A brief overview of infectious diseases impacting farmed shrimp and some experimental strategies on disease control

1. Infectious hypodermal and hematopoietic necrosis
virus (IHHNV)
Infectious hypodermal and hematopoietic necrosis virus
(IHHNV) or Penaeus stylirostris densovirus (PstDNV), was first
reported in 1981 in Hawaii affecting the species Litopenaeus
stylirostris and L. vannamei. This pathogen rapidly spread to
other countries in America (Mexico, Panama, Colombia, Ecuador
and Argentina), Asia (Indonesia, Malaysia, Philippines, Singapore
and Thailand) and French Polynesia. Its genome is organised
into three open reading frames (ORFs) encoding a non-structural
protein, an unknown protein and a capsid protein, respectively.
IHHNV causes infection to several shrimp species. It appears
that Penaeus monodon is not affected by IHHNV infection since
no clinical signs and no differences in size, weight or fertility
was found in IHHNV-positive animals. Clinical signs of IHHNV
infection depend on the species age and size, being the early
juvenile stages more susceptible to the disease.
In L. stylisrostris, acute IHHNV infection includes reduced
feeding and locomotion, erratic swimming and death. In L.
vannamei, acute IHHNV infection showed reduced growth
rate, marked size differences within a pond and deformity of
the rostrum, antennae and/or cuticle which is known as 'runt
deformity syndrome' (RDS). Histological lesions are Cowdry-
type A inclusion bodies in infected animals. This virus became
the main pathogen both in shrimp fisheries and aquaculture in the
1980s in Mexico.
It was estimated that its economical impact was between 0.5
and 1 billion US dollars. This virus is still present in wild and
farmed shrimp in Mexico and other countries.
Taura syndrome virus (TSV)
First reported in shrimp farms near Taura river, Ecuador in
1992, TSV soon spread to several countries in South, Central,
North America and Hawaii. Since 1999, TSV was also detected in
Asia (Taiwan, Thailand and Korea) which imported stocks of L.
INFECTIOUS DISEASES AND
CONTROL STRATEGIES IN SHRIMP
A brief overview of infectious diseases impacting farmed
shrimp and some experimental strategies on disease control
by César Marcial Escobedo-Bonilla, Instituto Politécnico Nacional - Centro Interdisciplinario
de Investigación para el Desarrollo Integral Regional (CIIDIR) Unidad Sinaloa
M
odern shrimp
aquaculture
began in 1933
in Japan with
the induced
spawning and
hatching of
Marsupenaeus
japonicus larvae.
This technology allowed the production of shrimp
larvae in hatcheries instead of using larvae from the
wild to stock grow-out ponds.
Shrimp farming is an important activity in
several low-income countries in Asia, America
and Africa as it generates employment and wealth.
Nonetheless, intensification of shrimp culture
increased the appearance of infectious diseases due
to deviations in environmental and physiological
factors.
Infectious diseases caused by viruses or bacteria
represent the biggest threat to development of
shrimp farming due to high mortalities. Pathogens
that have caused severe epizootics and high
mortalities to different stages and species of
shrimp include Baculoviruses, Parvo-like viruses,
Dicistrovirus, Ronivirus, Nimavirus and more
recently, a bacterium Vibrio parahaemolyticus
(Figure 1).
24 | January | February 2016 - International Aquafeed
FEATURE

2. vannamei from South America.
Its genome consists of a single, positive-sense RNA strand
around 10 kilobases long. It has two ORFs separated by a shorty
non-coding intergenic region. ORF 1 encodes a putative non-
structural polyprotein with several domains such as a helicase,
a protease and a RNA-dependent RNA polymerase; ORF 2
encodes three structural proteins VP2, VP1 and VP3.
In specific pathogen-free L. vannamei, larger animals are more
susceptible to infection and mortality than early juveniles. TSV
infection has three clinical stages. In the acute stage (three – five
days after onset of infection) animals display soft exoskeleton,
melanised multifocal necrosis and expanded chromatophores.
Here, animals become weak, have empty gut and often die (75 –
95 percent) during moulting. Cellular lesions include pyknosis,
karyorrhexis and necrosis in epithelia of cuticle, digestive tract,
gills, antennal gland and haematopoietic tissues.
The transition stage (four - eight days after onset of infection)
shows a reduction in cellular lesions and melanisation,
indicating the onset of the chronic phase. Here, surviving shrimp
(eight days after infection) show wound repair and regeneration
of epithelial tissues in affected organs. Mortality ceases and
SIZE 10
MAXUM
80
1600
NORGREN
52.19
[1325]
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BIN
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111.12 [2822]
18.00 [457]
1.93 [49]
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108.59
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36.91
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101.44
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88.00
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108.28
[2750]
278.03
[7062]
67.28
[1709]
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ET-261A.indd 1 2/12/15 4:52 PM
Figure 1 - (A) IHHNV, (B)TSV, (C) YHV, (D) WSSV and
(E) V. parahaemolyticus
B
C
D
E
A
International Aquafeed - January | February 2016 | 25
FEATURE

3. surviving shrimp molt shedding the necrotised cuticle.
Several shrimp species are susceptible to TSV infection except
for shrimp from the genus Farfantepenaeus (F. aztecus and F
duorarum) which seem to be resistant to TSV infection upon
experimental infection.
The economic impact of TSV during 1992 to 1996 was
estimated between 1.2 to 2 billion US dollars.
Yellow-head virus (YHV)
It appeared in 1992 in Thailand and later it spread to other
countries in Asia (Taiwan, Indonesia, Malaysia, China,
Philippines, India), Australia and America (USA and probably
Mexico).
YHV has up to six different genotypes including the gill-
associated virus (GAV) from Australia. Due to its morphology,
YHV first was thought to be a granulosis-like virus
(Baculoviridae). Later, its genome was found to be a RNA
molecule.
The YHV genome is a single linear (positive strand) RNA
molecule of 26652 nucleotides. It is organised into four distinct
ORFs. ORF1a has a 3C-like protease motif; ORF1b has a “SDD”
polymerase metal ion binding domain helicase. ORF2 encodes
putative nucleocapsid proteins (g7 and g2) and ORF3 encodes
putative surface glycoproteins (p18/20, p33 and g2.1). ORF4 is
very small and it has no known product.
Many shrimp species are susceptible to YHV. Clinical
signs include pale yellow body colouration, especially in
hepatopancreas and gills in P. monodon. Other clinical signs are
erratic swimming near pond shores and cumulative mortality
up to 100 percent within three - five days after onset of clinical
signs.
YHV causes systemic infection and replicates in organs such
as gills, foregut, lymphoid organ, connective tissues of nerves,
eyestalk, hepatopancreas and muscle. Cellular lesions include
pyknosis and kariorrhexis in epithelial cells in gills, connective
tissues and hematopoietic tissues.
The estimated losses caused by YHV from 1990 to 2007 are 500
million US dollars.
White spot syndrome virus (WSSV)
The virion is bacilliform, non-occluded, enveloped, with a tail-
like appendage at one end.
WSSV has one of the largest genomes (292 - 308 Kilobasepairs)
recorded for viruses. It contains up to 683 ORFs encoding
peptides from 51 to 6077 aminoacids representing 92 percent of
the total genome information.
WSSV was first recorded in Taiwan and soon after it spread to
several countries in Asia and America. It has a broad host range
including several penaeid shrimp, caridean shrimp, lobsters,
crayfish, crabs and other decapod crustaceans.
Clinical signs include white spots in the inner surface of cuticle,
probably formed by calcium carbonate accumulation due to
dysfunction of epithelial cells; reddish discoloration of the body
due to expansion of chromatophores; reduced feeding, lethargy
and delayed hemolymph clotting.
Cumulative mortality (100%) occurs three- ten days after onset
of clinical signs. Histopathology shows hypertrophied nuclei of
WSSV-infected tissues with intranuclear amphophilic inclusions
and marginated chromatin.
Since it first appeared in 1992 the economic impact of WSSV
on shrimp aquaculture is well over eight billion US dollars and
still is the most damaging viral pathogen for farmed shrimp
worldwide.
Bacterial infections are common in shrimp aquaculture. Many
diseases caused by Vibrio bacteria may kill wild and farmed
shrimp both from hatcheries and grow-out ponds. Worldwide,
several Vibrio species exist in the marine environment.
Recently, a novel disease known as early mortality syndrome
Figure 2 - Mortality curves from shrimp treated with (a) a diet
supplemented with Spirulina platensis, and (b) inoculated
with cidofovir. A slight delay in mortality was observed in the
Spirulina treatment, whereas a better antiviral effect was
shown in the cidofovir-treated shrimp. Nonetheless, either
of these treatments prevented WSSV infection or shrimp
mortality. From Rahman et al., (2006) Aquaculture 255: 600-
605.
Figure 3 - Mortality curves from shrimp treated by
hyperthermia. (a) shrimp inoculated with a low dose of WSSV
by oral route. (b) shrimp inoculated with a high dose of WSSV
by intramuscular route. In both experiments, the effect of
hyperthermia greatly reduced mortality in WSSV-infected
shrimp. From Rahman et al., (2006) Aquaculture. 261: 842-849.
26 | January | February 2016 - International Aquafeed
FEATURE

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Organised by the European Aquaculture society with the cooperation
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5. (EMS) or acute hepatopancreatic necrosis syndrome (AHPNS),
is caused by a singular type of Vibrio parahaemolyticus. This
bacterium contains an extrachromosomic plasmid (pVPA3-
1) encoding two toxins (PirA/PirB), related to Photorhabdus
luminiscens that are responsible for shrimp mortality.
EMS can produce high mortalities to affected shrimp early after
pond stocking (10 - 40 days). Surviving shrimp might undergo
stunting. EMS was first reported in 2009 in China and since
then, it has caused massive mortalities to farmed shrimp in Asian
countries such as Vietnam (2011), Thailand (2012) and Malaysia
(2012).
In 2013, EMS was also recorded in Mexico where it caused
severe production losses (up to 80 percent of total production in
Sinaloa, Sonora and Nayarit).
Several strategies have been developed and tested under
experimental conditions to tackle the negative impact of
infectious diseases caused by viruses or bacteria. These include:
Immunostimulants
Substances (peptidoglycans, β-glucans or lipopolysaccharides)
extracted from cell walls of bacteria (Bacillus sp.), fungi
(Saccharomyces cerevisiae, Schizophyllum commune), algae
(Sargassum polycystum) or herbs, which activate humoral
(antibacterial activity, agglutinins, cytokine-like factors,
modulators, clotting factors) and cellular (prophenoloxidase
system, encapsulation, nodule formation,
phagocytosis) defense responses in shrimp.
Experimental animals fed with
immunostimulants before or during
challenge with bacteria or viruses showed
reduced mortality compared to untreated
controls.
Antivirals
Substances from plants, algae or even
synthetic, have been tested in shrimp
with variable results. A diet containing
Spirulina platensis showed no antiviral
effect but only slightly delayed mortality
in WSSV-challenged shrimp. In contrast,
an extract from Indian Cynodon dactylon
supplemented to feed (2 percent), showed
100 percent protection upon a per os
WSSV infection.
An antiviral (bis [2-methylheptyl]
phthalate) extracted from the Indian plant Pongamia pinnata fed
before and during a per os WSSV challenge showed a dose-
dependent reduction of mortality (60 to 20 percent). A synthetic
antiviral (cidofovir) showed higher efficacy than the Spirulina-
supplemented diet to reduce and delay mortality of treated
shrimp. Nonetheless, cidofovir did not prevent WSSV infection
(figure 2).
Induction of a “quasi-immune” response and virus
neutralisation
This strategy is based on the rationale that some shrimp
surviving a virus outbreak may become resistant to a subsequent
pathogen infection. Therefore it indicates a sort of “memory” in
these animals.
Several studies evaluated the protective effect of inactive viral
particles or recombinant viral envelope proteins administered
to shrimp to prime their innate defense system. Results showed
reduced mortality of treated animals. In addition, monoclonal
or polyclonal antibodies directed against viral envelope proteins
have been used to inactivate viral particles through virus
neutralisation assays.
Three concentrations (10-1
, 10-2
and 10-3
) of a WSSV stock each
mixed with an equal volume of a purified monoclonal antibody
against WSSV VP28 showed a dose-dependent neutralisation
effect. Shrimp inoculated only with WSSV showed 100 percent
mortality at seven days post inoculation (dpi).
Shrimp treated with neutralised virus
concentrations 10-1
and 10-2
showed a slight delay
in time to mortality (100 percent at 11 dpi). Animals
treated with the 10-3
neutralised concentration
showed 20 percent mortality at 25 dpi.
Recombinant subunit peptides displayed 20 – 40
percent shrimp mortality depending on time of
WSSV challenge [3 to 21 days post treatment (dpt),
respectively]. Other experiments reported mortalities
between 48 percent with VP292 to 30 – 5 percent
with VP28 as these recombinant peptides were
administered twice during the experiments.
DNA vaccines
These tools are plasmids encoding WSSV envelope
proteins (VP15, VP28, VP35 and VP281). Shrimp
Figure 4 - Mortality curve from shrimp treated with dsRNA against WSSV
genes vp26 or vp28 using a high WSSV dose. RNAi against WSSV vp28 or
vp26 effectively reduced WSSV infection and shrimp mortality compared to
an unrelated dsRNA (LacZ) and controls. From Mejía-Ruíz et al., (2011) J Inv.
Pathol. 107: 65-68.
Low water
temperature is also
effective to inhibit
virus replication
in species living in
temperate or cold
water. In shrimp M.
japonicus, water
temperature at 15
ºC showed better
WSSV inhibition
than 33 ºC
28 | January | February 2016 - International Aquafeed
FEATURE

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International Aquafeed - January | February 2016 | 29
FEATURE

7. P. monodon treated with a vp28 DNA vaccine by intramuscular
route showed 10 percent mortality when challenged with WSSV
seven days post vaccination (dpv).
Mortality increased to 20, 80 and 95 percent when WSSV
challenge was done at 14, 25 and 50 dpv, respectively. A plasmid
containing WSSV vp28 gene and injected to WSSV-challenged P.
monodon showed 10, 24, 33 and 44 percent mortality at 7, 14, 21
and 30 dpv, respectively.
Oral delivery of a plasmid containing the WSSV gene vp28
expressed in attenuated S. typhimurium adsorbed into commercial
feed showed protection against WSSV challenge in crayfish
Cambarus clarkii. Crayfish mortality at 7, 15 and 25 dpv was 17,
33 and 43 percent, respectively.
Water temperature
Using high water temperature (hyperthermia) at 32 ºC before,
just after or even until 18 hours after WSSV inoculation reduced
virus replication and shrimp mortality (0 – 30 percent) compared
to controls at 27 ºC (100 percent) (figure 3). The route of WSSV
inoculation did not influence hyperthermia efficacy. Using
hyperthermia in alternate periods of 18 hours is still effective
against WSSV (0 – 40 percent mortality). Although hyperthermia
reduced virus replication in shrimp and crayfish, animals remain
infected as determined by PCR.
Low water temperature is also effective to inhibit virus replication
in species living in temperate or cold water. In shrimp M. japonicus,
water temperature at 15 ºC showed better WSSV inhibition than 33
ºC. Likewise, crayfish species (Pacifactacus leniusculus Astacus
astacus and P. clarkii) maintained at 4, 10 or 12 ºC showed zero
percent mortality upon WSSV infection. In contrast, WSSV-infected
animals maintained at 22 - 24 ºC had 100 percent mortality.
The mechanism of inhibiting virus replication is still unknown,
but it has been suggested that hyperthermia may induce
apoptosis of infected cells. Alternatively, another hypothesis is
that hyperthermia may impair the activity of cellular enzymes
essential for virus replication, thus inhibiting replication but
animals remain infected.
RNA interference (RNAi)
First described in the nematode Caenorhabditis elegans, RNE
interference is also found in fungi, plants and animals. In plants,
one biological function of RNAi was antiviral. RNAi can be a
useful tool against viral infections in animals.
Several RNAi studies have been done against shrimp viruses.
Sequence-specific RNAi has been used to inhibit replication
of TSV, IHHNV, YHV and WSSV. Double-stranded (ds)RNA
against a TSV protease strongly inhibited TSV replication (11
percent mortality) whereas controls had 100 percent mortality.
The antiviral effect of RNAi against IHHNV was demonstrated
to be both preventative and therapeutic, since dsRNA against
ORF1/2 or ORF3 administered either 12 hours before or 24 hours
after IHHNV challenge, effectively inhibited IHHNV replication.
RNAi treatment (dsRNA) against YHV protease in vivo showed
zero percent mortality at ten days post challenge.
In contrast, controls had greater than 90 percent mortality.
Several RNAi studies have been done against WSSV since this is
the most lethal pathogen in shrimp aquaculture. Variable efficacy
against WSSV replication (0 – 66 percent mortality) has been
reported depending on the genes targeted by dsRNA.
The antiviral effect against WSSV lasts up to 10 dpt (Figure 4).
Duration of antiviral effect reduced as time between treatment
and WSSV challenge increased. Antiviral effect was extended
up to 30 days post challenge through continuous re-infection of
treated shrimp. Alternatively, continuous dsRNA administration
through feed may also increase the duration of the antiviral effect
in cultured shrimp.
Bacterial infections are common in
shrimp aquaculture. Many diseases
caused by Vibrio bacteria may kill wild
and farmed shrimp both from hatcheries
and grow-out ponds.
30 | January | February 2016 - International Aquafeed
FEATURE