Herbal therapy for controlling viral disease in aquaculture

Herbal therapy for
controlling viral disease
in aquaculture
Harapriya Behera
Department of fish pathology and health management
Fisheries college and research institute

Your Logo or Name Here 2
Sl.
No.
Particulars Name Designation & Department
1 Chairman Dr.K.Riji John Professor and Head, Department of
Fish Pathology and Health
Management
2 Member Dr.M.Rosalind George Professor and Head, Department of
Fish Pathology and Health
Management
3
Member Dr. S. David Kingston Professor and Head, Department of
Fish biology and Fisheries Resource
Management
ADVISORY
COMMITTEE

Your Logo or Name Here
1. Aquaculture
2. disease
3. Viral infections
4. What is phytotherapy
5. Why phyto therapy
6. Mode of action of phytotherapy
7. Phyto therapy application in finfish culture
8. Phytotherapy application in shellfish culture
9. Some common examples
10.Immunisation
11.Summary
Content
3

• Aquaculture is the farming or husbandry of aquatic plants and animals,
considered to be the world’s most diverse food production sector.
• Global fish production* peaked at about 171 million tonnes in 2016 (SOFIA,
2018). While India has 11.41 mmt in 2016-17 (India.com)
Aquaculture
4
Major Objectives of Aquaculture
a) controlled production of food to
improve the supply for human
consumption and provide health
security
b) Increase livelihood of millions of
people.

Senzisko,1973

 Intensifictaion is conducive for the development and spread of viral infectious diseases.
 Increased output requires expansion in new environments and dependence on external feeds
(including fish), water which gives rise to the development of vectors who acts as a carrier for
the virulence pathogens such as viruses.
 Improper biosecurity ( water exchange) , unsterilized equipment, entry of other personnel, poor
feeding pattern leads to development of unfavourable conditions.
 Viral diseases are very difficult to be controlled. Efficient treatment or vaccines exist for
eradication which is cost and time effective. However, vaccines is effective only against one kind
of pathogens and the effect of immunostimulants only last for a short period of time (ardo, 2013).
 And culturing genetically resistance species and SPR and SPF broodstocks are the ultimate
ways.
6

 Phyotherapy defines treatment from plants; Medicinal Herbalism.
 Herbal medicines include herbs, herbal material, herbal preparations, finished herbal products that
contains parts of plant as active ingredients. Medicinal herbs are significant sources of synthetic and
herbal drugs.
 Phytotherapy has come to be recognized as a handy and viable alternative to chemotherapy, as it is
economical, effective, non-resistance forming, renewable, eco-friendly and farmer-friendly (Raman
2017).
7
 Medicinal plants are the treasures of our planet.
In India the use of medicinal plants for treatment of
various diseases and ailments dates back to the times of
Rig Veda written between 4500-1600 B.C (Raman 2017).

Synthesized antiviral drugs cause drug
resistance (Gilbert et al., 2002) and occurrence of
new viral strains always create a problem for the
effective control and management of viral
diseases of animals and humans.
The herbs contain many immunologically active
components such as, which can enhance
immune functions against pathogenic
microorganisms (yin et al., 2008) and they also
posess antimicrobial activity (Citrasu, 2010).
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a. polysaccharides,
b. organic acids,
c. alkaloids,
d. glycosides and
e. volatile oils

Interaction between extract
and the envelop virus that
prevent the entry of the
virus into the host.
Breaks double stranded
DNA of ds DNA viruses
(Matkar et al., 2008).
Enahances non specific
immunity etc.( Citrasu,
2010)
Inhibits or blocks the
transcription of the virus to
reduce the replication in the
host cells (Carlucci et.al 1999).
Phytotherapy

 SVCV, is a bullet-shaped RNA virus belonging
to
genus Vesiculovirus
family Rhabdoviridae
responsible for the heavy mortality in common
carp and other cyprinid species.
 OIE, SVCV usually cause high mortality in the
cultivation of cyprinid fishes because it is
difficult to eradicate the virus from affected
ponds, all aquatic life should be destructed
once SVCV infection is established.
10

Coumarin is a natural chemical compound found in many plants, natural spices, and
foods such as cassia (bastard cinnamon or Chinese cinnamon), cinnamon, melilot
(sweet clover), green tea, peppermint, celery, lavender and carrots, as well as in beer,
tobacco, wine, and other foodstuff (PubChem Compound Database)
11

 Coumarin derivative, 7-(4- benzimidazole-butoxy)-coumarin,
synthesized by Shen et. al 2018
 inhibit cell death and reduce cellular morphological damage
 10mg/li IP in zebra fish with a maximum inhibitory rate more than
90%.
 reduce the morality
 activate IFN response
 balance of the redox state
Shen et. al

Arctigenin, a bioactive component in Fructus Arctii
the dried fruits of Arctium lappa arctigenin
derivatives
2 A-2Q posess ester and ether side chains at the
phenolic hydroxyl positions
4 A-6A- arctigenin-iminazole hybrid derivatives,
arctigenin derivatives were synthesized by
nucleophilic substitution with appropriate iminazole
derivatives.
EPC cells were infected with SVCV, accompanied by
the treatment of 2Q and 6 A for 48 and 72 h, at the
concentration of 1.6 μg/mL and 2.0 μg/mL,
Greater burdock
(Umatenchedi)

Morphology of normal cells was intact, round and plump, and retained
spherical shape with a clear edge (Fig. 7A and E). Inversely, cells
infected with SVCV appeared typical apoptotic features including cell
shrinkage, volume reduction and cell blebbing after 48 h infection (Fig.
7B). Furthermore, more serious features including rupturing, leaking
intracellular matrix were observed at 72 h after exposure to SVCV (Fig.
7F). Noticeably, cells treated with 2Q and 6 A kept the normal
morphology without appearing a mass of cell deformation and rupture
similar to those in the normal cells (Fig. 7C–D and 7G-H).

 inhibition SVCV G protein expression
 inhibit apoptotis features including cell membrane rupture and nuclear
fragmentation
 Morphology of normal cells and 6A treated cells w intact, round and
plump, and retained spherical shape with a clear edge (SEM)
Chen et. al 2018

 VHSV, virus belonging to the genus Novirhabdovirus,
within the family Rhabdoviridae (Walker et al., 2000).
 Infection with VHSV may cause disease and mortality in all
life stages of susceptible fish.
 Some survivors of epizootics will become long-term carriers
of the virus.
 It can be controlled either by good husbandry practices or
by genetic alteration of animal.

Olive tree leaves, are rich
in biophenols (BPs), such
as oleuropein (Ole),
verbascoside, ligstroside,
tyrosol or hydroxytyrosol.
These compounds are skin
photoprotective and have
demonstrated antimicrobial
activity by inhibiting the
growth of a wide variety of
bacteria, fungi and viruses
(Hirschman, 1972).
Ole has been claimed in a
U.S. patent to have potent 17
Chemical structure of
oleuropein, the major
component of the olive leaf
extract.

• Data has been found from the research of Micol et. al 2005
that ole and LExt of Olive tree can act as therapeutic agents
for other enveloped virus rhabdovirus such as the viral
haemorrhagic septicaemia which is an OIE notifiable disease
and cause mass mortality of fresh and marine fishes.
• And also virus accumulates of possibly toxic substances both in
18

 Decrease in the amount of newly formed virus particles or loss of infectivity of the
newly formed virus particles, due to destruction of viral protein by LExt.
 Nearly complete absence of viral proteins was observed in the EPC cell monolayers
treated with LExt at 36 h post-infection.
 Ole interacts with the viral envelope producing VHSV particles with a reduced
membrane-fusion capacity.
Micol et. al
2005

grouper iridovirus of Taiwan (TGIV) having Causative
agent enveloped (dsDNA)
 The most promising method in prevention of Iridovirus
infection is the enhancement of resistance of the fish,
which can be achieved by the application of vaccines
and immunomodulating compounds.
20

 Novriadi and Haw in 2015 reported commercially available herbal bioconditioners (AquaHerb)
produced from Rhizophora mucronata, Pinus armandii and Melaleuca alternifolia was able to
modulate the immune system and significantly conferred resistance to Tiger grouper Epinephelus
fuscoguttatus against iridovirus infection in comparison to control.
 AquaHerb contain with several active principle components, such as alkaloids, flavonoids, terpenoids
and steroids which have been reported as antistress, antimicrobial properties, appetite stimulation,
tonic and immunostimulation.
21
Loop-root mangrove Chinese white pine Tea tree

Histogram of the mean survival (%) of groupers injected with
Iridovirus followed by immersion with AquaHerb (herbal
bioconditioners). Survival was counted after 8 days post
AquaHerb immersion. Control groupers were not immersed

 Modulate the immune system
 Eliminate the Iridovirus from fish tissue
 Decrease in cumulative mortality

 Koi herpesvirus disease (KHVD) is a herpesvirus infection
and also known as carp interstitial nephritis and gill necrosis
virus (CNGV) Cyprinid herpes virus belongs to family
Alloherpesviridae,
 Morbidity of affected populations can be 100%, and mortality
70‒80%, but the latter can be as high as 90 or 100%.
 Methods to control and prevent KHVD should mainly rely on
avoiding exposure to the virus coupled with good hygiene
and biosecurity practices

• Clinacanthus nutans is a Thai herbal medicinal plant in the family Acanthaceae.
• Ethanol crude extract of the fresh aerial part (leaves and stem) of C. nutans used
as antiviral agent.
25
Sabah snake grass
Vishamooli
1. Pre-infection was done with 5, 1, 0.5 and 0.1 mg/ml
conc. The virus was mixed with each concentration
of extract and with 0.5% DMSO then incubated at
24°C for 4hr.
2. treated with 50μl of CyHV-3 virus.
3. Post infection was done by serially diluted extract
with 2% L-15 in four concentrations; 2.5, 1, 0.5 and
0.1 mg/ml. After inoculation for 0, 24 and 72 hr, each
concentration of extract was added into the tanks.

 no mortalities or abnormal behaviours in fish in the 5 mg/ml
 interfere with viral replication process at an early stage of infection before
24 hr, but not after 72 hr.
 These results were possibly due to the rapid spread of viral infection in fish.
Haetrakul et.al
2017

 Infectious Pancreatic Necrosis is an acute to subacute, highly contagious
disease of young salmonid fishes.
 IPN is a birnavirus. It has double stranded ribonucleic acid.
 Morbidity rate among young susceptible salmonids usually reach 100%.
Mortality rate may reach 95% to 97% among the most susceptible fish.
Infectious pancreatic necrosis virus

• Geranyl aromatic derivatives from the
resinous exudates from the plant
Heliotropium filifolium are a group of the
semi-synthetics terepenoid compounds
known as Filifolinyl senecionate.
• Filifolinyl senecionate was extracted by
dichloromethane.
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Indian turnsole (Tetkotukki)
I. CHSE-214 cells were grown in 12-well plates.
II. After 24 hr infected with IPNV
III. After 1 h of infection at 15˚C with agitation every
15 min, the inoculum was removed
IV. 1mL of infection medium was added.
V. Filifolinylsenecionate was added at 0 and 2 h.p.i.
at three different concentrations (150, 200 and
250mg/mL) and were incubated until 10 h.p.i.

 Filifolinyl senecionate at 200 mg/mL, capable of inhibiting the replication of viruses ester
could be interacting with the viral RNA during the viral cycle.
 inhibits protein expression of genomic RNA, the effect of the compound on the expression of
the viral structural polypeptides VP1, VP2, VP3 and VP4
 inhibition of the synthesis of viral messenger RNA, which occurs at this time of the infective
cycle,
Modak et. al 2009

Nodavirus infection
30
 Positive-sense ssrna:
 Viral encephalopathy and retinopathy otherwise known as viral nervous necrosis is a
neuropathological condition.
 Dark fish are affected, light fish are normal. A change in colouration is an important
indicator of disease. Species differ in how they are affected
 50–100% cumulative mortality over a period of 48 hours.

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Sirokurunja
Cells were cultured routinely in 75 cm2 culture flask.
SIGE cells were inoculated in 24-well plate to give a confluence of 60%
to 70%.
After removal of culture medium 1 mL of GNNV suspension virus
infection was confirmed by cytopathic effect (CPE) on SIGE cells.
Gymnemagenol (20 μg mL−1) was added to SIGE cells inoculated in a
well of a 24-well plate containing culture medium and incubated for 1 h
at 28℃.
 Gymnemagenol, extracted from Gymnema sylvestre leaves
 Gymnemagenol, a triterpene glycoside saponin, is a aglycone or
genin or a sapogenin belonging to the saponin family.
 The extracted compound (gymnemagenol) was identified as 3β,
16β, 28, 29-tetrahydroxyolean-12-ene.

 Gymnemagenol exhibited concentration-dependent inhibition of GNNV proliferation in virus
infected SIGE cells in the culture medium
 Gymnemagenol at 20 μg mL−1 inhibited 50% cell viability at the end of 6 d, and at the end of
7 d 47% cells were still viable.
 This indicates that gymnemagenol was very effective in inhibiting the proliferation of GNNV
in infected SIGE cells.
Khanna et.al 2011

• Genus Whispovirus within the Nimaviridae family having large circular
double-stranded DNA genome
• The major targets of WSSV infection are tissues of ectodermal and
mesodermal embryonic origin, especially the cuticular epithelium and
subcuticular connective tissues. So it is known as Systemic ectodermal
mesodermal baculo virus (SEMBV)
• Vitamin C, ,ß-glucan and Sea weed extracts (fucoidan) may improve
resistance to WSD (Chang et al., 2003; Chotigeat et al., 2004). Shows
high mortality upto 100% within 3-5 days.
33

• Glucomannan and acemannan hasten wound healing, activate macrophages, arouse the immune
system and have antibacterial, and antiviral effects.
• Also, aloe contains anthraquinones, like aloe emodin,which generally work on viruses, preventing
virus adsorption and replication (Hu et al., 2003)
Aloevera
34
Aloe vera (L.) Burm. f. is a perennial plant of the
Xanthorrhoeaceae family that has been widely
studied as a healing agent in humans (Paez et al.,
2000). Aloe gel consists mainly of water (98%) and
polysaccharides (pectins, cellulose hemicellulose,
glucomannan, acemannan, and mannose
derivatives). Katralai

 4g powdered leaf of Aloe vera kg feed−1 showed high values of immunity.
 Lowest survivality was succumbed.
 Showed some protective effect against V. parahaemolyticus and WSSV.
Trejo-Flores et.al 2016

• Argemone mexicana is one of the most significant
herbs in traditional system of medicine and found
mostly along roadside and fields in India.
A. mexicana seeds contain 22–36% of pale yellow
non-edible oil, called argemone oil or katkar oil,
which contains the alkaloids, sanguinarine and
dihydrosanguinarine. Four quaternary isoquinoline,
alkaloids, dehydrocorydalmine, jatrorrhizine,
columbamine, and oxyberberine.
Argemone mexicana
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Mexican Prickly Poppy.

 Control group succumbed to death 100 % within four days while ethyl acetate extract group had
effectively reduced to cumulative mortality 17.43 and 7.11% respectively in stem and root treated
groups.
 Ethyl acetate extract of A. Mexicana enriched diets helped to improve the THC when fed with
400mg kg -1 feed.
 Proprophenol oxidase activity (Propo) observed was 0.088 OD at 90 mins incubation in the
heamolymph of control diet fed L. Vannamei after 4th day of post WSSV challenge. The Propo
level gradually increased 0.128 OD in treated diet fed L. Vannamei groups.
Palanikumar et. al 2017

Psidium guajava bark, root and leaves were
extracted with hexane, ethyl acetate and
methanol extracts.
Presence of coumarins, phenols, quinone,
alkanoids and tanins in ethyl acetate
extracts are responsible for antiviral activity
and successfully controlled WSSV
replication.
Psidium guajava
39
 Gujava roots and barks were dried and hexane, ethyl acetate, methanol extract
was prepared.
 when 5μl of WSSV suspension (300 μg of protein) was injected into animal with
10 μl of ethyl acetate extracts of barks.
Koya

In positive control where extracts were not given mortality of Feneropeneaus indicus
was 100% after 3 days of post infection with WSSV virus where as in treatment tank
mortality decreased upto 80%.
Velumurugan et.al 2011

Gracilaria tenuistipitata
41
 Gracilaria tenuistipitata are known to be
queen of sea having a great antimicrobial
activity.
 The Gracilaria extract and carrageenan
contain repeating unit of galactose
molecule with 1,3-linkage and 1,4-
linkages and ester sulphate.
Litopenaeus vannamei were treated with Ɣ-carrageenan (400 mg L-1) Hot water extract of Gracilaria
tenuistipitata 600 mgL-1 for 3 h, challenged with WSSV, and released into aerated normal seawater
at 120 h post-WSSV challenge.
And given booster with the same concentration for 3h with challenge .

 The survival rate of shrimp immersed in seawater containing 400 mg L -1or 600 mg L1 extract was
significantly higher than that of control shrimp group.
 The hyaline cell , granular cell ,Prophenol Oxidase activity, Respiratory burst activity, Super Oxide
Dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L-1 extract were
significantly higher than those of the challenged control shrimp at 12- 120 h post-challenge in
booster treatment.
Lin et. al 2012

Pongamia pinnata
43
Ethanolic extract of the leaves of Pongamia pinnata
was fed to Penaeus monodon by impregnating with
the commercial pelletized shrimp feed at 300 μg/g of
body weight of shrimp/day.
spectroscopic studies of the antiviral WSSV
compound from the leaves of P. pinnata revealed
that it is bis(2-methylheptyl)phthalate.
Pongamaram

 Treated groups showed 80% survival
 Extracts showed antimicrobial activity against Vibrio sp., Pseudomonas sp.,
and Streptococcus sp. and also antiviral activity against WSSV.
Rameswaram and Ramasamy 2007

Ethyl acetate, methanol, ethanol and distilled water extracts of a different species
of Indian traditional medicinal plants Ocimum americanum and Ocimum sanctum
showed antiviral activity against white spot syndrome virus (WSSV) in shrimp
(Balasubramanian, et al., 2007)
2% of the Cyanodon dactylon as a whole plant extracts showed a significant
survivality, enhances immunity of tiger prawns, enhance ProPo concentration than
the control.(Balasubramanian et al. 2008).
Aqueous extract from Ceriops tagal, a mangrove plant, imparted total protection
to shrimp from WSSV (Sudheer et al., 2011).
The aqueous extract of Sargassum weighti (seaweed) showed significant anti-
WSSV property against Penaeus indicus and crab, Paratelphusa hydrodomous
(Ghosh et. al 2014)
45

• YHV is a serious pathogen that causes a harmful disease called “yellow head disease”.
Additionally, YHV rapidly kills black tiger shrimp and causes mass mortality in several other types
of crustaceans, including Metapenaeus ensis, P. vannamei, P. stylrostris, P. aztecus and P.
duorarum.
• .
Yellow head virus
46

 Lignins are naturally found in the middle lamella of higher plants, such as Angiosperms,
Gymnosperms an Monocotyledons. Commercial lignin, available in the form of Kraft lignin.
possesses fewer sulfur groups and anionic charges.
 kraft lignin, a byproduct of the pulping industry, is a safe compound for black tiger shrimp or other
penaeid shrimp, indicated by its high 96-hrs LD50.
47

 Feed supplemented with 1–10 g/L kraft lignin effectively enhanced phagocytic activity but did
not enhance YHV inhibition in black tiger shrimp.
 However, pre-incubation of this by-product with harmful YHV particles conferred notable
protection against yellow head disease in experimental shrimp
Srisapoome et.al
2018

 Phyllanthus spp. was examined the ability of ethanol extracted polyvinylpyrolidone complexed
extracts to impact YHV (Direkbusarakom et al 1995)
 The extract were fed to groups of black tiger shrimp (15–20 g), in triplicate for 7 days. After 7 days,
the shrimp were exposed to the YHV by a water-borne exposure. The shrimp survived more than
95% while the control survived only 25%.
50
Killanelli

 80% methanolic extract of Rhus verniciflua stokes bark showed significant anti-viral activity
against fish pathogenic infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic
septicemia virus (VHSV) in a cell-based assay measuring virus-induced cytopathic effect (CPE).
Extracts have active ingredients as fustin and sulfuretin (Kang et. al 2012)
 Ethanol extract of Psidium guajava was tested for anti-viral activity against the fish pathogenic
viruses, IHNV, IPNV, and OMV using plaque reduction in CHSE-214 cell lines (Dierebusarakom et
.al 1998)
 Dierebusarakom et al. (1996) found that 18 Thai traditional herbs presented antiviral activity
against OMV and IHNV, while Orchocarpus siamensis and star goosebarry (Phyllanthus acidus)
inhibited the replication of OMV and IPNV in cells.
 Harikrishnan et al. (2010) reported that intraperitoneal administration of the leaf extracts of Punica
granatum at 50 or 100 mg kg−1 dose enhanced the innate immune responses and disease
resistance after 8 weeks in P. olivaceus against natural LDV infection.
51

Rotenone is a traditional herb widely used to kill fish in shrimp ponds,
because it is highly toxic for fish even at low concentration and is rapidly
degraded in the natural environment.
 Viruses are sensitive to different constituents of tulsi extracts that has
active component namely apigenin, linalool and ursolic acid, which
exhibited a broad spectrum of antiviral activity against dna viruses viz.
Herpes viruses (HSV), adenoviruses (ADV), hepatitis B virus and RNA
viruses.
Herbal compounds have the ability to inhibit the generation of oxygen
anions and to scavenge free radicals. The herbal antioxidant effect has been
shown to be similar to that of superoxide dismutase, metal-ion chelators
and xanthine oxidase inhibitors. The best example is the herb Picrorhiza
kurroa used as an antistress compound for shrimps (citarasu et al. 1998).
52

 Immunostimulants boost the defense mechanisms and conferrs protection of animals from
infectious diseases. Immunostimulants enhance the innate (or non-specific) immune response
(Sakai, 1999).
 The major components of the innate immune system are macrophages, monocytes, granulocytes
and humoral elements, like lysozyme (Magnadóttir, 2006), complement, antiprotease,
meloperoxidase, reactive oxygen species, reactive nitrogen species, phagocytosis, respiratory
burst activity, nitric oxide, total hemocytes, glutathione peroxidase, and phenoloxidase, against
bacterial, fungal, viral, and parasitic diseases (Harikrishnan et al., 2011a).
 Variety Herbs contain many types of active components, like polysaccharides, alkaloids or
flavonoids (Ardó et al., 2008) and different chemical compounds which have a great antimicrobial
activities.
 Several plants or their byproducts contain phenolic, polyphenolic, alkaloid, quinone, terpenoid,
lectine, and polypeptide compounds, many of which are effective alternatives to antibiotics,
chemicals, vaccines, and other synthetic compounds (Harikrishnan et al., 2011a).
53

Plant Species Immunity References
Ginger (zingiber officinale) Rainbow trout Non-specific parameters of
neutrophils Shakya 2016
Mixture of herbs angelica
membranaceus and A. Sinensis
Common carp Of phagocytic cells and plasma
lysozyme activity. Respiratory
burst activity of
Jian and wu, 2004
Purple coneflower and garlic Nile tilapia Non specific immunity Aly and mohamed, 2010).
Mistletoe (viscum album
coloratum)
Nile tilapia Lysozyme activity, respiratory
burst activity, alternative
complement activity and
phagocytic activity
Park and choi 2012
Lactuca indica Kelp grouper Total immunoglobulin, Harikrishnan et al., 2011c
Green tea ethanol extract Black rockfish Lipid utilization, lysozyme
activity and stress recovery,
and reduced total cholesterol
levels
Hwang et al., 2013
54

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62

Summary
 The concepts of using medicinal herbs have
received much attention as an alternative
strategy for producing immunomodulating
compounds because of their relatively low capital
cost and they are risk-free to the environmental
contamination.
 Revealed that the antiviral properties of plants
demonstrates that plants may inhibit one type of
virus but have no activity against most others.
Therefore, these results suggest that the
administration of several herbal or their
combination have been found to have more non-
specific immuno-stimulating effects against viral
infection
 Extracts can be applied as a prophylactic agent
in common carp and also used in handling of
equipments.
 Novel strategies such as the use of medicinal
plants need to be taken to reduce production
losses (huynh et al.,
63

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