is not to use wild,
Hepatopancreatic microsporidiosis or captured, live animals (e.g., live polychaetes, clams,
Enterocytozoon hepatopenaei oysters, etc.) as feeds for broodstock. Better would
Hepatopancreatic microsporidiosis (HPM) is be pasteurization (heating at 70oC for 10 minutes).
caused by Enterocytozoon hepatopenaei (EHP). It Another alternative would be to use gamma
was first reported as an unnamed microsporidian irradiated frozen feeds. Alternatively, polychaetes
from growth retarded black tiger shrimp Penaeus could be selected and tested for freedom from
monodon from Thailand in 2004. It also has smaller shrimp pathogens and then reared as broodstock
spores (approximately 1 μm in length) and is currently feed in biosecure settings designed to maintain their
known to infect both P. monodon and P. vannamei. freedom from shrimp pathogens.

Fig 8. Shrimp, P. monodon and P. vannamei affected with growth retardation and thereby showing wide range of size
variation due to hepatopancreatic microsporidiosis,
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

White faeces syndrome shell syndrome. Microscopy of squash preparations of

gut or faecal strings reveal masses of vermiform bodies
White faeces syndrome (WFS) has been reported in that superficially resemble gregarines. Bacteriological
shrimp farms since last decade, however during recent studies indicate that total bacteria and Vibrio spp. are
times, it has become widely prevalent in P. vannamei significantly higher in haemolymph and intestine of WFS
farms throughout the shrimp farming countries. This affected shrimp. Six species of fungi (Aspergillus flavus,
disease has been reported from both cultured black tiger A. ochraceus, A. japonicus, Penicillium spp., Fusarium
shrimp and pacific white shrimp. White faeces syndrome spp., and Cladosporium cladosporioides) have been
usually occurs after 60 days of culture (DOC) and it isolated from shrimp naturally infected with white faeces
may be accompanied by high shrimp mortality. Ponds syndrome. Histopathological examination reveals diffused
affected with white faeces syndrome show white faecal haemocyte encapsulation and dilated hepatopancreatic
strings floating on the pond surface while the shrimps tubules accompanied by necrosis. Furthermore it has
show white/golden brown intestine, reduced feed been estimated that the Thai production losses due to
consumption, growth retardation and often associated WFS in 2010 was estimated to be of the order of 10–15%.
with loose shell. The disease can cause moderate to The cause of white faeces syndrome and treatment is
severe economic loss by reducing the shrimp survival by uncertain. However reduced stocking density, proper water
20–30 percent. WFS has been found to be associated with exchange together with better management practices will
presence of vermiform like gregarine bodies, vibriosis, be helpful in evading WFS.
Enterocytozoan hepatopenaei, blue green algae and loose

Fig 9. Shrimp showing white gut, white faecal strings

floating on pond surface, and aggregated, trans-
formed microvilli (ATM) in microscopic wet mount of
shrimp white faeces.

20
Running Mortality Syndrome (RMS) Vibrio azureus. The hepatopancreas was largely normal
as revealed by histological techniques. However, some
Running mortality syndrome (RMS) is named by shrimp
samples showed enlargement of nucleus and increased
farmers for continuous low-level mortalities during the
inter hepatopancreatic tubular space with haemocytic
culture period, resulting in low survival and productions.
infiltration. Muscle necrosis was indicated by haemocytic
The syndrome is widely prevalent in the vannamei farms
infiltration. Lymphoid organ (LO) tubules had constricted
since 2011 in Andhra Pradesh (AP) and Tamil Nadu (TN).
lumen. Bioassay experiments carried out by feeding RMS
Generally mortalities start after a month or 40 days of
affected shrimp tissue to healthy shrimp (13-14 g) did not
culture (DOC); a portion of shrimp continue to survive
elicit any disease in the experimental shrimp. Due to low
and can grow to fully harvestable size. Affected shrimp
survivability, the FCR becomes very high and thus the
show patches of whitish musculature in the abdominal
farmers face considerable loss. RMS affected shrimp
segments as a clinical sign. Investigations carried out at
showed recovery and appeared healthy and active after
CIBA have revealed no association with known shrimp
6 days of transferring to wet lab in water with optimal
viral infection. Shrimp from RMS affected ponds tested
parameters. Co-habitation experiment with healthy shrimp
negative for WSSV, IHHNV, IMNV, TSV, YHV, MBV, HPV
and the infected animals also failed to induce RMS. The
and PvNV. Bacteriological examination of haemolymph
study could not attribute any infectious aetiology to RMS.
samples of RMS affected shrimp indicated predominance
of Vibrio spp., such as Vibrio parahaemolyticus and

21
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Fig 10. a Clinical signs of running mortality syndrome: moribund shrimp swimming sluggishly in the edge of the pond,
dead floating shrimp collected from pond with net

22
Fig 10. b Shrimp showing muscle necrosis (white patches) and histological section showing necrosis of muscle tissue.

23
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Vibriosis What are the symptoms of Vibriosis?

Epizootics due to vibrio infections in occur in all General signs of vibriosis in shrimp include lethargic,
life stages of shrimp, but are more common in abnormal swimming behaviour, loss of appetite,
hatcheries. It is a severe systemic bacterial disease. red discoloration, brown gills, soft shell, atrophied
Major epizootics of vibriosis have been reported in hepatopancreas and necrosis of the sub cuticular
P. japonicus from Japan, P. monodon from the Indo- tissue in the tail and appendages region. In severely
Pacific region and P. vannamei from Ecuador, Peru, affected shrimp, the gill covers appear flared up and
Colombia and Central America. In Latin America eroded and extensively melanised black blisters
vibriosis is also called Sea Gull Syndrome or can be seen on the carapace/abdomen. Moribund
Sindroma de Gaviota. shrimp appear hypoxic and often come to the pond
surface or edge.
What is the causative agent of Vibriosis?
Symptoms of luminescent bacterial disease (LBD)
Vibriosis is caused by gram-negative bacteria in the
include lethargy, slow larval metamorphosis and
family Vibrionaceae such as V. harveyi, V mimicus,
body malformations, bioluminescence, muscle
V. splendidus, V. vulnificus, V. parahaemolyticus, V.
opacity, melanisation, empty midgut and anorexia.
alginolyticus.

Fig 11. Shrimp exhibiting signs of vibriosis: flared up branchiostegites (gill covers), blister on the branchiostegites, necrosis
of appendages, melanisation of carapace (black spots).

24
In Latin America V. harveyi is reported to cause pathogens, in heavily stocked culture systems,
Bolitas negricans in penaeid shrimps which is the Vibrio-related diseases spread rapidly. Stressful
spanish term for small ball and the diseased shrimp environment conditions like poor water quality,
show balled epidermal tissue blocking the digestive nutrition, improper handling, overcrowding and
tract. There will be high mortalities in PL’s and young parasitic infestations act as predisposing factors.
juveniles.
How to prevent / control vibriosis?
How is vibriosis diagnosed?
Strict adoption of BMPs, maintaining optimal stocking
Vibriosis can be diagnosed based on gross signs densities would help in preventing occurrence of
and confirmed by isolation of bacterial pathogen vibriosis. When cases of vibriosis are detected, use
from haemolymph by standard microbiological of medicated feeds such as oxytetracycline @ 1.5g/
methods.   Kg, fed at 2-10% of body weight for 10-14 days along
with proper water and pond management may be
How vibriosis is transmitted?
helpful. Antibiotics should be used with utmost
Vibrio species normal microflora of marine and care. Sufficient withdrawal period (about 25-30
brackishwater ecosystems and are widely distributed days) should be allowed for the antibiotic to become
in aquaculture facilities. Being opportunistic inactive or harmless. 

Black gill disease in shrimps. Presence of excessive levels of toxic

substances such as nitrite, ammonia, heavy metals,
Affected shrimps have gills with black to brown
crude oils etc. in the culture water may lead to black
discoloration, in acute cases necrosis and atrophy
gill disease. High organic load, heavy siltation and
of the gill lamellae may be apparent. The blackening
reducing conditions in rearing pond can also cause
is due to the deposition of melanin at sites of
this disease in shrimps. Infection with certain
massive haemocyte accumulation, followed by
bacterial, fungal and protozoan pathogens can also
dysfunction and destruction of gill processes. A
cause black gill condition in shrimp. Treatment of the
small percentage of shrimp population in ponds
black gill disease depends upon the cause of the
occasionally suffer with black gill disease. However,
disease. Preventive or corrective measure may be
during recent vannamei crops, incidences of black
adopted to avoid or reduce the biotic / abiotic factors
gill disease are on the rise. A number of abiotic and
in the rearing pond to control the disease condition.
biotic reasons have been attributed to the black gill

Fig 11. P. vannamei shrimp affected with

black gill disease.

25
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Protozoan fouling Zoothamnium, Epistylis, Acineta and Ephelota

are involved. Maintaining good water quality and
Affected shrimps are restless and their locomotion
reducing organic load and silt in water exchange
and respiratory functions are hampered. In
with good quality water would help in prevention of
heavily infected juvenile and adult shrimps, one
protozoan fouling.  Formalin of affected individuals
can observe fuzzy mat-like appearance due to
can be used for controlling protozoan infestation.
ciliate fouling.  Protozoans such as Vorticella,

Fig 12. Tiger shrimp affected with protozoan fouling

26
3.2. Finfish Diseases in India mortality in farmed red sea bream (Pagrus major)
and more than 30 other species of cultured marine
Considering the significance and recorded to be
fish belonging mainly to the orders Perciformes
present in India, description of only two viral diseas-
and Pleuronectiformes. The infection has also been
es affecting brackishwater finfish is described here.
detected in Asian sea bass. It affects all the stages
Iridovirus (Rana virus) infection of fish but the susceptibility of juveniles is generally
Iridovirus infection is a significant cause of higher than that in adults.

Fig 13. Iridovirus infection with typical skin lesions (Source: https://nas.er.usgs.gov/queries/greatlakes/FactSheet.
aspx?SpeciesID=2657&Potential=N&Type=0&HUCNumber=DHuron ) and histopathological changes

What is the causative agent of iridoviral disease? kidney, liver and intestine should be collected.
However, the spleen and/or kidney tissues are the
The disease is caused by double stranded DNA
most appropriate organ for pathogen detection by
virus of genera Lymphocystivirus and Ranavirus.
immunofluorescent antibody test (IFAT). The fish
Ranaviruses causes systemic disease in infected
sample should be stored at 4°C for use within 24
fish and are associated with high morbidity and
hours or -80°C for longer periods. The disease is
mortality.
characterised by the appearance of abnormally
What are the symptoms of iridoviral disease? enlarged cells stained deeply with Giemsa solution
Outbreaks of the disease have been mostly reported in the histopathological observations of the spleen,
in the summer season at water temperatures of heart, kidney, intestine and gill of infected fish.
25°C and above. Depending on host fish species, Electron microscopy confirms the presence of
fish age, water temperature, and other culture virions (200-240 nm in diameter) in these cells.
conditions, mortality rates ranges between 0 and PCR is able to detect Iridovirus infection with
100%. Affected fish become lethargic, exhibit severe high degree of sensitivity in short time. Recently
anaemia, petechiae of the gills, and enlargement of real time PCR has been developed which shown
the spleen. improved rapidity, sensitivity, reproducibility, and
the reduced risk of carry over contamination over
How is iridoviral disease diagnosed?
normal PCR. An antibody based enzyme-linked
The sample should be collected from moribund immunosorbent assay (ELISA) has also been
fish. Gill and visceral organs such as spleen, heart, developed to detect iridovirus infection.

27
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

How iridoviral disease is transmitted?

The principal mode of transmission of Iridovirus infection is horizontal via water. The vertical mode of
transmission has not yet been established.

How to prevent / control iridoviral disease?

Strict adoption of biosecurity protocols, screening fish prior to inducting them as broodstock in finfish
hatcheries and stocking screened seed in grow-out culture, strict adoption of BMPs help in preventing
occurrence of iridoviral disease.

Viral nervous necrosis

Viral nervous necrosis (VNN) also known as in cultured and wild population of brackishwater/
viral encephalopathy and retinopathy (VER) is a marine fish species such as Lates calcarifer, Mugil
devastating disease of marine fish species cultured cephalus, Chanos chanos and Epinephelus tauvina
worldwide. It affects more than 50 wild and cultured etc. VNN has also been reported from low saline and
marine fish species, especially the larval and freshwater environments.
juvenile stages which records high mortality. In
India, betanodavirus infection has been observed

Fig 14. Sea bass affected with VNN

28
What is the causative agent of viral nervous necrosis by electron microscopy, viral antigens or antibodies
disease? by serological methods such as indirect fluorescent
antibody test, and enzyme linked immune-sorbent
The disease is caused by piscine nodavirus of the assay or detection of viral nucleotides by molecular
genus betanodavirus of the family Nodaviridae. The techniques such as RT-PCR and by tissue culture are
virus contains single stranded, bipartite positive different ways for pathogen detection. The RT-PCR
sense RNA genome. targeting capsid protein gene sequence is the most
sensitive test and has become the main diagnostic
What are the symptoms of viral nervous necrosis
method for fish nodaviruses using blood, sperm, as
disease?
well as nervous and ovarian tissues.
The major clinical signs of VNN are common
How is viral nervous necrosis disease transmitted?
behavioural changes such as lack of appetite, erratic,
spiral or belly-up swimming and dark coloration of Betanodaviruses are quite resistant to
body. Clinically, the affected animals show spiral or environmental conditions, which make it possible
looping swim pattern, swim bladder hyperinflation to get translocated by commercial activities via
and later wasting. The severity of disease is more influent water, juvenile fish, utensils, vehicles, etc.
in juveniles with equal higher rate of mortality. In Translocation of species for stocking purpose from
sea bass, the earliest onset of clinical signs of the one location to another may be another way but it
disease is during 16-21 day post hatch. Vacuolation is yet to be validated. Latent infection among wild
is seen in the grey matter of brain and retina of eye. fishes also serves as source of infection. Apart
Necrosis is observed in the spinal cord, brain and from all these means of horizontal transmission,
retina while intra-cytoplasmic inclusion in nervous vertical transmission is highly suspected to take
cells. place from infected spawners to fry. Instances of
asymptomatic/ sub-clinical infection among wild
How is viral nervous necrosis disease diagnosed?
fishes may possibly act as potential carriers.
Viral nervous necrosis can be diagnosed by
demonstrating characteristic lesions in the brain
and/or retina by light microscopy. Detection of virions

29
 30
1

7
4

5
2

8
6

11
10
(CMS)
Vibriosis

ease (AHPND)
etic necrosis (IHHN)

Taura syndrome (TS)

White spot disease (WSD)

Yellow head disease (YHD)

Shrimp Diseases

Infectious myonecrosis (IMN)

White faeces syndrome (WFS)

Probable causative agent

Enterocytozoon hepatopenaei (EHP)

Acute hepatopancreatic necrosis dis-
Necrotising hepatopancreatitis (NHP)

Chronic (running) mortality syndrome

Hepatopancreatic microsporidiosis or
+
Infectious hypodermal and haematopoi- +

Exoskeletal deformities
+

Irregular, pitted, melanised lesions on

cuticle
+

Bent rostrums
+
+
+
+
+
+

+
+
Shrimp Lethargic
Table 1. Filed level Diagnostic table on diseases of shrimp

Black gills
+

+
+
+

+
+
Fouling by epicommensals
+

+
+

Black spots on exoskeleton

+
Flaring up of brancheostegites
+

+
+
Gathering at surface/edges of pond /tank
Bluish body coloration

+
+
+

+ Reddish discoloration

+
+
Soft-shell (exoskeleton)

+
+
Loose shell (exoskeleton)
+

White spots on exoskeleton

+
Hepatopancreas swollen and light yellow

+
+
Granular hepatopancreas

+
Empty gut

+
Whitish necrotic areas in distal abdominal
segments

+
Whitish midgut line

+
White faecal strands

+
+
+
+

+
Reduced feeding
4. Level 1 Diagnosis of table on diseases of shrimp and finfish

+
Shrimp feed voraciously for two to three
days and then stop feeding abruptly

+
+
Reduced growth/ stunted growth

+
Mortality within 30-40 days of stocking

+
+

+
Mass mortality - 70-100% within 3-10 days

+
+
+

+
+
Daily mortality

+
+
+
+

+
+
+
Low-level mortalities
Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture
 31
Vibriosis
Iridoviral disease
or Viral nervous necrosis (VNN)
Probable causative agent

Epizootic ulcerative syndrome (EUS)

Diseases of Finfish in India

Viral encephalopathy and retinopathy (VER)

+
+
+
+

lethargy
+

gill anaemia or haemorrhage

+
+

pop eye&/or ocular haemorrhage

Table 2. Filed level Diagnostic table on diseases of finfish

+
+
external haemorrhages
+
internal haemorrhages
+
whirling/corkscrew behaviour
eratic swimming/jumping from water/

+
scraping or other motor skill disfunction

+
+
skin/body lesions /ulcers
external growths or cysts

+
gathering at surface/edges of waterbody
swollen or colour change of internal
organs

+
+
(kidney/spleen/other hapatic)
secondary bacterial/fungal/parasitic

+
infection

+
ulceration /lesions in tissue

+
reduced feeding

+
+
mass mortality
 5. Disease Prevention and Control in Brackishwater aquaculture

A number of “component causes” (risk factors) starting with pond preparation to provide the aquat-
along with the “necessary cause” might become ic animal with a clean pond base and appropriate
“sufficient cause” to produce disease outbreaks. stable water quality. Before initiating a second crop,
It is extremely difficult to control disease once it the pond has to be prepared by drying, removing the
strikes and so far not successful stories have been organic matter accumulated from the earlier crop to
documented in treating diseases during aquacul- ensure sustained production. After the harvest, the
ture operation. Hence “prevention is always better pond bottom is allowed to dry and crack, primarily
than cure”. Disease prevention in brackish water to oxidize the accumulated organic components.
aquaculture and achieving sustainability of shrimp The pond bottom should be thoroughly dried to al-
farming largely depends on the implementation of low the soil should crack to a depth of 25 - 50 mm.
biosecurity principles and best management prac- Tilling pond enhances drying and accelerates de-
tices (BMPs) in the farms. BMP encompasses policy, composition of organic matter and oxidation. Where
regulatory and programme frameworks in response complete drying is not possible, formalin, potassium
to managing risks associated with diseases. BMPs permanganate, benzyl chromium chloride, provo-
are simple and practical but science based Interna- done iodine etc can be used disinfection. Liming of
tional Principles for Responsible Shrimp Farming aquaculture ponds is done to neutralize soil acidity
which covers farm management practices that in- and increase total alkalinity and total hardness con-
clude farm selection, farm design, water use, brood- centrations in water.
stock and post-larvae, feed management, health
Pathogen exclusion or biosecurity is an important
management, food safety and social responsibility.
means of prevention of dreaded diseases such as
BMPs minimize the potential of farm-raised fishery
WSD. Biosecurity is a broad concept and the appli-
products from being contaminated with pathogens,
cation of biosecurity concepts to shrimp aquacul-
chemicals, or unapproved or misused animal drugs.
ture will contribute significantly to reduce losses due
to diseases and make the sector more sustainable
Risk factors occur throughout the shrimp cropping
and environmentally responsible. Biosecurity means
cycle and include pond preparation, stocking densi-
“life protecting”, but its use appears to be restricted
ties, seed quality, water management, pond bottom
to issues related to preventing the introduction, es-
management, feed management and several others.
tablishment or spread of unwanted biological organ-
Effective management of these factors can help in
isms or agents. The principles of biosecurity should
reducing the risks of disease occurrence. BMP is the
be considered to keep the pathogen not only out of
ability to prevent losses to disease through effec-
the culture environment but also out of the country
tive elimination of pathogens and their carriers. The
and the region. Disinfection of aquaculture facilities
basic elements of a BMP programme in a shrimp
is a common disease management practice to en-
hatchery include the physical, chemical and biologi-
sure biosecurity. Methods for disinfection of aqua-
cal methods necessary to protect the hatchery from
culture establishments have been outlined in OIE
all diseases of high risk. The most important BMP for
Aquatic Manual. Following practice will help in en-
hatcheries would be use of specific pathogen free
suring biosecurity from WSSV.
(SPF) or high health (HH) shrimp broodstocks. BMPs
in aquaculture include a number of components

32
1. Source water: Ideally farms must have reservoirs and disinfect separately. Disinfect by evenly
of adequate capacity of seawater for operation distributing calcium hypochlorite to provide a
of hatchery or aquafarm. Source water should be minimum final free chlorine concentration of 10
filtered first through coarse screens to remove ppm within the entire system’s water. Allow the
larger aquatic animals and debris and then system to stand for a minimum of 24–48 hours
pumped into the supply/settling canal. Then, the at this minimal chlorine concentration by adding
water is passed through a series of progressively hypochlorite if required.
finer screens, through a fine mesh (150–250 μm
4. Disinfection of effluent water: Chlorinate (50 ppm
mesh size) bag screen before being introduced
chlorine) for 24-48h. Vigorously aerate reservoir at
into the reservoir.
least 48 h for de-chlorination to remove residual
2. Water should be chlorinated with appropriate chlorine. Alternatively, ozone treatment may be
dose of chlorine (10 ppm) to kill any potential carried out if available at levels of 0.08–1.0 mg per
vectors or carriers in the source water collected in litre to significantly reduce microbial load.
the reservoir. For one ha reservoir / pond of one
5. Following harvest after a crop, the deposits of
meter depth, 150-160 kg of calcium hypochlorite
organic debris in the pond bottom should be
providing 65% active chlorine would give a final
removed or treated, ploughed and tilled. Level
concentration of 10ppm. Personnel handling such
the ponds and ensure that there are no wet
chemicals should take precautions to protect
patches in the ponds, especially at the centre or
their skin and eyes. Since commercial bleach
near the sluice gates. All parts of ponds should
powders vary in active chlorine content, dosages
be thoroughly sun dried for at least three weeks.
need to be adjusted accordingly. Reservoir should
be vigorously aerated at least for 48 h to remove 6. Disinfection of dried earthen ponds can be further
residual chlorine. carried out with quicklime (calcium oxide) at a
rate of 4000–5000 kg per ha. Quicklime causes
3. Disinfection of WSD affected grow-out ponds:
desiccation / dehydration of organic matter.
Do not discharge water from WSD affected
ponds. Remove aeration devices and implements

33
 6. Anatomy of shrimp and fish and sampling for disease investigation

Anatomy of shrimp called uropods. On the lateral side, a pair of legs on

each of the first five abdominal segments, pleopods
Shrimp can be divided into two parts, head and
or swimming leg helps the shrimp to swim.
abdomen. The head region is called cephalothorax.
The head is covered by a hard cuticle called carapace. Major internal organs of shrimp include
The carapace contains spines and a large rostrum hapatopancreas, heart, digestive system, lymphoid
extending straight. Towards the upper end of head on organ, nerves and arteries. The hepatopancreas is
both sides, two compound eyes are present. Other an important part, which secretes digestive enzymes
important organs in the frontal part include antenna, and functions both as liver and pancreas. Heart of
antennule and maxilliped. The down part of the head shrimp contains hemolymph which becomes green
contains 5 pairs of walking legs called as periopods. in colour because of a copper containing pigment
The abdomin is divided into six segments. At the end called haemocyanin. Digestive system includes
of the abdominal segment, a apine like structure mouth, mandibles, cardiac stomach, pyloric
called telson is flanked by two fan like structures stomach, midgut, hindgut and anus.

Fig.15 a. General Morphology of shrimp

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 Fig.15 b. Anatomy of shrimp

Anatomy of Fish the  caudal fins, have no direct connection with the
spine. They are supported by the muscles which
The body of fish is divided into a head, trunk and tail,
compose the main part of the trunk. The heart has
although the divisions between the three are not al-
two chambers and pumps the blood through the re-
ways externally visible. The main skeletal element is
spiratory surfaces of the gills and on round the body
the vertebral column, composed of articulating ver-
in a single circulatory loop. The eyes are adapted for
tebrae. The ribs attach to the spine and there are
seeing underwater and have only local vision. There
no limbs or limb girdles. The main external features
is an inner ear but no external or middle ear.
of the fish, the fins, are composed of either bony or
soft spines called rays which, with the exception of

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Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

Fig.16. Morphology and anatomy of finfish

Sampling for disease investigation are essential. For investigating disease, moribund
sample (infected animals those are about to die) are
Accurate disease diagnosis is an important aspect best samples for diagnosis. However, individuals
for health management. Since disease investigation from the same pond may also be collected fixed
facilities may not be available in shrimp / fish appropriately and provided to the diagnostic
farming areas, it would be essential that the samples laboratory.
will have to be sent to diagnostic laboratories or the
referral laboratories for diagnosis of the diseases. For For shrimp, samples of pleopods or haemolymph
investigation of disease in aquaculture, background or gills are ideal testing for WSSV, IHHNV, TSV
information on the disease and appropriate samples and YHV. For testing IMNV, telson is preferred. For

36
testing hepatopancreatic viruses, AHPND and EHP, and shrimp diseases. Sampling should be carried
hepatopancreas, stomach, midgut etc need to be out in such a way as to provide the best possible
collected. Lymphoid organ is a preferred sample for likelihood that the sample will be representative
many of the shrimp viruses. Samples can be collected for the population (Table). Assumptions of 2%
either on alcohol, RNAlater for molecular diagnostics and 5% prevalences are most commonly used for
and in Davidson’s fixative for histopathological surveillance of presumed exotic pathogens, with a
examination. Proper sampling procedure is very 95% confidence limit).
much important for appropriate diagnosis of fish

Table 3. Sample sizes needed to detect at least one infected host in a population of a given size, at a given
prevalence of infection.

Population Prevalence (%)

size 0.5 1.0 2.0 3.0 4.0 5.0 10.0
50 46 46 46 37 37 29 20
100 93 93 76 61 50 43 23
250 192 156 110 75 62 49 25
500 314 223 127 88 67 54 26
1000 448 256 136 92 69 55 27
2500 512 279 142 95 71 56 27
5000 562 288 145 96 71 57 27
10000 579 292 146 96 72 57 27
100000 594 296 147 97 72 57 27
1000000 596 297 147 97 72 57 27
>10000000 600 300 150 100 75 60 30

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 7. CIBA’s Referral laboratory services

As a National referral laboratory for brackishwater aquatic animal diseases (NRLD), CIBA with its capacity in
diagnosing all OIE listed aquatic animal pathogens including emerging pathogens such as Acute Hepatopa-
ncreatic Necrosis Disease (AHPND) and Enterocytozoon hepatopenaei (EHP), has addressed several issues
in aquatic animal health sector. The NRLD of CIBA has been providing valuable services to various agencies
such as Animal Quarantine and Certification Services, Southern Region (AQCS-SR), Chennai, Aquatic Quar-
antine Facility (AQF), Rajiv Gandhi Centre for Aquaculture (RGCA), for screening live imported aquaculture
inputs such as Artemia cyst, exotic and specific pathogen free (SPF) P. vannamei brooders and PLs etc.

Table 4. Protocols used for the screening of shrimp pathogens in the National Referral Laboratory for Brack-
ishwater Aquatic Animal Diseases (NRLD) of CIBA

S.No. Shrimp and Fish Pathogens Reference

1. White Spot Syndrome Virus (WSSV) OIE*; Kimura et al, 1996
2. Infectious Hypodermal and Haematopoietic Necrosis Virus OIE
(IHHNV)
3 Monodon baculovirus OIE
4 Hepatopancreatic Parvo virus (HPV) OIE
5. Yellow Head Virus (YHV) OIE
6. Taura Syndrome Virus (TSV) OIE
7. Infectious Myonecrosis Virus (IMNV) OIE
8. Acute Hepatopancreatic Necrotic Disease (AHPND) Sirikharin et. al, 2014; Itsathitphaisarn
et al. 2016
9. Necrotising Hepatopancreatitis Bacteria (NHPB) IQ 2000 kit (Gene Reach)
10. Enterocytozoon hepatopenaei (EHP) Tangprasittipap et al. (2013)
11. Viral Nervous Necrosis (VNN) OIE
12. Iridovirus Jeong et al., 2006

*http://www.oie.int/international-standard-setting/aquatic-manual/access-online/

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Field guide for diagnosis, prevention and control of diseases of shrimp and finfish in brackishwater aquaculture

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