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Effect of Hibiscus rosa-sinensis and Cassia

auriculata on immune response of Cirrhinus
mrigala infected with Aeromonas hydrophila
Ragin Maria Cheriyan 1 and B. Karpagam*2

1PG Student, Department of Zoology Nirmala College for Women (Autonomous), Coimbatore- 641018, Tamilnadu, India.
2Assistant Professor, Department of Zoology Nirmala College for Women (Autonomous), Coimbatore-641018,

ABSTRACT

An investigation was carried out to evaluate the effect of Hibiscus rosa-sinensis and Cassia auriculata
flower powders as supplemented feed on the relative percent survival and antigen antibody titer. Four
experimental feeds were prepared by adding 5 grams and 10 grams of flower powder to the basal diet
and one control feed without plant powder. The Cirrhinus mrigala fishes were supplemented with these
feeds for 45 days. After 30 days of feeding the fishes were infected with Aeromonas hydrophila and
survival rate and antibody titer were noted on 38th and 45th day after treatment. The data obtained
were analyzed using one way analysis of variance (ANOVA). The fishes fed with 10% Hibiscus rosa-sinensis
flower powder supplemented feed showed greater effect in terms of survival rate and antigen antibody
titer. This study indicated that inclusion of flower powder in fish feed, in better survival and antigen
production. The formulation of plant based diet for fish will provide new opportunities.
KEYWORDS: flower powder, supplementation, Aeromonas hydrophila, relative percent survival rate,
and the antigen antibody titer.

INTRODUCTION

Aquaculture ascertained by FAO (1990) as the meadows of aquatic organisms containing

crustaceans, fishes and aquatic plants with some species of invasion in the culturing process to reinforce
productions such as furnishing, feeding and conservation from predators. From ancient times, the
aquaculture has been conducted and it is spread all over the world constantly it is revolutionized from a
tradition into science (FAO, 1990). It increases its growing rate of 6% annually and it has been gaining
importance over capture fisheries since 1990s (Reverter et al., 2014). Disease outbreaks are increasing
being recognized as a potential constraint on aquaculture production and trade and cause massive financial
loss either through mortality or reduced profit margins (Plumb and Hanson 2011; Mehana et al., 2015).
Bacteria, the major group of pathogens, poses one of the most significant threats to successful fish production
throughout the world (Roberts, 1989). Aeromonas hydrophila is a ubiquitous organism present in the aquatic
environment causing diseases in fish under stress (Doukas et al., 1998). A. hydrophila is a gram negative,
motile rod recorded as an opportunistic pathogen in freshwater fish species and it is considered to have
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widespread geographical distribution (Davis et al., 1978). A. hydrophila has been recognized as the
causative agent of hemorrhagic septicemia/ motile aeromonas septicemia, skin ulceration, fin/tail rot and
afternoon disease (Haley et al., 1967). Often, healthy fish suddenly develop swimming abnormalities, pale gills,
bloat (abdominal distension) and dermal/ocular ulcerations.

Many plant powders and extracts with antimicrobial , antibacterial and immunostimulant properties
have been used as therapeutic agents against fish pathogens. Against bacteria, they disrupt the bacterial cell
wall, block the synthesis of proteins and DNA, inhibit enzyme secretion and hinder the bacterial signaling
mechanism via quorum sensing (Citarasu, 2010). Hibiscus rosa-sinensis is a widely grown evergreen
ornamental herbs, shrubs and trees of the tropics and sub-tropics and are reported to possess various
medicinal properties (Hirunpanich et al., 2006, Chang et al., 2006, Herrara, 2004, Palaniswamy, 2003 and
Telefor et al., 1998). Just like Hibiscus rosa-sinensis, Cassia auriculata is also showed antibacterial
antioxidants activities (Anushia et al., 2009). The investigation activity at enhancing the immune system of
Cirrhinus mrigala challenging with Aeromonas hydrophila using plant flower powders like H. rosa-sinensis
and C. auriculata.

MATERIALS AND METHODS

Experimental fish collection and maintenance

The experimental animal selected for present study was mrigal fish (Cirrhinus mrigala). The
fingerlings of Cirrhinus mrigala was collected from Aliyar dam, near Pollachi, Coimbatore district, Tamil
Nadu. They were acclimatized for 2-3 weeks in the laboratory and was then transferred in to the
experimental tanks. They were fed with commercial feed during this period. The tanks were maintained
with care and was kept clean. The excess food and fecal matter were removed on a daily basis. The water
was changed once in a week keeping 50% of the habitat water.

Collection of plant samples

The flowers selected for the present study are Hibiscus rosa-sinensis and Cassia auriculata. The
flowers from these plants were collected from in and around Coimbatore, Tamil Nadu.

Processing and preparation of flower powder

Fresh flowers of H. rosa sinensis and C. auriculata were collected, washed and shade dried. The

dried flowers were finely powdered and stored in air tight containers at room temperature for further use.

Preparation of feed
Fish feed was prepared by adding equal proportions of wheat flour and coconut oil cake in the ratio
of 1:1:1 and corn flour as a binder. These substances were mixed thoroughly with hot water and it was
steamed for 25-30 minutes and then cooled at room temperature for 30 minutes. Pellets were prepared by
using domestic appliances with 0.5 mm diameter. It was dried by keeping in the sun. Four experimental

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diets were prepared by adding 5 grams and 10 grams of flower powders separately and the feed without

plant powder was kept as control.

TABLE 1: INGREDIENTS USED IN FEED PREPARATION

Diet

C T1 T2 T3 T4
Composition
Wheat flour 50 50 50 50 50

Coconut oil 50 50 50 50 50

cake

Corn flour 1 1 1 1 1

C.auricula - 5g 10g - -

H.rosa-sinensis - - - 5g 10g

Selection of Pathogens

The fish pathogen Aeromonas hydrophila was provided from the department microbiology, PSG

Institute of research and management, Coimbatore. The pathogen was maintained on agar slopes at 4°C and

was used for infecting the healthy fish. A. hydrophila was cultured in a nutrient agar broth for 24 hrs at 37°C

in an incubator. The cultured broth was then centrifuged at 3000 rpm for 10 minutes. The supernatant was

discarded and the pelleted bacteria were washed thrice with phosphate buffer saline (PBS) and prepared to

108 cfu/ml as determined using a Neubaur haemocytometer slide (Rao et al., 2006). This bacterial suspension

was used for further experiments.

Experimental design

The fishes were randomly distributed into 5 experimental tubs. Each tub consists of 8 fishes. The

fish were fed with experimental feed at the rate of 5% of body weight once in a day before 9:00 for 45 days.

Group C was fed with basal diet and it acts as the control. The remaining groups were fed with 5g of C.

auriculata (T1), 10g of C. auriculata (T2), 5g of H. rosa-sinensis (T3), 10g of H. rosa-sinensis (T4).

Pathogen challenge test

After 30 days of feeding trail, fishes in the control and treatments were injected intraperitoneally with 0.1 ml (or)

100 l of 10-4 cfu/ml A.hydrophila suspension. Mortality was recorded until 15 days after

post challenge. Behavioral alterations, feeding response and mortality were observed daily and dead fish

were removed. The experiment was conducted in triplicate. The behavioral changes and survival were
observed.

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Relative percent survival

1- (% of Mortality in treated group)
Relative percent survival (RPS) = ------------------------------------------------- ------ × 100
(% of Mortality in control group)

Antigen-antibody titer (agglutination test)

Serum antibody titer was measured using the agglutination protocol described by Klesius et al., 2000
after 7 days of infection. The agglutination test was assayed in well microtiter plates. Serum (15 l) was
diluted at 1:1 ratio in saline PBS in the first well and was serially diluted in the wells of the first row till the 11th
well of the microtitre plate leaving the 12th well as a negative control. Similarly, other serum samples
were also diluted serially in each row of the microtitre plate. 50 ml of the antigen was added to all the wells.
Gently shake the microtitre plate for efficiently mixing of the reagents. Incubate the titer plates for one hour
at room temperature. The highest dilution of the sample which shows detectable (macroscopic)
agglutination was recorded and expressed as log2 antibody titre of the serum.

Statistical analysis

The results of the present study were subjected to statistical analysis. The data obtained on immunological
parameters were analyzed using one way ANOVA and the level of significance was defined at p<0.05.

RESULTS
RELATIVE PERCENT SURVIVAL

The relative percent survival was 100% in T4 fishes on 38th day after treatment when compared to
control. On 45th day of treatment, 100% survival was observed in all the treatments whereas control showed
minimum relative percent survival of 2% (Figure -1).

RELATIVE PERCENT SURVIVAL

120
100 100 100 100 100
SURVIVAL
100

80 68
PERCENT
RELATIVE

52
60
40
19
20
2 2
0
Control T1 T2 T3 T4

TREATMENTS

38th day 45th day

Figure 1- Effect of on the Hibiscus rosa -sinensis and Cassia auriculata on the relative

percentage of survival of Cirrhinus mrigala

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ANTIGEN-ANTIBODY TITER

The antibody response to Aeromonas hydrophila by various experimental groups fed with different
concentrations of flower powders of Hibiscus rosa-sinensis and Cassia auriculata were found out by using
antibody titer plate shown in figure-2.

The antibody response produced by experimental fishes were higher than the control group. The
antibody response of T4 fishes was 0.903 whereas control showed a minimum antibody titer value of 0.602on
38th day after infection. On 45th day after treatment more antibody response (1) was found in T4 when
compared to control.

ANTIGEN ANTIBODY TITER

1.2
1
1 0.903 0.903
0.845
0.778 0.778
0.8 0.698 0.698
0.602 0.602
0.6

0.4

0.2

0
Control T1 T2 T3 T4

TREATMENTS

38th day 45th day

Figure 2- Effect of on the Hibiscus rosa -sinensis and Cassia auriculata on the antigen antibody
titer of Cirrhinus mrigala

DISCUSSION

The flower powders used in this study would have enhanced the immune response in all
experimental groups. The present study revealed that fishes fed with T4 and T2 showed higher survival rate
when compared to control. This might be due to the action of the flower extracts in the fish feed. The
secondary metabolites found in the flower extracts might be responsible for a significant increase in the
survival rate of the fishes in experimental groups.

The results of present study correlated with the findings of Iruthayam et al., 2014 who reported that Mystus
montanus fingerlings fed with medicinal plants showed increased resistance and survival against Aeromonas
hydrophila. Sahu et al., (2007 a and b), inferred that L. rohita fed with garlic and mango kernel showed
increased survival rate against Aeromonas hydrophila when compared to control. Similarly, Baba et al.,
2016 stated that a positive effect on the survival rate of common carp observed when treated with Avena
sativa extract against Aeromonas hydrophila. Sanjana et al.,2019 stated that The fishes fed with 5%
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Ocimum basilicum showed greater effect in terms of survival and at 10% it showed better antigen antibody
titer.

To Aeromonas hydrophila by the experimental fishes fed with Hibiscus rosa-sinensis and Cassia
auriculata extracts at different concentrations showed significant enhancement when compared to control.
The antibody titer was significantly higher in experimental groups and this increase in the production of
antibody might be due to the immuno stimulatory effect of flower extracts in the experimental feed. The
phytochemicals found in the flower extracts were able to resist against bacteria which was in enhanced
antibody production.

The results of the present study were similar to the findings Pratheepa et al.,2014 who reported that the
Euphorbia hirta plant leaf extract enhance antibody response but the extract which are in higher
concentration (25 to 50g) were only able to stimulate higher antibody production. Hema Priya et al., (1997)
suggested that the leaf extracts of Phyllanthus niruri and Acalypha indica stimulated the antibody response
in Tilapia (Oreochromis mossambicus). Similar results were observed by Metalbal et al., 2000 who
reported that Ocimum sanctum positively determined the immunomodulatory effect stimulate the antibody
response and disease resistance in Oreochromis mossambicus against the infection of Aeromonas
hydrophila.

CONCLUSION

The results on present study indicated the beneficial role of selected plant flowers of H. rosa-sinensis and C.
auriculata as immunostimulants against the pathogen A. hydrophila in C. mrigala . The study revealed that H.
rosa-sinensis at 10% concentration enhanced the survival rate of the fish against the pathogen. It has also
improved the rate of oxygen consumption in flower extract treated groups. In general, the immunostimulant
was found to produce antibodies in greater amount against the pathogen. The study thus states that H. rosa
sinensis and C. auriculata are suitable as supplemented feeds for fishes at farm level as they have been
identified as effective immune stimulants against certain pathogens.

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