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Chinese herbs (Astragalus radix and

Ganoderma lucidum) enhance immune
response of carp, Cyprinus carpio, and
protection against Aeromonas hydrophila. Fish
Shellfish Immunol

ARTICLE in FISH &AMP SHELLFISH IMMUNOLOGY · OCTOBER 2008

Impact Factor: 3.03 · DOI: 10.1016/j.fsi.2008.08.015 · Source: PubMed

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 Fish & Shellfish Immunology 26 (2009) 140–145

Contents lists available at ScienceDirect

Fish & Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune

response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila
Guojun Yin a, L. Ardó b, K.D. Thompson c, A. Adams c, Z. Jeney b, G. Jeney b, *
a
Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center,
Chinese Academy of Fishery Sciences, Quitang 1, Wuxi 214081, China
b
Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8, Szarvas H-4440, Hungary
c
Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, UK

a r t i c l e i n f o a b s t r a c t

Article history: The effect of Chinese herbs (Astragalus radix and Ganoderma lucidum) on immune response of carp was
Received 28 June 2008 investigated. Fish were fed diets containing Astragalus (0.5%), Ganoderma (0.5%) and combination of two
Received in revised form 19 August 2008 herbs (Astragalus 0.5% and Ganoderma 0.5%) for 5 weeks. Other groups of fish were vaccinated (i.p.)
Accepted 22 August 2008
against Aeromonas hydrophila/Aeromonas salmonicida (Shering Plough, Essex, U.K.) at the beginning of
Available online 6 September 2008
the experiment and fed the same diets as described above. Control fish (negative control) and fish
vaccinated only (positive control) were fed basal diets without supplements of herbs. The respiratory
Keywords:
burst activity, phagocytosis, lysozyme activity and circulatory antibody titres in plasma were monitored.
Immune response
Astragalus radix Following 5 weeks after feeding, fish were infected with A. hydrophila and mortalities were recorded.
Ganoderma lucidum The results of this study showed that feeding non-vaccinated and vaccinated carp with combination of
Aeromonas hydrophila Astragalus and Ganoderma stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood
Common carp and lysozyme and circulatory antibody titres in plasma in vaccinated carp. Fish challenged with A.
Cyprinus carpio hydrophila had variable survival. The best survival (60%) was in vaccinated group fed with both herbs,
Vaccine while almost 90% of control fish (negative control) and 60% of fish vaccinated only (positive control) died.
Immunostimulant Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction the non-specific defence mechanisms and to elevate the specific

Several Aeromonads are noted as causing major problems for
carp aquaculture. Although motile Aeromonads are fish pathogens
it is important to note that these bacteria also compose part of the
normal intestinal microflora of healthy fish [1] and consequently
stress is often considered to be a contributing factor in disease
outbreaks caused by these bacteria. Aeromonas hydrophila is more
abundant in waters with a high organic load than in relatively
unpolluted water [2].
Vaccines are being developed against A. hydrophila but these are
not yet commercially available. A. hydrophila is such a heteroge-
neous species, having variable antigens, that vaccine development
is extremely complex.
Using immunostimulants in combination with fish vaccine is an
attractive method for increasing the protective capabilities of fish,
and by boosting the potency of the vaccine smaller doses can be
given [3]. In our research we used herbs to give early activation to
* Corresponding author. Tel.: þ86 66515317; fax: þ86 66312142.
E-mail address: jeneyg@haki.hu (G. Jeney).
immune response.
Chinese herbs have been used as traditional medicine and
immune booster for human beings for thousands of years in China.
Recently, growing interest has been paid to the immune stimu-
lating function of some herbs in aquaculture, non-specific immu-
nity such as bacteriolytic activity and leucocyte function was
improved by mixtures of Chinese herbs in shrimp (Penaeus
chinensis) and tilapia [4,5]. Phagocytosis by white blood cells
and lysozyme activity in the serum of crucian carp were both
increased by feeding four different herbs: Rheum officinale,
Andrographis paniculata, Isatis indigotica, Lonicera japonica [6], NBT
positive cells and lysozyme activities were increased in jian carp
(Cyprinus carpio var. Jian) by feeding herbal mixture of traditional
Chinese medicine [7].
In this study extracts of two Chinese herbs from Astragalus root
(Astragalus radix extracted from Astragalus membranaceus) and
from Ganoderma mushroom (Ganoderma lucidum) were chosen
because of their recorded ability to enhance the immune system.
Analysis shows that A. radix contains polysaccharides, mono-
saccharides, flavonoid and alkaloid, together with choline, betaine,
folic acid, various amino acids, mucoitin, gum, cellulose, and 14
trace minerals, including selenium, zinc, and iron, which are

1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2008.08.015
 G. Yin et al. / Fish & Shellfish Immunology 26 (2009) 140–145
essential micronutrients for man and animals. Recently published
research has found that some components such as: poly-
saccharides, organic acids, alkaloids, glucosides and volatile oil can
enhance immune function [8,9]. In previously reported experi-
ments with tilapia we showed that A. radix had a positive influence
on the immune system by acting as a booster [10].
G. lucidum is an important medicinal herb containing poly-
saccharides. Ganoderma polysaccharides have been reported to be
effective in modulating immune functions, inhibiting tumour
growth [11], preventing oxidative damage [12], protecting liver,
reducing serum glucose levels, while producing no toxic effects
[13]. In the study reported here, carp (C. carpio) were fed with
different doses of two extracts of Chinese herbs: A. radix and G.
lucidum to investigate the effect of these substances on the non-
specific and specific immune response of non-vaccinated and
vaccinated carp and to determine resistance against A. hydrophila.
After feeding the herbs, increased activity of the non-specific
defence mechanism was monitored. Parameters measured
included, neutrophil oxidative activity by reduction of ferricyto-
chrome c and phagocytic activity. These non-specific parameters
and the specific immune response were also observed when fish
were injected with vaccine. Specific immune response activity was
demonstrated by increased circulating antibody titres. Furthermore
we determined resistance to A. hydrophila by challenge the fish
with virulent pathogen.
2. Materials and methods
2.1. Fish
Carp (C. carpio) (62.8  5.40 g) were held in a recirculation
system of the Research Institute for Fisheries, Aquaculture and
Irrigation (HAKI, Szarvas, Hungary). Fish were fed with a dry feed,
produced in the experimental milling facility of the Institute and
kept in 2000 l fibreglass tanks at water temperature of 22–23  C.
2.2. Herbal extracts
Astragalus extract containing 40% Astragalus polysaccharide and
Ganoderma extract containing 30% polysaccharides were commer-
cial products from Xuancheng Baicao Plants Industry and Trade
Ltd., China.
2.3. Experimental design and sampling procedure
All experiments were carried out in the recirculation system of
the HAKI Institute, Szarvas, Hungary.
Batches (eight groups) of 60 five-month old carp with an
average initial weight of 62.8  5.40 g were held in 100 l fibreglass
tanks. Water temperature and pH were constant (22–23  C; pH 8.5)
during the experimental period, and dissolved oxygen was main-
tained at 80–90% of saturation. Water flow was maintained at 7 l/
min. Fish were fed ad libitum 6 times a day with a pelleted feed
containing either A. radix (0.5%), or Ganoderma (0.5%), or combi-
nation of Astragalus (0.5%) and Ganoderma (0.5%) for 5 weeks. At the
beginning of experiment four groups of fish were vaccinated (i.p.
0.1 ml/fish) using vaccine developed against A. hydrophila/A. sal-
monicida (Shering Plough, Essex, U.K.) and fed the same diets as
described above. Control fish (negative control) and fish vaccinated
only (positive control) were fed basal diets without supplements of
herbs.
Blood samples (five fish/group) were collected from caudal vein
1, 2, 3, 4 and 5 weeks after the feeding. Heparin was used as an
anticoagulant. Individual fish were sampled only once to avoid the
influence on the assays due to multiple bleeding and handling
stress on the fish.
141
2.4. Separation of leucocytes from the blood
Leucocytes for assay were separated from each blood sample by
density-gradient centrifugation. One millilitre of histopaque 1.119
(Sigma) containing 100 ml of bacto hemagglutination buffer, pH 7.3
(Difco, USA) was dispensed into siliconised tubes. One millilitre of
a mixture of 1.077 density histopaque and hemagglutination buffer
and 1 ml of blood was carefully layered on the top. The sample
preparations were centrifuged at 700 g for 15 min at 4  C. After
centrifugation, plasma was collected and stored at 80  C for future
analysis. Separated leucocytes were gently removed and dispensed
into siliconised tubes, containing phenol red free Hanks Balanced
Salt Solution (HBSS, Sigma). Cells were then washed twice in HBSS
and adjusted to 1 107 viable cells ml1.
2.5. Respiratory burst activity
Respiratory burst activity of isolated leucocytes was quantified
by reduction of ferricytochrome c [14]. Briefly, 100 ml of leucocyte
suspension and an equal volume of cytochrome c (2 mg l1 in
phenol red free HBSS) containing phorbol 12-myristate 13-acetate
(PMA, Sigma) at 1 mg ml1 were placed in triplicate in microtitre
plates. In order to test specificity, another 100 ml of leucocyte
suspensions and solutions of cytochrome c containing PMA and
superoxide dismutase (SOD, Sigma) at 300 U ml1 were prepared in
duplicate in microtitre plates. Samples were then mixed and
incubated at room temperature for 15 min. Extinctions were
measured at 550 nm against a cytochrome c blank in a multiscan
spectrophotometer. Readings were converted to nmol O 2 by sub-
tracting the O.D. of the PMA/SOD treated supernatant from that
treated with PMA given alone for each fish, and converting O.D. to
nmol O 2 by multiplying 15.87. Final results were expressed as
5
nmol O 2 produced per 10 blood leucocytes.
2.6. Phagocytosis assay
Phagocytic activity of blood leucocytes was determined spec-
trophotometrically by the method as described in Ref. [15]. This
assay involves the measurement of congo red-stained yeast cells
that have been phagocytised by cells. To perform the assay, 1000 ml
of the leucocyte solution was mixed with 2000 ml of the congo red-
stained and autoclaved yeast cell suspension (providing a yeast cell:
leucocyte ratio of 20:1). The mixtures were incubated at room
temperature for 60 min. Following incubation, 1 ml ice-cold HBSS
was added and 1 ml of histopaque (1.077) was injected into the
bottom of each sample tube. The samples were centrifuged at 850 g
for 5 min to separate leucocytes from free yeast cells. Leucocytes
were harvested and washed twice in HBSS. The cells were then
resuspended in 1 ml trypsin–EDTA solution (5.0 g l1 trypsin and
2.0 g l1 EDTA, Sigma) and incubated at 37  C overnight. The
absorbance of the samples was measured at 510 nm using trypsin–
EDTA as a blank.
2.7. Lysozyme assay
Plasma lysozyme activity was measured spectrophotometrically
according to the method as described in Ref. [16]. The lysozyme
substrate was a 0.02% (w/v) suspension of Micrococcus lysodeikticus
made up in phosphate buffer (0.05 M, pH 6.2). Lyophilised hen egg
white lysozyme was used as a standard. A new standard curve was
prepared for each assay. Standard solutions as well as samples were
added to the substrate at 25  C. The results were expressed as
mg ml1 equivalent of hen egg white enzyme activity.
142 G. Yin et al. / Fish & Shellfish Immunology 26 (2009) 140–145
2.8. ELISA assay for determining antibody titres
Antibody titres were measured against A. hydrophila. Serum
antibody titres were determined using an indirect ELISA [17]. Plates
were coated with suspensions of A. hydrophila adjusted to an OD610
of 1.0. Serum samples from vaccinated fish were serially diluted
(twofold dilutions) in PBS, and a monoclonal antibody (anti-carp
IgM monoclonal antibody, Aquatic diagnostic Ltd, Stirling, Scot-
land) was used as the second antibody.
2.9. Challenges with virulent pathogen
The susceptibility of the fish fed herbs to a bacterial challenge
was examined in vivo A. hydrophila (strain OB 212 Bacteriology Unit,
Institute of Aquaculture University of Stirling, Scotland) was used as
the challenge strain. Groups of 30 fish fed with herbs alone or in
combination of herbs and vaccine were challenged 5 weeks after
the start of treatments. Carp were injected intraperitoneally with
a 1-day growth of virulent pathogen adjusted to 1 106 cells per
fish. The fish were observed regularly at 4 h intervals for behav-
ioural changes and mortalities. All the dead fish were removed and
bacterial swabs were taken from the kidneys, and cultured on
tryptone soya agar (TSA) plates. Bacteria isolated from the fish were
confirmed as A. hydrophila using conventional methods.
2.10. Statistics
Results are presented as the average (standard error) for five
fish, and were compared at each time point using one-way ANOVA
and Dunn’s multiple range tests (SigmaStat 3.2). Significant differ-
ences between experimental groups were expressed at a significant
level of P < 0.05. Mortality (obtained at the end of experiment) in
each treated group was compared statistically with control group
using Chi-square test (SigmaStat 3.2). Significant differences
between control and treated groups were expressed at a significant
level of P < 0.001 and P < 0.05.
3. Results
The effect of herbs and combination of herbs and vaccination on
respiratory burst activities of isolated phagocytic cells is shown in
Fig. 1. Respiratory burst activities were elevated in the group fed
with Ganoderma on week 1 and in fish fed with Astragalus on week
3 compared to the negative control (Fig. 1a). Otherwise there was
Fig. 1. Respiratory burst activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the
same diets (b). Data is expressed as the mean of five fish  SEM. Significant differences (P < 0.05) from the untreated control are indicated by asterisks. Significant differences
(P < 0.05) from the vaccinated group only (positive control) are indicated by letter a.
no effect on respiratory burst activities in fish fed with herbs only
during the whole experiment, although in some cases fish fed with
Astragalus had lower values of respiratory burst activities, the
differences were not significant comparing to control. When using
the combination of herbs and vaccine in fish, elevation of respira-
tory burst activities was noticed on weeks 3 and 4 compared to
control (Fig. 1b), whereas significant inhibition of respiratory burst
activities was observed on week 3 in vaccinated groups fed with
herbs compared to positive control (vaccinated only). At the end of
experiment, vaccinated fish fed with Astragalus and combination
of Astragalus and Ganoderma showed significantly higher values of
respiratory burst activities compared to both controls – negative
and positive (Fig. 1b).
Elevated phagocytic activity was noted after 3 weeks in fish fed
with herbs (Fig. 2a), while in vaccinated groups fed with Gano-
derma and the combination of two herbs, elevated levels were
noted on week 1 and at the end of experiment compared to both
controls (Fig. 2b). On the second and the third weeks vaccinated
fish fed with herbs phagocytosis was significantly lower when
compared to both controls. There were no significant differences
among groups fed with different herbs and fish treated with the
combination of vaccine and herbs.
Plasma lysozyme activities were significantly higher after 1
week in groups fed with herbs compared to control (Fig. 3a),
whereas in vaccinated fish significant differences were measured
only in groups fed with Ganoderma (second week), Astragalus (third
week) and in fish fed with the combination of herbs a significant
difference was observed at the end of experiment (Fig. 3b).
Following vaccination all the vaccinated fish were found to have
circulatory antibody 1 week post-vaccination (Fig. 4). Antibodies
were not detected in control fish and fish fed with herbs only,
whereas all fish immunised with the vaccine were responding well.
Significant differences (P < 0.05) were noted between immunised
fish and control throughout the whole experiment, the highest
antibody titres were measured at the end of experiment. However,
there were no significant differences among fish vaccinated only
and fish treated with the combination of vaccine and herbs.
Fish infected with A. hydrophila started to die after 36 h after
injection. Mortalities due to A. hydrophila first occurred in control
fish reaching almost 50% after 2 days post-infection and a cumula-
tive mortality over the 6-day experimental period was 87% (Fig. 5a).
Cumulative mortalities in fish fed with herbs were at significantly
lower level comparing to control, reaching 58% and 60%. Fish
treated with vaccine only started to die after 2 days post-infection,
 G. Yin et al. / Fish & Shellfish Immunology 26 (2009) 140–145 143

Fig. 2. Phagocytic activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same
diets (b). Legends are the same as on Fig. 1.

cumulative mortality reached 50% at 4-day experimental period.
The same tendency was observed in vaccinated fish fed with
Astragalus. The significantly lowest mortality comparing to control
was noted in vaccinated fish fed with combination of two herbs
with a cumulative mortality 38%.
4. Discussion
Both herb extract used alone or in combination with vaccine can
modulate the non-specific defence mechanism. The present results
showed that fish fed with Chinese herbs alone or in combination with
vaccine significantly enhanced respiratory burst activity of phagocytic
cells, phagocytosis and lysozyme activities in plasma. Similar results
were obtained in jian carp (C. carpio var. Jian) and large yellow croaker
(Pseudosciaena crocea) fed with combination of Astragalus root and
Chinese Angelica root (Radix Angelicae sinensis) [18,19].
Extracellular respiratory burst activities in carp fed with herbs
alone or in combination with vaccine were elevated. However,
there were no significant differences among groups fed with
different herbs and fish treated with combination of vaccine and
herbs. Injection with glucan increased head kidney macrophages’
extracellular respiratory burst activity [20]. Extracellular activity
was very high in rainbow trout fed with dietary glucan [21].
Rainbow trout fed with ginger (Zingiber officinale) extract had
significantly higher extracellular activity of phagocytic cells in
blood, while in trout fed with nettle and mistletoe extracts the
production of extracellular superoxide anion was on the same level
as in the control fish [22]. In our previous study we could not detect
such differences in respiratory burst activity in tilapia fed with
Astragalus extract and in the case of fish fed with Scutellaria there
was significant inhibition of extracellular superoxide anion
production [10]. Other dietary components, namely vitamins E, C
and A, have been shown to have little effect upon macrophage or
phagocytic cells respiratory burst activity [23–25]. Changes in
superoxide anion production due to the multiple injections of
b-glucan were independent of different dosages [26].
Fishes treated with immunostimulants usually show enhanced
phagocytosis. Several studies have reported that oral administra-
tion of yeast products (MacroGard; Vitastim; Saccharomyces cer-
evisiae) [21,27,28], chitin [29], plant extracts of four Chinese herbs
(R. officinale, A. paniculata, I. indigotica, L. japonica) increased
phagocytosis of white blood cells of crucian carp [6], naturally
occurring tetrapeptide tuftsin enhanced phagocytosis in Labeo
rohita [30]. In this study, phagocytic activity of blood leucocytes
was increased in carp fed with herbs only on weeks 3 and 4, while
in vaccinated fish, elevated phagocytic activities were measured on
week 5 compared to control. Astragalus has been reported to
increase the phagocytosis of the blood cells of soft-shelled turtles

Fig. 3. Plasma lysozyme activity in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends
are the same as on Fig. 1.
144 G. Yin et al. / Fish & Shellfish Immunology 26 (2009) 140–145
Fig. 4. Circulatory antibody titres against Aeromonas hydrophila in vaccinated carp fed
diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and
vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
(Pelodiscus sinensis) [31] and alveolus macrophage of pneumoco-
niosis rats [32]. Astragalus polysaccharide (APS) is the major active
component of A. radix. The role of APS on the specific and non-
specific immune responses has been reviewed [33]. APS modulates
the functions of the immune cells including T cells, B cells, NK cells
and macrophage [34]. Polysaccharides of Ganoderma have been
reported to be effective in modulating immune functions, inhibit-
ing tumour growth [35]. However, there is no data available on use
of Ganoderma in fish. On the second and the third weeks vaccinated
fish fed with herbs showed significantly lower phagocytosis when
compared to both controls. In our previous study it was found that
feeding tilapia with Scutellaria extract with higher doses (0.5 and
1.0%) caused reduction of function in phagocytic cells, while, when
fish were fed with low dose of Scutellaria (0.1%) there was no
stimulation on phagocytic activities [10]. Other studies, where
higher doses of immunostimulants were used together with
vaccine have been found to be even suppressive [36,37].
Fig. 5. Cumulative mortalities after artificial challenging with Aeromonas hydrophila in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a)
and vaccinated carp fed the same diets (b). Significant differences from the untreated control are indicated by asterisks (*P < 0.05; **P < 0.001).
As a first line of defence, various peptides, such as lysozymes are
present in serum where they prevent adherence and colonisation
by microorganisms [38]. The supplemented feeds also enhanced
the lysozyme activity in all the treatments. Similarly many authors
have reported that administration of b-glucan enhances lysozyme
activity in Atlantic salmon and turbot [26,39–42]. Other immu-
nostimulants, such as chitosan, levamisole and chitin also elevated
lysozyme levels in plasma of carp [43].
In our previous study with tilapia elevated lysozyme activity
was measured when fish were fed with Astragalus, while in groups
fed with Scutellaria there were no significant changes in plasma
lysozyme activities [10]. Lysozyme is a cationic enzyme that breaks
b-1,4 glycosidic acids and N-acetyl glucosamine in the peptido-
glucan of bacterial cell walls. This action is known to attack mainly
Gram positive bacteria as well as some Gram negative bacteria in
conjunction with complement [38]. Robertson et al. [20] showed an
increased protection against fish bacterial infection, which corre-
lated with an increment in serum lysozyme levels, phagocytic
activity and bactericidal activity of head kidney leucocytes. Such
enhancement in lysozyme levels could also be correlated with
enhanced phagocytic activity.
When using herb extracts with vaccine the specific immune
response was also elevated, although there were no significant
differences among vaccinated only group and vaccinated fish fed
with herb extracts. The antibody titres against A. hydrophila were
not significantly different to the control in L. rohita [30]. While in
tilapia it was found that medicinal plants Azadirachta indica
enhanced the primary and secondary antibody responses in inverse
dose dependant mode [44].
In this study after challenge with A. hydrophila, mortalities were
significantly reduced in all groups compared to controls, with the
lowest mortality in vaccinated fish fed with both herbs. It was
considered that we would be able to use the herbs without the
addition of a specific vaccine for inducing protection in carp against
A. hydrophila. The dose of bacteria we used for challenge was very
high, resulting in 90% mortality of control fish. Sharifpour [45]
observed that intramuscular injection of A. hydrophila at a concen-
tration of 5.3  106 cells ml1 caused infection in carp, and fish
started to die within 12 h with one quarter of the injected fish dying
between 12 and 24 h post-infection. Sarder et al. [46] injected
tilapia intraperitoneally with A. hydrophila suspension
(5  106 cells ml1) and fish started to die within 12 h. Mortality
due to infections with A. hydrophila was reduced significantly by
 G. Yin et al. / Fish & Shellfish Immunology 26 (2009) 140–145 145

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