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Effect of dietary supplemented andrographolide on growth, non-specific immune parameters and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton)
Fish & Shellfish Immunology 35 (2013) 1433e1441
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Effect of dietary supplemented andrographolide on growth, non-specific immune parameters and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton) Kusunur Ahamed Basha, Ram Prakash Raman*, Kurcheti Pani Prasad, Kundan Kumar, Ezhil Nilavan, Saurav Kumar Central Institute of Fisheries Education, Panch Marg, Off Yari Road, Versova, Mumbai 400 061, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2013 Received in revised form 3 August 2013 Accepted 8 August 2013 Available online 22 August 2013
The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Andrographolide Non-specific Immune system Immunostimulant Aeromonas hydrophila
1. Introduction Aquaculture fish production increased significantly over the past few decades necessitating intensive fish culture practices. Since intensification is one of the prime necessities to cope with the present and future demand and hence, increase in productivity per unit space is performed by increasing the rearing density. Intensification leads to a number of other associated stressors like overcrowding, transport, handling, grading and poor water quality tend to adversely affect the health of the cultured fish [1]. These conditions produce poor physiological environment increasing the susceptibility of fish to infectious agents paving the way for the outbreak of a number of diseases due to an increasing range of
* Corresponding author. Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Mumbai 400 061, India. Tel.: þ91 9594073424; fax: þ91 2226361573. E-mail addresses: [email protected], [email protected] (R.P. Raman). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.005
pathogens. The disease outbreaks are increasingly being recognized as a potential constraint on aquaculture production trade, and cause massive economical loss through mortality or inferior meat quality, resulting in reduced profit margins [2]. Bacterial diseases have been reported to be a principal limiting factor in both wild and cultured fishes [3]. The most common and frequently encountered bacterial pathogen in India is Aeromonas hydrophila which causes severe damage to carp production [4]. A. hydrophila is associated with wide range of freshwater fishes. It is an important pathogen in causing stress related diseases in fish with the common symptoms of ulceration, exophthalmia and abdominal distension [5e7]. However, a potent drug and effective vaccine development against A. hydrophila continues to be a challenge to fish pathologists [8]. The most common approach to treat bacterial diseases is the application of antibiotics. However, accumulation of chemicals and antibiotics in the environment and in the fish flesh have led to the imposition of stringent regulations that limit the use of antibiotics [9]. A crucial approach in disease management should be based on application of preventive measures and one of the promising
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alternative or tool in fish disease management that have been evolved in response to these problems is “immunostimulants” which has overcome the lacuna of vaccines and probiotics [10]. An immunostimulant is a chemical, drug or action that enhances the defence mechanisms or immune response [11], thus rendering the animal more resistant to diseases. Non-specific defence mechanism plays an important role at all stages of fish infection. Fish particularly depend mostly on these non-specific mechanisms than do mammals [12]. Phytotherapy is the oldest form of health care known to man-kind. Bioactive substances present in herbs are well-known to have an antimicrobial and immunomodulatory properties. Herbs are an interesting alternative because they are inexpensive, renewable, locally available, user friendly and can be easily prepared [13]. Recently, there has been an increasing interest in the modulation of the non-specific immune system of fish, as a prophylactic measure against disease. Many of the medicinal plants such as Ocimum sanctum [14], Acalypha indica, Phyllanthus niruri, Azadirachta indica, Piper betle, Mentha piperita [15], Allium sativum [16], Astragalus membranaceus, Lonicera japonica [17] and Withania somnifera [18] have been shown to trigger innate immune system and enhance disease resistance against pathogenic organisms. An extensive work on the use of immunostimulatory herbs in fish was conducted by various researchers and they opined that the herbal extracts can be used in fish culture as an alternate to the chemotherapeutic agents [19e22]. Andrographis paniculata (AP) also called Kalmegh or “King of Bitters” belongs to family Acanthaceae, native to India and Sri Lanka. Mostly the leaves and roots are used for medicinal purposes. Diterpenoid lactone andrographolide (C20H30O5, melting point 230e 239 C) is the principle medicinal compound found in A. paniculata, which is mainly concentrated in leaves and can be easily isolated from the crude plant extracts as crystalline solid [23]. The various bioactivities of andrographolide such as hepatoprotective against various inducers [24], antimicrobial [25], antioxidant [26], antidiabetic [27], anti-inflammatory [28,29], anticancer [30], antitumour [31] and immunomodulator [32] properties have been evaluated. The ethyl alcohol extract and purified diterpene andrographolides are reported to stimulate both specific and non-specific immune responses in mice [33]. In the recent years herbs based growth promoters, immunostimulants and therapeutics which are ecofriendly are given more importance in aquaculture system. Thus keeping these aspects in view, the present study was undertaken to investigate the effect of andrographolide on the growth, nonespecific immune parameters and disease resistance against Aeromonas hydrophila in Indian major carp Labeo rohita. 2. Materials and methods
before cooking. Further, completely cooled dough was mixed with vitamins, mineral mixture and six of the experimental diets contained with andrographolide in different concentrations T0 (0.0%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) as listed in Table 1. The dough was pressed through a hand pelletizer to get uniform sized pellets. These pellets were dried at 40 C for 12 h. After drying, pellets were packed in polythene bags, sealed airtight and labelled according to the different concentrations of treatments, for further use. The feed was having (34.89e36.66%) of crude protein, (404.3e408.04 kcal 100 g1) of isocaloric, (9.40e 10.8%) of lipid content, (25.05e26.14%) of carbohydrate and (3.01e 3.58%) of ash content. 2.3. Experimental design One hundred and eighty L. rohita fingerlings were equally and randomly distributed in six experimental groups in triplicate following a completely randomized design (CRD). The experimental trial for effects of andrographolide on growth, non-specific immune parameters in L. rohita was conducted with feeding the various level of andrographolide as control T0 (without andrographolide), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for a period of 42 days. The fish were fed with the experimental diet at the rate of 3% of body weight twice a day at 09:00 and 17:00 h to approximate satiation for 42 days. The physicochemical parameters of water such as temperature (24.3e 25.8 C), pH (7.2 0.4), dissolved oxygen (6.6e6.9 mg L1), ammonia (0.01 0.005 mg L1), nitrate (0.02e0.06 mg L1) and nitrite (0.001e0.004 mg L1) were maintained in optimum condition during the experimental period. Six fish were sampled from each treatment group on 14th, 28th and 42nd day and blood was drawn for different immunological assays. After 42 days, nine fishes from each treatment were segregated for challenge study particular injection with A. hydrophila and post challenge study was continued up to 56 days. At the end of 56 days, growth parameters and relative percentage survival rate were estimated for disease resistance against A. hydrophila. 2.4. Collection of blood and serum Each fish was anesthetized with clove oil (Merck, Germany) at 50 ml per litre of water before collecting blood samples from fish. Blood was drawn from caudal vein of fish by using 1.0 ml hypodermal syringe and 24 gauge needles, which was rinsed with 2.7% EDTA solution before use. The collected blood was immediately transferred to the test tube coated with thin layer of EDTA (as an anticoagulant) and shaked well in order to prevent clotting of blood. Serum was collected without using anticoagulant and was
2.1. Experimental animal and regime Labeo rohita fingerlings with an average weight of 9.50 0.50 g were procured from Malad Fish Farm, Maharashtra, India. The fishes were acclimatized for 15 days in laboratory condition in 1000 L FRP tanks at 25e27 C, under continuous aeration. The fishes were fed with control pelleted diet at the rate of 3% of body weight twice a day. 2.2. Preparation of experimental diet The andrographolide (EC 50%) was procured from Neocare Naturals Limited, Hyderabad, India and used for preparation of six isonitrogenous experimental diet following the method of Rao et al. [34] with slight modification. Experimental diets were prepared by mixing all the ingredients in required quantity along with water to form dough and calculated amount of oil was added to the dough
Table 1 Composition of experimental diets (in 100 g). Ingredients (g)
T0 (control)
T1
T2
T3
T4
T5
Fish meal Soya flour Corn flour Wheat flour Rice bran GOC CMC Veg. oil Vit & minerala Andrographolide (EC 50%)
25 20 12 15 10 12 1 4 1 e
25 20 12 15 10 11.9 1 4 1 0.1
25 20 12 15 10 11.8 1 4 1 0.2
25 20 12 15 10 11.6 1 4 1 0.4
25 20 12 15 10 11.2 1 4 1 0.8
25 20 12 15 10 10.4 1 4 1 1.6
a Composition of vitaminemineral mix (Agrimin) (quantity kg1), Vitamin A e 6,25,000 IU; Vitamin D3 e 62,500 IU; Vitamin E e 250 mg; Nicotinamide e 1 g; Cu e 312 mg; Co e 45 mg; Mg e 6 g; Fe e 1.5 g; Zn e 2.13 g; I e 156 mg; Se e 10 mg; Mn e 1.2 g; Ca e 247.34 g; P e 114.68 g; S e 12.2 g; Na e 5.8 mg; K e 48.05 mg.
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separated from blood by keeping the tubes in slanting position for about 2 h and thereafter it was centrifuged at 1370 g for 15 min at 4 C, followed by collection of straw coloured serum with micropipette and stored at 20 C for further analysis. 2.5. Growth parameters Fish were weighed on the 0th day of the experiment and 56th day of sampling. The weight was taken using an electronic balance (Citizen, India). The growth performance of fish was evaluated in terms of percent weight gain (%), feed conversion ratio (FCR), protein efficiency ratio (PER) and specific growth rate (SGR) using the following formulae:
Weight gainð%Þ ¼ ðFinal weight Initial weightÞ 100=Initial weight Specific growth rateð%Þ ¼ ½LnðFinal weightÞ LnðInitial weightÞ 100=total days of experiment Feed conversion ratio ¼ fFeed givenðdry weightÞ=
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buffer (A450 ¼ 0.5e0.7) was taken, to which 50 ml of diluted serum sample was added. The content of cuvette was mixed well for 15 s and measured using a spectrophotometer at 450 nm. The reading of lysis of the bacteria was immediately recorded at interval of 15, 30 and 270 s. A unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 per minute and lysozyme activity was expressed as U/min. 2.6.4. Serum antiprotease activity Total antiprotease activity was determined according to Bowden et al. [38] with minor modifications. 10 mL undiluted serum was initially incubated with trypsin solution in duplicate (Trypsin bovine pancreas, in 0.01 M Tris HCl, pH 8.2, Himedia, India), then 500 mL 2 mM BAPNA (sodium-benzoyl-DL-arginine-p-nitroanilide HCl, Himedia, India) substrate was added and the volume was made up to 1 ml with 0.1 M Tris HCl (pH 8.2). It was incubated at 22 C for 25 min. The reaction was stopped with 30% acetic acid and optical density was read at 415 nm in a microplate reader against a blank. The inhibitory capacity of antiprotease was expressed in terms of percentage trypsin inhibition as described by Zuo and Woo [39]. The percentage of trypsin inhibition was calculated by (trypsin blank (OD) sample (OD)/trypsin blank (OD) 100).
Body weight gainðwet weightÞg Protein efficiency ratio ¼ Net weight gainðwet weightÞ= Crude protein fed
2.6. Non-specific immune parameters 2.6.1. Nitroblue tetrazolium assay The respiratory burst activity of the neutrophils was measured by nitroblue tetrazolium (NBT) assay following the method of Secombes [35]. 100 ml of blood was placed into the wells of a flat bottom micro titre plate and incubated at 37 C for 1 h to allow adhesion of cells. The supernatant was discarded and the wells were washed three times with PBS. After washing, 100 ml of 0.2% NBT were added and incubated for 1 h. The cells were then fixed with 100% methanol for 2e3 min and washed three times with 70% methanol. The plates were air-dried and 120 ml of 2 N potassium hydroxide and 140 ml dimethyl sulphoxide were added to each well. The OD was recorded in an ELISA (BioTek Power Wave 340, India) reader at 620 nm. 2.6.2. Myeloperoxidase activity Total myeloperoxidase content present in serum was measured according to Quade and Roth [36] with slight modification. About 15 ml of serum was diluted with 135 ml of Hank’s Balanced Salt Solution (HBSS) without Ca2þ or Mg2þ in 96 well plates. 25 ml of 20 mM 3,30 -5,50 tetramethyl benzidine hydrochloride (TMB) (Himedia, India) and 25 ml of 5 mM H₂O₂ (Qualigens, India) (both substrates of MPO and prepared on same day) were added. The colour change reaction was stopped after 2 min by adding 50 ml of 4 M sulphuric acid (H2SO4). Plate was centrifuged at 400 g for 10 min, and 150 ml of the supernatants, present in each well, were transferred to new 96 well plates. The OD was recorded at 450 nm in a microplate reader (BioTek Power Wave 340, India). 2.6.3. Serum lysozyme activity Serum lysozyme activity was measured using colorimetric method by Anderson and Siwicki [37]. In a cuvette, 3 ml of Micrococcus luteus (ATCC 7468, India) suspension in phosphate
2.6.5. Phagocytic activity (PA) Phagocytic activity was detected using Staphylococcus aureus (Bangalore Geni, India) as described by Anderson and Siwicki [37]. A sample (0.1 ml) of blood was placed in a microtiter plate well, 0.1 ml of S. aureus 1 107 cfu ml1 (A450 ¼ 0.5e0.6) cells suspended in phosphate buffered saline (pH 7.2) were added and mixed well. The bacteriaeblood solution was incubated for 20 min at room temperature. 5 ml of this solution was taken on to a clean glass slide and a smear was prepared. The smear was air-dried, then fixed with ethanol (95%) for 5 min and airdried. The air-dried smear was stained with 7% Giemsa for 10 min. Two smears were made from each fish. The total of 100 neutrophils and monocytes from each smear were observed under the light microscope and the numbers of phagocytizing cells were counted. Phagocytic activity equals the number of phagocytizing cells divided by the total number of phagocytes counted.
PA ¼ Number of phagocytizing cells 100=Total number of phagocyte cells counted 2.7. Culture of pathogen A. hydrophila (ATCC 7966) was cultured in nutrient broth (Himedia) at 37 C for 24 h. The cultures were centrifuged at 3000 g for 10 min. The supernatants were discarded and the pellets were suspended in phosphate buffered saline (PBS, pH 7.4). The final bacterial concentration was adjusted to1.8 106 cfu ml1 by serial dilution. 2.7.1. Challenge study After 42 days of feeding with andrographolide supplemented diet, nine fishes from each treatment group were selected randomly and injected intraperitoneally with the A. hydrophila suspension of 0.2 ml (1.8 106 cfu ml1) and maintained for 14 days. The fishes were observed regularly for any overt signs of disease including behavioural abnormalities and mortality. The causative agent was confirmed by re-isolating A. hydrophila from the moribund fish.
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Table 2 Weight gain (BWG), feed conversion ratio (FCR), protein efficiency ratio (PER) and specific growth rate (SGR) in L. rohita under different treatment groups fed with various levels of andrographolide (values are mean SE). Treatment
BWG (in %) a
T0 T1 T2 T3 T4 T5
56.95 79.54e 88.06f 74.88d 67.19c 60.39b
0.632 0.469 0.611 0.888 0.716 0.646
FCR
PER f
3.46 2.52b 2.33a 2.67c 3.00d 3.26e
0.014 0.030 0.027 0.014 0.027 0.030
SGR a
0.93 1.27e 1.38f 1.20d 1.07c 0.98b
0.024 0.015 0.016 0.006 0.009 0.009
0.75a 0.97e 1.05f 0.93d 0.85c 0.78b
0.006 0.004 0.005 0.008 0.007 0.006
Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
2.8. Calculation of relative percentage survival Survival at the end of 14 days post infection was calculated using the following formula
Relative percentage survivalðRPS%Þ ¼
Number of surviving fishes after challenge 100 Number of fishes injected with bacteria
2.9. Statistical analysis The data were statistically analysed by statistical package SPSS version 16 in which data were subjected to one-way ANOVA and Duncan’s multiple range test (DMRT) was used to determine the significant differences between the means. Comparisons were made at 5% probability level.
3.2. Immunological studies The fishes were orally supplemented with dietary andrographolide at various levels showed significant (p < 0.05) difference in nonspecific immune responses for 14th, 28th and 42nd day of sampling. The NBT activity (OD at 620 nm) of the experimental groups were found to be significantly (p < 0.05) different in the treatment groups when compared with control and observed highest in T2 group on 14, 28 and 42 days of sampling. Moreover, the NBT activity noticeably showed increasing trend from 14 days to 42 days of sampling (Fig. 1). The myeloperoxidase activity (OD at 450 nm) of the experimental groups varied significantly (p < 0.05) in the treatment groups and showed decreasing trend from 14 to 28 days followed by an increasing trend from 28 days to 42 days of sampling. The highest myeloperoxidase activity was found to be in treatment group T3 followed by T2 and T5 (Fig. 2). The lysozyme activity (U/min) showed an increasing trend during sampling periods in dietary fed andrographolide groups from 14 days to 42 days and differed significantly (p < 0.05) among the treatment groups. Moreover, the highest lysozyme activity was found in T2 group followed by T3 in all sampling days (Fig. 3). The serum antiprotease activity (%) was found to be significantly (p < 0.05) higher in T1 group followed by T4 group and differed when compared with control. The serum antiprotease activity was observed in increasing trend during sampling periods (Fig. 4). The phagocytic activity (%) was found to be significantly (p < 0.05) different in andrographolide groups compared to control and showed increasing trend from 14 to 42 days of sampling (Fig. 5). The highest phagocytic activity was observed in T2 group in all the sampling periods. 3.3. Relative percentage survival
3. Results 3.1. Growth performance The fishes fed with various levels of andrographolide showed significant (p < 0.05) difference in growth parameters. The body weight gain (in %) was significantly (p < 0.05) higher in T2 group when compared with control. However, FCR was found to be significantly (p < 0.05) different among the treatment groups and specific growth rate was significantly (p < 0.05) higher in the treatment groups T2 and T3 when compared to control. The protein efficiency ratio (PER) was significantly (p < 0.05) higher in treatment group T3 and differed with control (Table 2).
Relative percentage survival of L. rohita after challenge with A. hydrophila in different experimental groups is presented in Fig. 6. The treatment groups fed with andrographolide supplemented diet showed significantly (p < 0.05) high disease resistance against A. hydrophila infection when compared with control group. The highest percentage survival was recorded in T2 (74.06%) followed by T1 (62.95%) and T3 (51.84%) groups. 4. Discussion Herbs containing bioactive compounds promote health, increase the body’s natural resistance to infection, and facilitate in
Fig. 1. NBT activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 2. Myeloperoxidase activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
prevention and treatment of various diseases [40e42]. To develop alternative practices for growth promotion and disease management in aquaculture, attention has also been focussed on identification of novel drugs, especially from plant sources. This study evaluated the effect of andrographolide on growth performance, non-specific immune parameters and disease resistance against A. hydrophila in L. rohita fingerlings. Several herbs have been tested for their growth promoting activity in aquatic animals [43]. The present study demonstrated that diets supplemented with andrographolide enhanced growth and disease resistance in L. rohita during the experimental periods. The dietary inclusion of algae has been shown to improve the growth performance of Acanthopagrus schlegeli [44]. The methanolic herbal extracts (Solanum trilobatum, A. paniculata and Psoralea corylifolia) helped to enhance growth and improved the health against bacterial infection in Penaeus monodon post larvae and A. paniculata was found to be more effective compared to other two herbal extracts [45]. The dietary inclusion of methanolic extracts of A. paniculata showed increase in growth performance of Oreochromis mossambicus [46]. Rainbow trout (Oncorhynchus mykiss) fed with ginger had significantly increased growth, feed conversion, and
protein efficiency [47]. Similarly, supplementing diets with acetone extract of ginger was reported to enhance the growth of tilapia (O. mossambicus) [48]. NBT activity is an indicator of oxygen dependent bactericidal activities. The increase in NBT level in the present study was found beneficial for fish in protecting them from invading pathogens. A variety of agents, including bacteria, bacterial products [49,50], levamisole [51], glucans [52], yeast RNA [53], garlic [16] azadirachtin [21,22] are known to stimulate phagocytes. It is well accepted that fish phagocytes after activation are able to generate superoxide anion and its reactive derivatives (i.e. hydrogen peroxide and hydroxyl radicals) during a period of intense oxygen consumption, called the respiratory burst [35]. These reactive oxygen species are considered toxic for fish bacterial pathogens [54]. In the present study, higher respiratory burst activity was observed in all treatment groups when compared to control. The similar results were also observed in L. rohita fed with varying levels of garlic and mango kernel [16,55] and levamisole [51] but vary with diet containing u-3 fatty acid [52]. Myeloperoxidase (MPO) is a peculiar and specific hemeprotein released by neutrophils. It is secreted and functional during
Fig. 3. Serum lysozyme activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 4. Antiprotease activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
activation of neutrophils, which plays an important role in the defence of an organism. MPO is abundantly stored and expressed in primary azurophilic granules of neutrophils. It utilizes hydrogen peroxide during respiratory burst to produce hypochlorous acid [56]. In the present study, the andrographolide supplemented dietary fed groups showed higher myeloperoxidase activity in comparison to control. Siwicki [57] reported that Cyprinus carpio injected with levamisole showed increased myeloperoxidase activity. The present findings support the reports of Kumari and Sahoo [58], Clarius batrachus fed with b-1, 3 glucan and in L. rohita injected with curcumin [59]. Similarly, higher myeloperoxidase activity was observed in Oplegnathus fasciatus fed with vitamin-E [60] and in Carassius auratus fed with azadirachtin [22]. Phagocytic activity is a key indicator of enhanced non-specific immune response. In the present study, the treatment groups fed with andrographolide supplemented diet was found to be significantly higher phagocytic activity compared to control with an
increasing trend in subsequent sampling periods. The present findings support the report of C. carpio fed with oligodeoxynucleotides supplemented diet [61]; greasy groupers (Epinephelus tauvina) fed with herbal diet containing purified active component of Ocimum sanctum, Withania somnifera, and Myristica fragrans [41], chinese sucker (Myxocyprinus asiaticus) fed with traditional chinese medicinal plant extracts [62] and also in C. auratus fed with azadirachtin supplemented diet [22]. The higher phagocytic activities in the treatment groups might be due to activation of phagocytic cells mostly neutrophils and monocytes in the circulation and andrographolide might have also activated the complement factors via the alternative pathway, which acts as opsonin leading to enhancement of phagocytosis. Lysozyme activity functions as a primary defence factor of nonspecific humoral immunity in preference to cellular defence mechanisms. Its ability to disrupt the cell walls of certain pathogens makes lysozyme a natural antagonist to harmful invaders like
Fig. 5. Phagocytic activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 6. Relative percentage survival (RPS) under different treatments fed with various levels of andrographolide in L. rohita (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
parasite, bacteria and virus. Lysozyme occurs prominently in fish serum and mucus [63]. Neutrophils are considered the source of lysozyme and the enzyme appears to be much more bactericidal than lysozyme of higher vertebrates [64]. An increased level has been considered to be a natural protective mechanism in fish [65]. In the present study, the serum lysozyme activity increased significantly in treatment groups fed with andrographolide at various levels. It was reported that herbal medicines could enhance the lysozyme activity in fish, as documented in chinese sucker [62] and jian carp Cyprinus carpio var. Jian [66] treated with traditional chinese medicinal plant extracts; in Oreochromis niloticus supplemented with Astragalus radix root [67] and C. auratus fed with azadirachtin [21,22]. Medicinal herbs containing potent bioactive substances may also influence digestive process by enhancing or impairing enzyme activity and improving or diminishing the digestibility of nutrients [68]. Principally, a1 protease inhibitor and a2 macroglobulin play a major role in restricting the ability of bacteria to invade and grow in fish by acting against proteases from pathogenic organisms [64]. In the present study, the andrographolide supplemented diet fed groups showed enhanced antiprotease activity in treatment groups during sampling days. This is in conformity with the findings of Rao and Chakrabarti [69] who reported that Catla catla fed with Achyranthes aspera (0.5%) diet for four weeks provided resistance against bacterial pathogens. Similar findings were also reported by Kaleeswaran et al. [70] who fed Cynodon dactylon mixed diet to C. catla. The treatment groups fed with dietary andrographolide (EC 50%) showed significantly enhanced resistance against A. hydrophila infection. This might be due to activation and enhancement of the non-specific immune system of experimental fish by andrographolide (EC 50%). Similar results in L. rohita fed with diets containing Mangifera indica kernel showed higher relative percentage survival after challenging with A. hydrophila [55]. Similarly, tilapia (O. niloticus) fed with two Chinese medicinal herbs [17] and ethanolic extracts of Psidium guajava [71]; and also in C. auratus fed with azadirachtin [22] showed higher RPS after challenged with A. hydrophila. In conclusion, the present study reports the effect of andrographolide on growth and non-specific immune parameters of L. rohita fingerlings. The activation of non-specific immune responses in L. rohita fingerlings could be due to the chemical entity especially diterpenoid lactone andrographolide that activated non-specific
cell receptors of vital immune cells and stimulated lymphoid cell colony and tissues. Further significant positive impact on various growth parameters and higher percentage survival rate of L. rohita challenged with A. hydrophila infection through 0.10% andrographolide supplemented diet, strengthened the immunostimulatory effect. The activation of non-specific immune profiles has direct evidence on health status of the host that finally improved the survival rate of the target species with proper use of immunostimulants. However, in vitro efficacy of andrographolide of various sources as well as the effect of andrographolide through different modes of administration should be further investigated in order to ascertain its molecular mechanism. Acknowledgements The authors would like to express their gratitude to Dr. W. S. Lakra, Director, Central Institute of Fisheries Education, Mumbai, India for kind encouragement and providing all necessary infrastructural facilities for carrying out the present work. They are thankful to Neocare Naturals Limited, Hyderabad, India for providing the andrographolide. References [1] Li P, Lewis DH, Galtin DM. Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass (Morone chrysops Morone saxatilis) to Streptococcus iniae infection. Fish Shellfish Immunol 2004;16:561e9. [2] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection. Fish Shellfish Immunol 2003;15:71e90. [3] Lightner DV. Diseases of cultured Penaeid shrimp. In: McVey JP, editor. CRC handbook of mariculture. Crustac Aquac, 1. Boca Raton, Florida, USA: CRC Press; 1983. p. 289e320. [4] Karunasagar I, Rosalind GM, Karunasagar I. Immunological response of the Indian major carps to Aeromonas hydrophila vaccine. J Fish Dis 1991;14: 413e7. [5] Amin NE, Abdallah IS, Elallawy T, Ahmed SM. Motile Aeromonas septicaemia among Tilapia nilotica (Sarotherodon niloticus) in upper Egypt. Fish Pathol 1985;20:93e7. [6] Miyazaki T, Jo Y. A histopathological study of motile aeromonad disease in ayu, (Plecoglosis altivelis). Fish Pathol 1985;20:55e60. [7] Rahman MH, Kusuda R, Kawai K. Virulence of starved Aeromonas hydrophila in cyprinid fish. Fish Pathol 1997;32:163e8. [8] Anbarasu K. Impact of immunosuppressors and potentiators on Aeromonas hydrophila bacterin immunised bagrid catfish, (Mystus gulio) (Hamilton) (Doctoral dissertation). Tiruchirapalli, India: Bharathidasan University; 2000. [9] Alderman DJ, Hasting TS. Antibiotic use in aquaculture: development of antibiotic resistance potential for consumer health risks. Int J Food Sci Technol 1998;33:139e55.
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Effect of dietary supplemented andrographolide on growth, non-specific immune parameters and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton) Kusunur Ahamed Basha, Ram Prakash Raman*, Kurcheti Pani Prasad, Kundan Kumar, Ezhil Nilavan, Saurav Kumar Central Institute of Fisheries Education, Panch Marg, Off Yari Road, Versova, Mumbai 400 061, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2013 Received in revised form 3 August 2013 Accepted 8 August 2013 Available online 22 August 2013
The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Andrographolide Non-specific Immune system Immunostimulant Aeromonas hydrophila
1. Introduction Aquaculture fish production increased significantly over the past few decades necessitating intensive fish culture practices. Since intensification is one of the prime necessities to cope with the present and future demand and hence, increase in productivity per unit space is performed by increasing the rearing density. Intensification leads to a number of other associated stressors like overcrowding, transport, handling, grading and poor water quality tend to adversely affect the health of the cultured fish [1]. These conditions produce poor physiological environment increasing the susceptibility of fish to infectious agents paving the way for the outbreak of a number of diseases due to an increasing range of
* Corresponding author. Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Mumbai 400 061, India. Tel.: þ91 9594073424; fax: þ91 2226361573. E-mail addresses: [email protected], [email protected] (R.P. Raman). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.005
pathogens. The disease outbreaks are increasingly being recognized as a potential constraint on aquaculture production trade, and cause massive economical loss through mortality or inferior meat quality, resulting in reduced profit margins [2]. Bacterial diseases have been reported to be a principal limiting factor in both wild and cultured fishes [3]. The most common and frequently encountered bacterial pathogen in India is Aeromonas hydrophila which causes severe damage to carp production [4]. A. hydrophila is associated with wide range of freshwater fishes. It is an important pathogen in causing stress related diseases in fish with the common symptoms of ulceration, exophthalmia and abdominal distension [5e7]. However, a potent drug and effective vaccine development against A. hydrophila continues to be a challenge to fish pathologists [8]. The most common approach to treat bacterial diseases is the application of antibiotics. However, accumulation of chemicals and antibiotics in the environment and in the fish flesh have led to the imposition of stringent regulations that limit the use of antibiotics [9]. A crucial approach in disease management should be based on application of preventive measures and one of the promising
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alternative or tool in fish disease management that have been evolved in response to these problems is “immunostimulants” which has overcome the lacuna of vaccines and probiotics [10]. An immunostimulant is a chemical, drug or action that enhances the defence mechanisms or immune response [11], thus rendering the animal more resistant to diseases. Non-specific defence mechanism plays an important role at all stages of fish infection. Fish particularly depend mostly on these non-specific mechanisms than do mammals [12]. Phytotherapy is the oldest form of health care known to man-kind. Bioactive substances present in herbs are well-known to have an antimicrobial and immunomodulatory properties. Herbs are an interesting alternative because they are inexpensive, renewable, locally available, user friendly and can be easily prepared [13]. Recently, there has been an increasing interest in the modulation of the non-specific immune system of fish, as a prophylactic measure against disease. Many of the medicinal plants such as Ocimum sanctum [14], Acalypha indica, Phyllanthus niruri, Azadirachta indica, Piper betle, Mentha piperita [15], Allium sativum [16], Astragalus membranaceus, Lonicera japonica [17] and Withania somnifera [18] have been shown to trigger innate immune system and enhance disease resistance against pathogenic organisms. An extensive work on the use of immunostimulatory herbs in fish was conducted by various researchers and they opined that the herbal extracts can be used in fish culture as an alternate to the chemotherapeutic agents [19e22]. Andrographis paniculata (AP) also called Kalmegh or “King of Bitters” belongs to family Acanthaceae, native to India and Sri Lanka. Mostly the leaves and roots are used for medicinal purposes. Diterpenoid lactone andrographolide (C20H30O5, melting point 230e 239 C) is the principle medicinal compound found in A. paniculata, which is mainly concentrated in leaves and can be easily isolated from the crude plant extracts as crystalline solid [23]. The various bioactivities of andrographolide such as hepatoprotective against various inducers [24], antimicrobial [25], antioxidant [26], antidiabetic [27], anti-inflammatory [28,29], anticancer [30], antitumour [31] and immunomodulator [32] properties have been evaluated. The ethyl alcohol extract and purified diterpene andrographolides are reported to stimulate both specific and non-specific immune responses in mice [33]. In the recent years herbs based growth promoters, immunostimulants and therapeutics which are ecofriendly are given more importance in aquaculture system. Thus keeping these aspects in view, the present study was undertaken to investigate the effect of andrographolide on the growth, nonespecific immune parameters and disease resistance against Aeromonas hydrophila in Indian major carp Labeo rohita. 2. Materials and methods
before cooking. Further, completely cooled dough was mixed with vitamins, mineral mixture and six of the experimental diets contained with andrographolide in different concentrations T0 (0.0%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) as listed in Table 1. The dough was pressed through a hand pelletizer to get uniform sized pellets. These pellets were dried at 40 C for 12 h. After drying, pellets were packed in polythene bags, sealed airtight and labelled according to the different concentrations of treatments, for further use. The feed was having (34.89e36.66%) of crude protein, (404.3e408.04 kcal 100 g1) of isocaloric, (9.40e 10.8%) of lipid content, (25.05e26.14%) of carbohydrate and (3.01e 3.58%) of ash content. 2.3. Experimental design One hundred and eighty L. rohita fingerlings were equally and randomly distributed in six experimental groups in triplicate following a completely randomized design (CRD). The experimental trial for effects of andrographolide on growth, non-specific immune parameters in L. rohita was conducted with feeding the various level of andrographolide as control T0 (without andrographolide), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for a period of 42 days. The fish were fed with the experimental diet at the rate of 3% of body weight twice a day at 09:00 and 17:00 h to approximate satiation for 42 days. The physicochemical parameters of water such as temperature (24.3e 25.8 C), pH (7.2 0.4), dissolved oxygen (6.6e6.9 mg L1), ammonia (0.01 0.005 mg L1), nitrate (0.02e0.06 mg L1) and nitrite (0.001e0.004 mg L1) were maintained in optimum condition during the experimental period. Six fish were sampled from each treatment group on 14th, 28th and 42nd day and blood was drawn for different immunological assays. After 42 days, nine fishes from each treatment were segregated for challenge study particular injection with A. hydrophila and post challenge study was continued up to 56 days. At the end of 56 days, growth parameters and relative percentage survival rate were estimated for disease resistance against A. hydrophila. 2.4. Collection of blood and serum Each fish was anesthetized with clove oil (Merck, Germany) at 50 ml per litre of water before collecting blood samples from fish. Blood was drawn from caudal vein of fish by using 1.0 ml hypodermal syringe and 24 gauge needles, which was rinsed with 2.7% EDTA solution before use. The collected blood was immediately transferred to the test tube coated with thin layer of EDTA (as an anticoagulant) and shaked well in order to prevent clotting of blood. Serum was collected without using anticoagulant and was
2.1. Experimental animal and regime Labeo rohita fingerlings with an average weight of 9.50 0.50 g were procured from Malad Fish Farm, Maharashtra, India. The fishes were acclimatized for 15 days in laboratory condition in 1000 L FRP tanks at 25e27 C, under continuous aeration. The fishes were fed with control pelleted diet at the rate of 3% of body weight twice a day. 2.2. Preparation of experimental diet The andrographolide (EC 50%) was procured from Neocare Naturals Limited, Hyderabad, India and used for preparation of six isonitrogenous experimental diet following the method of Rao et al. [34] with slight modification. Experimental diets were prepared by mixing all the ingredients in required quantity along with water to form dough and calculated amount of oil was added to the dough
Table 1 Composition of experimental diets (in 100 g). Ingredients (g)
T0 (control)
T1
T2
T3
T4
T5
Fish meal Soya flour Corn flour Wheat flour Rice bran GOC CMC Veg. oil Vit & minerala Andrographolide (EC 50%)
25 20 12 15 10 12 1 4 1 e
25 20 12 15 10 11.9 1 4 1 0.1
25 20 12 15 10 11.8 1 4 1 0.2
25 20 12 15 10 11.6 1 4 1 0.4
25 20 12 15 10 11.2 1 4 1 0.8
25 20 12 15 10 10.4 1 4 1 1.6
a Composition of vitaminemineral mix (Agrimin) (quantity kg1), Vitamin A e 6,25,000 IU; Vitamin D3 e 62,500 IU; Vitamin E e 250 mg; Nicotinamide e 1 g; Cu e 312 mg; Co e 45 mg; Mg e 6 g; Fe e 1.5 g; Zn e 2.13 g; I e 156 mg; Se e 10 mg; Mn e 1.2 g; Ca e 247.34 g; P e 114.68 g; S e 12.2 g; Na e 5.8 mg; K e 48.05 mg.
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separated from blood by keeping the tubes in slanting position for about 2 h and thereafter it was centrifuged at 1370 g for 15 min at 4 C, followed by collection of straw coloured serum with micropipette and stored at 20 C for further analysis. 2.5. Growth parameters Fish were weighed on the 0th day of the experiment and 56th day of sampling. The weight was taken using an electronic balance (Citizen, India). The growth performance of fish was evaluated in terms of percent weight gain (%), feed conversion ratio (FCR), protein efficiency ratio (PER) and specific growth rate (SGR) using the following formulae:
Weight gainð%Þ ¼ ðFinal weight Initial weightÞ 100=Initial weight Specific growth rateð%Þ ¼ ½LnðFinal weightÞ LnðInitial weightÞ 100=total days of experiment Feed conversion ratio ¼ fFeed givenðdry weightÞ=
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buffer (A450 ¼ 0.5e0.7) was taken, to which 50 ml of diluted serum sample was added. The content of cuvette was mixed well for 15 s and measured using a spectrophotometer at 450 nm. The reading of lysis of the bacteria was immediately recorded at interval of 15, 30 and 270 s. A unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 per minute and lysozyme activity was expressed as U/min. 2.6.4. Serum antiprotease activity Total antiprotease activity was determined according to Bowden et al. [38] with minor modifications. 10 mL undiluted serum was initially incubated with trypsin solution in duplicate (Trypsin bovine pancreas, in 0.01 M Tris HCl, pH 8.2, Himedia, India), then 500 mL 2 mM BAPNA (sodium-benzoyl-DL-arginine-p-nitroanilide HCl, Himedia, India) substrate was added and the volume was made up to 1 ml with 0.1 M Tris HCl (pH 8.2). It was incubated at 22 C for 25 min. The reaction was stopped with 30% acetic acid and optical density was read at 415 nm in a microplate reader against a blank. The inhibitory capacity of antiprotease was expressed in terms of percentage trypsin inhibition as described by Zuo and Woo [39]. The percentage of trypsin inhibition was calculated by (trypsin blank (OD) sample (OD)/trypsin blank (OD) 100).
Body weight gainðwet weightÞg Protein efficiency ratio ¼ Net weight gainðwet weightÞ= Crude protein fed
2.6. Non-specific immune parameters 2.6.1. Nitroblue tetrazolium assay The respiratory burst activity of the neutrophils was measured by nitroblue tetrazolium (NBT) assay following the method of Secombes [35]. 100 ml of blood was placed into the wells of a flat bottom micro titre plate and incubated at 37 C for 1 h to allow adhesion of cells. The supernatant was discarded and the wells were washed three times with PBS. After washing, 100 ml of 0.2% NBT were added and incubated for 1 h. The cells were then fixed with 100% methanol for 2e3 min and washed three times with 70% methanol. The plates were air-dried and 120 ml of 2 N potassium hydroxide and 140 ml dimethyl sulphoxide were added to each well. The OD was recorded in an ELISA (BioTek Power Wave 340, India) reader at 620 nm. 2.6.2. Myeloperoxidase activity Total myeloperoxidase content present in serum was measured according to Quade and Roth [36] with slight modification. About 15 ml of serum was diluted with 135 ml of Hank’s Balanced Salt Solution (HBSS) without Ca2þ or Mg2þ in 96 well plates. 25 ml of 20 mM 3,30 -5,50 tetramethyl benzidine hydrochloride (TMB) (Himedia, India) and 25 ml of 5 mM H₂O₂ (Qualigens, India) (both substrates of MPO and prepared on same day) were added. The colour change reaction was stopped after 2 min by adding 50 ml of 4 M sulphuric acid (H2SO4). Plate was centrifuged at 400 g for 10 min, and 150 ml of the supernatants, present in each well, were transferred to new 96 well plates. The OD was recorded at 450 nm in a microplate reader (BioTek Power Wave 340, India). 2.6.3. Serum lysozyme activity Serum lysozyme activity was measured using colorimetric method by Anderson and Siwicki [37]. In a cuvette, 3 ml of Micrococcus luteus (ATCC 7468, India) suspension in phosphate
2.6.5. Phagocytic activity (PA) Phagocytic activity was detected using Staphylococcus aureus (Bangalore Geni, India) as described by Anderson and Siwicki [37]. A sample (0.1 ml) of blood was placed in a microtiter plate well, 0.1 ml of S. aureus 1 107 cfu ml1 (A450 ¼ 0.5e0.6) cells suspended in phosphate buffered saline (pH 7.2) were added and mixed well. The bacteriaeblood solution was incubated for 20 min at room temperature. 5 ml of this solution was taken on to a clean glass slide and a smear was prepared. The smear was air-dried, then fixed with ethanol (95%) for 5 min and airdried. The air-dried smear was stained with 7% Giemsa for 10 min. Two smears were made from each fish. The total of 100 neutrophils and monocytes from each smear were observed under the light microscope and the numbers of phagocytizing cells were counted. Phagocytic activity equals the number of phagocytizing cells divided by the total number of phagocytes counted.
PA ¼ Number of phagocytizing cells 100=Total number of phagocyte cells counted 2.7. Culture of pathogen A. hydrophila (ATCC 7966) was cultured in nutrient broth (Himedia) at 37 C for 24 h. The cultures were centrifuged at 3000 g for 10 min. The supernatants were discarded and the pellets were suspended in phosphate buffered saline (PBS, pH 7.4). The final bacterial concentration was adjusted to1.8 106 cfu ml1 by serial dilution. 2.7.1. Challenge study After 42 days of feeding with andrographolide supplemented diet, nine fishes from each treatment group were selected randomly and injected intraperitoneally with the A. hydrophila suspension of 0.2 ml (1.8 106 cfu ml1) and maintained for 14 days. The fishes were observed regularly for any overt signs of disease including behavioural abnormalities and mortality. The causative agent was confirmed by re-isolating A. hydrophila from the moribund fish.
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Table 2 Weight gain (BWG), feed conversion ratio (FCR), protein efficiency ratio (PER) and specific growth rate (SGR) in L. rohita under different treatment groups fed with various levels of andrographolide (values are mean SE). Treatment
BWG (in %) a
T0 T1 T2 T3 T4 T5
56.95 79.54e 88.06f 74.88d 67.19c 60.39b
0.632 0.469 0.611 0.888 0.716 0.646
FCR
PER f
3.46 2.52b 2.33a 2.67c 3.00d 3.26e
0.014 0.030 0.027 0.014 0.027 0.030
SGR a
0.93 1.27e 1.38f 1.20d 1.07c 0.98b
0.024 0.015 0.016 0.006 0.009 0.009
0.75a 0.97e 1.05f 0.93d 0.85c 0.78b
0.006 0.004 0.005 0.008 0.007 0.006
Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
2.8. Calculation of relative percentage survival Survival at the end of 14 days post infection was calculated using the following formula
Relative percentage survivalðRPS%Þ ¼
Number of surviving fishes after challenge 100 Number of fishes injected with bacteria
2.9. Statistical analysis The data were statistically analysed by statistical package SPSS version 16 in which data were subjected to one-way ANOVA and Duncan’s multiple range test (DMRT) was used to determine the significant differences between the means. Comparisons were made at 5% probability level.
3.2. Immunological studies The fishes were orally supplemented with dietary andrographolide at various levels showed significant (p < 0.05) difference in nonspecific immune responses for 14th, 28th and 42nd day of sampling. The NBT activity (OD at 620 nm) of the experimental groups were found to be significantly (p < 0.05) different in the treatment groups when compared with control and observed highest in T2 group on 14, 28 and 42 days of sampling. Moreover, the NBT activity noticeably showed increasing trend from 14 days to 42 days of sampling (Fig. 1). The myeloperoxidase activity (OD at 450 nm) of the experimental groups varied significantly (p < 0.05) in the treatment groups and showed decreasing trend from 14 to 28 days followed by an increasing trend from 28 days to 42 days of sampling. The highest myeloperoxidase activity was found to be in treatment group T3 followed by T2 and T5 (Fig. 2). The lysozyme activity (U/min) showed an increasing trend during sampling periods in dietary fed andrographolide groups from 14 days to 42 days and differed significantly (p < 0.05) among the treatment groups. Moreover, the highest lysozyme activity was found in T2 group followed by T3 in all sampling days (Fig. 3). The serum antiprotease activity (%) was found to be significantly (p < 0.05) higher in T1 group followed by T4 group and differed when compared with control. The serum antiprotease activity was observed in increasing trend during sampling periods (Fig. 4). The phagocytic activity (%) was found to be significantly (p < 0.05) different in andrographolide groups compared to control and showed increasing trend from 14 to 42 days of sampling (Fig. 5). The highest phagocytic activity was observed in T2 group in all the sampling periods. 3.3. Relative percentage survival
3. Results 3.1. Growth performance The fishes fed with various levels of andrographolide showed significant (p < 0.05) difference in growth parameters. The body weight gain (in %) was significantly (p < 0.05) higher in T2 group when compared with control. However, FCR was found to be significantly (p < 0.05) different among the treatment groups and specific growth rate was significantly (p < 0.05) higher in the treatment groups T2 and T3 when compared to control. The protein efficiency ratio (PER) was significantly (p < 0.05) higher in treatment group T3 and differed with control (Table 2).
Relative percentage survival of L. rohita after challenge with A. hydrophila in different experimental groups is presented in Fig. 6. The treatment groups fed with andrographolide supplemented diet showed significantly (p < 0.05) high disease resistance against A. hydrophila infection when compared with control group. The highest percentage survival was recorded in T2 (74.06%) followed by T1 (62.95%) and T3 (51.84%) groups. 4. Discussion Herbs containing bioactive compounds promote health, increase the body’s natural resistance to infection, and facilitate in
Fig. 1. NBT activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 2. Myeloperoxidase activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
prevention and treatment of various diseases [40e42]. To develop alternative practices for growth promotion and disease management in aquaculture, attention has also been focussed on identification of novel drugs, especially from plant sources. This study evaluated the effect of andrographolide on growth performance, non-specific immune parameters and disease resistance against A. hydrophila in L. rohita fingerlings. Several herbs have been tested for their growth promoting activity in aquatic animals [43]. The present study demonstrated that diets supplemented with andrographolide enhanced growth and disease resistance in L. rohita during the experimental periods. The dietary inclusion of algae has been shown to improve the growth performance of Acanthopagrus schlegeli [44]. The methanolic herbal extracts (Solanum trilobatum, A. paniculata and Psoralea corylifolia) helped to enhance growth and improved the health against bacterial infection in Penaeus monodon post larvae and A. paniculata was found to be more effective compared to other two herbal extracts [45]. The dietary inclusion of methanolic extracts of A. paniculata showed increase in growth performance of Oreochromis mossambicus [46]. Rainbow trout (Oncorhynchus mykiss) fed with ginger had significantly increased growth, feed conversion, and
protein efficiency [47]. Similarly, supplementing diets with acetone extract of ginger was reported to enhance the growth of tilapia (O. mossambicus) [48]. NBT activity is an indicator of oxygen dependent bactericidal activities. The increase in NBT level in the present study was found beneficial for fish in protecting them from invading pathogens. A variety of agents, including bacteria, bacterial products [49,50], levamisole [51], glucans [52], yeast RNA [53], garlic [16] azadirachtin [21,22] are known to stimulate phagocytes. It is well accepted that fish phagocytes after activation are able to generate superoxide anion and its reactive derivatives (i.e. hydrogen peroxide and hydroxyl radicals) during a period of intense oxygen consumption, called the respiratory burst [35]. These reactive oxygen species are considered toxic for fish bacterial pathogens [54]. In the present study, higher respiratory burst activity was observed in all treatment groups when compared to control. The similar results were also observed in L. rohita fed with varying levels of garlic and mango kernel [16,55] and levamisole [51] but vary with diet containing u-3 fatty acid [52]. Myeloperoxidase (MPO) is a peculiar and specific hemeprotein released by neutrophils. It is secreted and functional during
Fig. 3. Serum lysozyme activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 4. Antiprotease activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
activation of neutrophils, which plays an important role in the defence of an organism. MPO is abundantly stored and expressed in primary azurophilic granules of neutrophils. It utilizes hydrogen peroxide during respiratory burst to produce hypochlorous acid [56]. In the present study, the andrographolide supplemented dietary fed groups showed higher myeloperoxidase activity in comparison to control. Siwicki [57] reported that Cyprinus carpio injected with levamisole showed increased myeloperoxidase activity. The present findings support the reports of Kumari and Sahoo [58], Clarius batrachus fed with b-1, 3 glucan and in L. rohita injected with curcumin [59]. Similarly, higher myeloperoxidase activity was observed in Oplegnathus fasciatus fed with vitamin-E [60] and in Carassius auratus fed with azadirachtin [22]. Phagocytic activity is a key indicator of enhanced non-specific immune response. In the present study, the treatment groups fed with andrographolide supplemented diet was found to be significantly higher phagocytic activity compared to control with an
increasing trend in subsequent sampling periods. The present findings support the report of C. carpio fed with oligodeoxynucleotides supplemented diet [61]; greasy groupers (Epinephelus tauvina) fed with herbal diet containing purified active component of Ocimum sanctum, Withania somnifera, and Myristica fragrans [41], chinese sucker (Myxocyprinus asiaticus) fed with traditional chinese medicinal plant extracts [62] and also in C. auratus fed with azadirachtin supplemented diet [22]. The higher phagocytic activities in the treatment groups might be due to activation of phagocytic cells mostly neutrophils and monocytes in the circulation and andrographolide might have also activated the complement factors via the alternative pathway, which acts as opsonin leading to enhancement of phagocytosis. Lysozyme activity functions as a primary defence factor of nonspecific humoral immunity in preference to cellular defence mechanisms. Its ability to disrupt the cell walls of certain pathogens makes lysozyme a natural antagonist to harmful invaders like
Fig. 5. Phagocytic activity under different treatments fed with various levels of andrographolide in L. rohita during different sampling days (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
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Fig. 6. Relative percentage survival (RPS) under different treatments fed with various levels of andrographolide in L. rohita (values are mean SE). Mean values with different superscript with in a column for a parameter is significantly different, (p < 0.05).
parasite, bacteria and virus. Lysozyme occurs prominently in fish serum and mucus [63]. Neutrophils are considered the source of lysozyme and the enzyme appears to be much more bactericidal than lysozyme of higher vertebrates [64]. An increased level has been considered to be a natural protective mechanism in fish [65]. In the present study, the serum lysozyme activity increased significantly in treatment groups fed with andrographolide at various levels. It was reported that herbal medicines could enhance the lysozyme activity in fish, as documented in chinese sucker [62] and jian carp Cyprinus carpio var. Jian [66] treated with traditional chinese medicinal plant extracts; in Oreochromis niloticus supplemented with Astragalus radix root [67] and C. auratus fed with azadirachtin [21,22]. Medicinal herbs containing potent bioactive substances may also influence digestive process by enhancing or impairing enzyme activity and improving or diminishing the digestibility of nutrients [68]. Principally, a1 protease inhibitor and a2 macroglobulin play a major role in restricting the ability of bacteria to invade and grow in fish by acting against proteases from pathogenic organisms [64]. In the present study, the andrographolide supplemented diet fed groups showed enhanced antiprotease activity in treatment groups during sampling days. This is in conformity with the findings of Rao and Chakrabarti [69] who reported that Catla catla fed with Achyranthes aspera (0.5%) diet for four weeks provided resistance against bacterial pathogens. Similar findings were also reported by Kaleeswaran et al. [70] who fed Cynodon dactylon mixed diet to C. catla. The treatment groups fed with dietary andrographolide (EC 50%) showed significantly enhanced resistance against A. hydrophila infection. This might be due to activation and enhancement of the non-specific immune system of experimental fish by andrographolide (EC 50%). Similar results in L. rohita fed with diets containing Mangifera indica kernel showed higher relative percentage survival after challenging with A. hydrophila [55]. Similarly, tilapia (O. niloticus) fed with two Chinese medicinal herbs [17] and ethanolic extracts of Psidium guajava [71]; and also in C. auratus fed with azadirachtin [22] showed higher RPS after challenged with A. hydrophila. In conclusion, the present study reports the effect of andrographolide on growth and non-specific immune parameters of L. rohita fingerlings. The activation of non-specific immune responses in L. rohita fingerlings could be due to the chemical entity especially diterpenoid lactone andrographolide that activated non-specific
cell receptors of vital immune cells and stimulated lymphoid cell colony and tissues. Further significant positive impact on various growth parameters and higher percentage survival rate of L. rohita challenged with A. hydrophila infection through 0.10% andrographolide supplemented diet, strengthened the immunostimulatory effect. The activation of non-specific immune profiles has direct evidence on health status of the host that finally improved the survival rate of the target species with proper use of immunostimulants. However, in vitro efficacy of andrographolide of various sources as well as the effect of andrographolide through different modes of administration should be further investigated in order to ascertain its molecular mechanism. Acknowledgements The authors would like to express their gratitude to Dr. W. S. Lakra, Director, Central Institute of Fisheries Education, Mumbai, India for kind encouragement and providing all necessary infrastructural facilities for carrying out the present work. They are thankful to Neocare Naturals Limited, Hyderabad, India for providing the andrographolide. References [1] Li P, Lewis DH, Galtin DM. Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass (Morone chrysops Morone saxatilis) to Streptococcus iniae infection. Fish Shellfish Immunol 2004;16:561e9. [2] Smith VJ, Brown JH, Hauton C. Immunostimulation in crustaceans: does it really protect against infection. Fish Shellfish Immunol 2003;15:71e90. [3] Lightner DV. Diseases of cultured Penaeid shrimp. In: McVey JP, editor. CRC handbook of mariculture. Crustac Aquac, 1. Boca Raton, Florida, USA: CRC Press; 1983. p. 289e320. [4] Karunasagar I, Rosalind GM, Karunasagar I. Immunological response of the Indian major carps to Aeromonas hydrophila vaccine. J Fish Dis 1991;14: 413e7. [5] Amin NE, Abdallah IS, Elallawy T, Ahmed SM. Motile Aeromonas septicaemia among Tilapia nilotica (Sarotherodon niloticus) in upper Egypt. Fish Pathol 1985;20:93e7. [6] Miyazaki T, Jo Y. A histopathological study of motile aeromonad disease in ayu, (Plecoglosis altivelis). Fish Pathol 1985;20:55e60. [7] Rahman MH, Kusuda R, Kawai K. Virulence of starved Aeromonas hydrophila in cyprinid fish. Fish Pathol 1997;32:163e8. [8] Anbarasu K. Impact of immunosuppressors and potentiators on Aeromonas hydrophila bacterin immunised bagrid catfish, (Mystus gulio) (Hamilton) (Doctoral dissertation). Tiruchirapalli, India: Bharathidasan University; 2000. [9] Alderman DJ, Hasting TS. Antibiotic use in aquaculture: development of antibiotic resistance potential for consumer health risks. Int J Food Sci Technol 1998;33:139e55.
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