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Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla
International Immunopharmacology 6 (2006) 782 – 790 www.elsevier.com/locate/intimp
Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla Rina Chakrabarti *, Rao.Y. Vasudeva Department of Zoology, University of Delhi, Delhi – 110 007, India Received 10 March 2005; received in revised form 15 September 2005; accepted 21 November 2005
Abstract Achyranthes aspera, an Indian medicinal plant (family: Amaranthaceae) was incorporated in artificial fish diet, and fed to catla Catla catla. After 4 weeks of feeding, fish were immunized with bovine serum albumin (BSA), spleen and blood were sampled on weekly intervals for four times after immunization. Antigen-specific antibody level in serum was determined by ELISA. Antigen clearance was determined in spleen by immuno-electron microscopy. Achyranthes has significantly ( P b 0.05) enhanced the BSAspecific antibody titers than the untreated control group throughout the study period. The efficiency of antigen clearance was also enhanced in C. catla treated with Achyranthes. D 2005 Elsevier B.V. All rights reserved. Keywords: Catla catla; BSA; ELISA; Antibody response; Antigen clearance
1. Introduction Immunostimulants activate several components of the immune system such as phagocytes, natural killer cells, T-lymphocytes, B-lymphocytes, complement and lysozyme. Such substances may also, but not necessarily, render animals more resistant to infectious diseases and reduce the risk of disease outbreaks if administrated prior to situations known to result in stress and impaired general performance, or prior to expected increase in exposure to pathogenic microorganisms and parasites. In addition, aquaculture may benefit from the use of such immunostimulants when they are used prior to, and during, developmental phases when the organisms are particularly susceptible to infectious agents [1]. Several reports are available on immunostimulants
* Corresponding author. Tel.: +91 11 27666496. E-mail address: [email protected] (R. Chakrabarti). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.11.020
originated from plant, animal and bacterial sources. Bath treatment of Catla catla catla spawn with 1% crude extract of neem, garlic and turmeric (1 : 1 : 1) [2], and oral treatment of Labeo rohita rohu with Catharanthus roseus extract [3] and Seriola quinqueradiata yellowtail with glycyrrhizin [4] enhanced immune response and disease resistance against experimental infection with Aeromonas hydrophila and Edwardsiella seriola.
Table 1 Composition of artificial diets Ingredients/100 g of feed
Experimental diet
Control diet
Fish meal (g) Wheat flour (g) Cod liver oil (g) Supradin — vitamin and mineral premix (g) Seed of Achyranthes aspera (g)
32.88 54.62 10 2
32.88 55.12 10 2
0.5
–
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
783
Fig. 1. Effect of Achyranthes aspera on BSA-specific serum antibody titers in Catla catla. Each value represents the mean F SE of four fish (*P b 0.01; **P b 0.05).
Achyranthes aspera L., (Division: Angiospermae; Class: Dicotyledones; Sub-class: Monochlamydeae; Series: Curvembryeae; Family: Amaranthaceae) is an indigenous medicinal plant of Asia, South America, and Africa that is commonly used by traditional healers for
the treatment of fever, especially malarial fever, dysentery, asthma, hypertension and diabetes [5–7]. Kushta Hartal warqi (= arsenic sulfide + Callicarpa arborea + A. aspera) is used in rheumatism and paralysis [8]. The roots of Achyranthes are reported to have application in
Fig. 2. Electron micrographs of spleen of control Catla catla on day-7 after immunization (7100, 3500, 7100, 4400).
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infantile diarrhea and cold [9]. Leaves are reported to possess hypoglycemic activity [10] and also employed against asthma [11]. Indian major carps (C. catla, L. rohita and Cirrhinus mrigala) are very prone to diseases and huge losses have been experienced by Indian aquaculture industry [12]. Since A. aspera holds a reputed position as medicinal herb in different systems of medicine in India, we were interested to see its effect on the immunity and disease resistance of fishes, and it was found that feeding with Achyranthes protected the fish from infection [13]. To know how rapidly the Achyranthes induces the elimination of the injected antigen, we have conducted the present experiment. 2. Materials and methods 2.1. Animals C. catla (150 F 20 g) were obtained from the local fish market, Delhi, and were acclimatized for three weeks in outdoor cemented tanks (170 L). Experiment was conducted under two feeding regimes, control diet and A. aspera incor-
porated diet. Four replicates were used for each feeding regime, seven fish were kept in each replicate. Temperature and pH ranged between 29–33 8C and 7.4–8.0, respectively, during the experiment. Dissolved oxygen level was maintained above 5 mg/L with the help of aerators throughout the experiment. 2.2. Experimental diet and feeding Dried seeds were collected from the spike of A. aspera grown in the field near the department. The seeds were washed with tap water to remove any dust and air-dried. 0.5% measured amount of seed was ground in mortar and pestle with water. The whole extract was added to the feed ingredients (fishmeal, wheat flour, cod liver oil, vitamin and mineral premix) to prepare the experimental diet. The prepared mixture was then passed through a manual feed maker using 1 mm sieve and the threads formed were air-dried. The dried threads were further broken into smaller pieces manually. Normal diet was prepared using same ingredients, except A. aspera seeds (Table 1). Test groups were fed with experimental diet containing A. aspera, and control groups were fed with normal diet. Feeding was started four weeks prior to the immunization and continued till the end of the experiment. Both groups of fish were fed at 1% of body weight at 9:00 AM.
Fig. 3. Electron micrographs of spleen of control Catla catla on day-14 after immunization (3500, 7100, 8900, 7100).
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
2.3. Antigen and immunization After four weeks (28 days) of feeding, fish were anaesthetized with MS-222 and injected intraperitoneally with 500 Al of bovine serum albumin (BSA, Fraction-V, Merck) dissolved (i.e. 10 mg of BSA/fish) in phosphate buffered saline (PBS). 2.4. Sampling Blood was collected from both control and Achyranthes treated test groups, on the 7th, 14th, 21st and 28th day after immunization, allowed to clot at room temperature and serum was obtained by centrifugation. At each sampling time, four fish were sacrificed from each group. After collecting the blood, spleen samples were aseptically dissected out for immuno-electron microscopy. 2.5. Determination of antigen-specific antibody titers by ELISA The wells of the microtiter plate (greiner Bio-One, Germany, ELISA plate, Microlon, 96W, Flat-bott, High binding) were coated with 100 Al of fish serum (2-fold serial dilutions) diluted in phosphate buffered saline (pH
785
7.4). The plates were incubated for 12 h at 4 8C. After incubation the wells were washed three times with PBS containing tween-20 (0.05%). The free binding sites of the wells were blocked by adding 300 Al of 5% gelatin dissolved in PBS per well and incubated for 12 h at 4 8C. The wells were washed with PBS-tween, and 100 Al of bovine serum albumin (BSA) in PBS was added to each well (100 ng BSA/well) and incubated for 2 h at 4 8C. The wells were washed with PBS-tween, and 100 Al of rabbit antiBSA serum diluted to 1 : 500 in PBS was added to each well and incubated for 1 h at 37 8C. The wells were washed with PBS-tween, and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase, diluted to 1 : 1000 in PBS, was added to all wells (100 Al/well) and incubated for 1 h at 37 8C. The wells were washed with PBS-tween, and 100 Al of substrate (containing 13 mg of o-phenylenediamine dihydrochloride and 10 Al of hydrogen peroxide in 10 ml of citrate–phosphate buffer, pH 5.0) was added to all wells and incubated for 10 min. After the development of color, the reaction was terminated by adding 50 Al of 1M oxalic acid per well. The optical density was measured at 492 nm in an automatic microplate reader (Microscan — MS5605A, Electronic Corporation of India Ltd.). The highest dilution of the serum that gave the OD N 0.1 was taken as the titer.
Fig. 4. Electron micrographs of spleen of control Catla catla on day-21 after immunization (7100, 8900, 4400, 7100).
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2.6. Determination of antigen clearance by immuno-electron microscopy (IEM) Few pieces of spleen (1 mm thickness) of catla were fixed in a solution containing 1% glutaraldehide and 2% paraformaldehide in 0.1 M phosphate buffer for overnight. Then tissues were washed with 0.1 M phosphate buffer and dehydrated with gradient ethanol (30–100%) at 4 8C. The tissues were infiltrated with a mixture of ethanol and LR White (1 : 1) followed by pure LR White (TAAB). Tissues were embedded in beam capsule filled with LR White and polymerized at 55 8C. Ultra thin sections (70 nm) were cut in a microtome (Reichert Jung Ultracut-E) and grids were prepared. The sections on the grid were blocked with 2% fish gelatin in 0.1 M phosphate buffer for 2 h. After blocking, the sections were washed and labeled by incubating with rabbit anti-BSA polyclonal antibodies (ICN Biochemicals, USA) diluted to 1 : 500 in phosphate buffer containing 1% fish gelatin and incubated for 12 h. The grids were washed thoroughly with 1% fish gelatin in phosphate buffer and labeled with secondary antibody, i.e. goat anti-rabbit IgG conjugated with 15 nm gold particles (TAAB), diluted to 1 : 100 in phosphate buffer containing 1% fish gelatin, and incubated for 2 h. The grids were washed with phosphate buffer containing 1% fish gelatin, and finally washed with double distilled water. After
labeling, the sections were stained with uranyl acetate and lead citrate and observed for labeled particles under electron microscope, (Fei-Philips Morgagni 268D Digital TEM) with image analysis system. 2.7. Statistics The data was statistically analyzed using students’ t-test in Microsoft Excel. The level of significance was P b 0.05. 3. Results 3.1. Antibody response In catla, BSA-specific antibody level was peaked on day-7 and then gradually decreased up to day-28. The trend was similar in both control and Achyranthes treated groups. The anti-BSA antibody level was always higher in Achyranthes treated group than the control throughout the study period (Fig. 1). The difference was significant ( P b 0.05) throughout the study period, except on day-28. The titers reduced by 97% in control group and 94% in test group on day-28, compared with their respective titers of day-7. In the control group, the average antibody titers were 896 103, 264 103, 40 103 and 26 103 on days 7, 14, 21 and 28, respectively. In
Fig. 5. Electron micrographs of spleen of control Catla catla on day-28 after immunization (5600, 5600, 5600, 5600).
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
Achyranthes treated group, the average titers were 2304 103, 704 103, 288 103 and 128 103 on days 7, 14, 21 and 28, respectively. The anti-BSA antibody titers of Achyranthes treated group was 2.5–7.2 times higher than the control in various days of sampling. 3.2. Antigen clearance The presence of gold particles locates the BSA in the immuno-labeled spleen sections. In control group catla, the amount of antigen (BSA) present in the spleen on day-7 was shown in Fig. 2. It was seen that on day-7 the particles were densely crowded throughout the sections, indicating the abundant presence of BSA. On day-14, the amount of antigen present in the control group catla was shown in Fig. 3. The labeling appeared densely throughout the sections indicating the ample presence of BSA. On day-21, the particles were still densely crowded, indicating the presence of BSA abundantly (Fig. 4). On day-28, the antigen presence in the spleen of control catla was shown in Fig. 5. It was noticed that the density of the particles was lower in spleen sections compared with the earlier days samples of the control catla. Nevertheless the particles were found abundantly throughout the sections, indicating the plentiful presence of BSA. All these results indicate that the injected antigen, i.e. BSA was still present in large quantities in
787
control catla even on day-28 after immunization, and the efficiency of antigen clearance was poor in untreated control catla. In Achyranthes treated catla, the amount of antigen (BSA) present in the spleen on day-7 was shown in Fig. 6. Though crowded particles were found on day-7, the density of particles was very low compared with the control one. From the figures it can be clearly seen that the antigen presence was very low even compared to the day-28 control. On day-14, the amount of antigen present in the Achyranthes treated catla was shown in Fig. 7. The labeling was very low and found rarely indicating the presence of low amounts of BSA. On day-21 and day-28, the amount of antigen present in the spleen of Achyranthes treated catla was shown in Figs. 8 and 9, respectively. From day-21 onwards the particles were rarely found throughout the sections, indicating the efficient clearance of injected BSA by the treatment with Achyranthes.
4. Discussion Antibody is the major humoral component of the specific immune system, which plays an adaptive role in neutralizing and killing of invaded pathogens with the help of other humoral and cellular components of the immune system. Enhancement in the production of
Fig. 6. Electron micrographs of spleen of Achyranthes treated Catla catla on day-7 after immunization (5600, 7100, 7100, 5600).
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specific antibodies will help in neutralization and quick removal of antigen that entered into the host. Achyranthes incorporated diets were fed to fish before immunization as a prophylactic treatment. Using BSA as antigen the immunostimulatory property of Achyranthes was determined in catla. It was found that Achyranthes has significantly enhanced the specific antibody response against BSA. Similarly, Achyranthes has enhanced the cRBC-specific hemagglutinating antibody titers and some other components of non-specific immunity in carps C. catla catla, L. rohita rohu and Cyprinus carpio common carp [14–17]. Similarly different plant sources were reported to enhance the antibody response in fishes. Intraperitoneal administration of leaf extract of Acalypha indica, Phyllanthus niruri and seed kernel of Azadirachta indica has enhanced the antibody response in tilapia (Oreochromis mossambicus) against sheep red blood cells (SRBC) [18]. Feeding with Spirulina had significantly increased antibody titers to keyhole limpet hemocyanin (KLH) in channel catfish Ictalurus punctatus [19,20]. When any foreign material enters in to the host, the host’s immune system recognizes and mounts its im-
mune response against it and clears the antigen from the body of the host. As the antigen (BSA) was injected into the fish, the immune system should mount a response and eliminate it from the system of the host as quickly as possible to confer safety of the host and to protect the host from the onset of infection. In earlier experiment it was observed that treatment with Achyranthes significantly protected the fish from a bacterial pathogen. Fingerlings of rohu, fed with either control or Achyranthes incorporated diets, were experimentally infected with A. hydrophila. High survival rate was observed in 0.5% Achyranthes treated group (72%) than the control (23%) [13]. In the present study it was observed that control fish were unable to clear the injected antigen from the system, even 28 days after immunization, as the labeling was seen abundantly throughout the sections (Figs. 2–5). These results indicate that the immune system of control fish is poor in clearing the invaded antigen, hence, high percentage of mortalities were observed in our earlier pathogen challenge experiment. Treatment with Achyranthes has enhanced the efficiency of catla that eliminated the injected BSA from the system. In Achyranthes treated
Fig. 7. Electron micrographs of spleen of Achyranthes treated Catla catla on day-14 after immunization (5600, 7100, 7100, 5600).
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
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Fig. 8. Electron micrographs of spleen of Achyranthes treated Catla catla on day-21 after immunization (5600, 7100, 7100, 7100).
Fig. 9. Electron micrographs of spleen of Achyranthes treated Catla catla on day-28 after immunization (7100, 5600, 5600, 4400).
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fish very low amount of particles were present on day14, and the particles were scarcely found on days 21 and 28, i.e. BSA was almost cleared from the system even on day-21. Since Achyranthes enhances the specific antibody response and also components of the non-specific immunity such as phagocytic activity, bactericidal activity, anti-proteases [13,15], the injected antigen would have been efficiently cleared. The results of earlier and present studies indicate the potentiality of A. aspera as an immunostimulant. Acknowledgements We are very much thankful to the Dr. T. K. Das and all staff of Electron Microscope Facility, All India Institute of Medical Sciences, New Delhi, for helping in the processing of the tissue samples. References [1] Raa J. The use of immune-stimulants in fish and shellfish feeds. In: Cruz-Sua´rez LE, Ricque-Marie D, Tapia-Salazar M, OlveraNovoa MA, Civera-Cerecedo R, editors. Avances en Nutricio´n Acuı´cola V. Memorias del V Simposium Internacional de Nutricio´n Acuı´cola. 19–22 November 2000. Me´rida, Yucata´n, Mexico, p. 47 – 56. [2] Dey RK, Chandra S. Preliminary studies to raise disease resistant seed (fry) of Indian major carp, Catla catla (Ham.) through herbal treatment of spawn. Fish Chimes March 1995:23 – 5. [3] Thuy NTT, Mukherjee SC, Prasad KP. Studies on the immunostimulatory effect of certain plant extracts on fish. The Sixth Indian Fisheries Forum, CIFE, Mumbai, India; 2002. Abstracts AH-13:153. [4] Edahiro T, Hamoguchi M, Kusuda R. Effect of Glycyrrhizine against Streptococcal infection of young yellowtail, Seriola quinqueradiata. Suisan Zoshoku 1990;38:239 – 43. [5] Girach RD, Khan ASA. Ethnomedicinal uses of Achyranthes aspera leaves in Orissa (India). Int J Pharmacogn 1992;30: 113 – 5. [6] Tang W, Eisenbrand G. Achyranthes bidentata BI. Chinese Drugs of Plant Origin. Berlin7 Springer-Verlag; 1992. p. 13 – 7.
[7] Liersch BKHR. Achyranthes. In: Haensel R, Keller K, Rimpler G, Schneider G, editors. Hagers Handbuch der Pharmazeutischen Praxis, V. Berlin7 Springer-Verlag; 1992. p. 54 – 9. [8] Aziz N, Gilani AH, Rindh MA. Kushta(s): unique herbo-mineral preparations used in South Asian traditional medicine. Med Hypotheses 2002;59:468 – 72. [9] Borthakur SK, Goswami N. Herbal remedies from Dimoria of Kamrup district of Assam in northeastern India. Fitoterapia 1995;66:333 – 40. [10] Akhtar MS, Iqbal J. Evaluation of the hypoglycemic effect of Achyranthes aspera in normal and alloxan diabetic rabbits. J Ethnopharmacol 1991;31:49 – 57. [11] Singh V. Traditional remedies to treat asthma in the North West and Trans-Himalayan region in J. & K. state. Fitoterapia 1995;66:507 – 9. [12] Raman RP, Rahman MK. Herbal therapy in aquaculture. The Sixth Indian Fisheries Forum, CIFE, Mumbai, India; 2002. Abstracts AH-17:157. [13] Vasudeva RY, Das BK, Jyotyrmayee P, Chakrabarti R. Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish Shellfish Immunol 2006;20:263 – 73. [14] Vasudeva RY, Romesh SM, Singh SA, Chakrabarti R. Potentiation of antibody production in Indian major carp Labeo rohita, rohu, by Achyranthes aspera as a herbal feed ingredient. Aquaculture 2004;238:67 – 73. [15] Vasudeva RY, Chakrabarti R. Enhanced anti-proteases in Labeo rohita fed with diet containing herbal ingredients. Indian J Clin Biochem 2004;19:132 – 4. [16] Vasudeva RY, Chakrabarti R. Stimulation of immunity in Indian major carp Catla catla with herbal feed ingredients. Fish Shellfish Immunol 2005;18:327 – 34. [17] Vasudeva RY, Chakrabarti R. Dietary incorporation of Achyranthes aspera seed influences the immunity of common carp Cyprinus carpio. Indian J Anim Sci 2005;75:1097 – 102. [18] Hemapriya VS, Logambal SM, Michael DR. Immunostimulation by leaf extracts of a few south Indian medicinal plants in Oreochromis mossambicus (Peters). Dev Comp Immunol 1997;21:175. [19] Duncan LP, Phillip HK. Dietary immunostimulants enhance non-specific immune responses in channel catfish but not resistance to Edwardsiella ictaluri. J Aquat Anim Health 1996;8:241 – 8. [20] Duncan LP, Phillip HK. Effects of feeding Spirulina on specific and nonspecific immune responses of channel catfish. J Aquat Anim Health 1996;8:308 – 13.
Achyranthes aspera stimulates the immunity and enhances the antigen clearance in Catla catla Rina Chakrabarti *, Rao.Y. Vasudeva Department of Zoology, University of Delhi, Delhi – 110 007, India Received 10 March 2005; received in revised form 15 September 2005; accepted 21 November 2005
Abstract Achyranthes aspera, an Indian medicinal plant (family: Amaranthaceae) was incorporated in artificial fish diet, and fed to catla Catla catla. After 4 weeks of feeding, fish were immunized with bovine serum albumin (BSA), spleen and blood were sampled on weekly intervals for four times after immunization. Antigen-specific antibody level in serum was determined by ELISA. Antigen clearance was determined in spleen by immuno-electron microscopy. Achyranthes has significantly ( P b 0.05) enhanced the BSAspecific antibody titers than the untreated control group throughout the study period. The efficiency of antigen clearance was also enhanced in C. catla treated with Achyranthes. D 2005 Elsevier B.V. All rights reserved. Keywords: Catla catla; BSA; ELISA; Antibody response; Antigen clearance
1. Introduction Immunostimulants activate several components of the immune system such as phagocytes, natural killer cells, T-lymphocytes, B-lymphocytes, complement and lysozyme. Such substances may also, but not necessarily, render animals more resistant to infectious diseases and reduce the risk of disease outbreaks if administrated prior to situations known to result in stress and impaired general performance, or prior to expected increase in exposure to pathogenic microorganisms and parasites. In addition, aquaculture may benefit from the use of such immunostimulants when they are used prior to, and during, developmental phases when the organisms are particularly susceptible to infectious agents [1]. Several reports are available on immunostimulants
* Corresponding author. Tel.: +91 11 27666496. E-mail address: [email protected] (R. Chakrabarti). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.11.020
originated from plant, animal and bacterial sources. Bath treatment of Catla catla catla spawn with 1% crude extract of neem, garlic and turmeric (1 : 1 : 1) [2], and oral treatment of Labeo rohita rohu with Catharanthus roseus extract [3] and Seriola quinqueradiata yellowtail with glycyrrhizin [4] enhanced immune response and disease resistance against experimental infection with Aeromonas hydrophila and Edwardsiella seriola.
Table 1 Composition of artificial diets Ingredients/100 g of feed
Experimental diet
Control diet
Fish meal (g) Wheat flour (g) Cod liver oil (g) Supradin — vitamin and mineral premix (g) Seed of Achyranthes aspera (g)
32.88 54.62 10 2
32.88 55.12 10 2
0.5
–
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
783
Fig. 1. Effect of Achyranthes aspera on BSA-specific serum antibody titers in Catla catla. Each value represents the mean F SE of four fish (*P b 0.01; **P b 0.05).
Achyranthes aspera L., (Division: Angiospermae; Class: Dicotyledones; Sub-class: Monochlamydeae; Series: Curvembryeae; Family: Amaranthaceae) is an indigenous medicinal plant of Asia, South America, and Africa that is commonly used by traditional healers for
the treatment of fever, especially malarial fever, dysentery, asthma, hypertension and diabetes [5–7]. Kushta Hartal warqi (= arsenic sulfide + Callicarpa arborea + A. aspera) is used in rheumatism and paralysis [8]. The roots of Achyranthes are reported to have application in
Fig. 2. Electron micrographs of spleen of control Catla catla on day-7 after immunization (7100, 3500, 7100, 4400).
784
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
infantile diarrhea and cold [9]. Leaves are reported to possess hypoglycemic activity [10] and also employed against asthma [11]. Indian major carps (C. catla, L. rohita and Cirrhinus mrigala) are very prone to diseases and huge losses have been experienced by Indian aquaculture industry [12]. Since A. aspera holds a reputed position as medicinal herb in different systems of medicine in India, we were interested to see its effect on the immunity and disease resistance of fishes, and it was found that feeding with Achyranthes protected the fish from infection [13]. To know how rapidly the Achyranthes induces the elimination of the injected antigen, we have conducted the present experiment. 2. Materials and methods 2.1. Animals C. catla (150 F 20 g) were obtained from the local fish market, Delhi, and were acclimatized for three weeks in outdoor cemented tanks (170 L). Experiment was conducted under two feeding regimes, control diet and A. aspera incor-
porated diet. Four replicates were used for each feeding regime, seven fish were kept in each replicate. Temperature and pH ranged between 29–33 8C and 7.4–8.0, respectively, during the experiment. Dissolved oxygen level was maintained above 5 mg/L with the help of aerators throughout the experiment. 2.2. Experimental diet and feeding Dried seeds were collected from the spike of A. aspera grown in the field near the department. The seeds were washed with tap water to remove any dust and air-dried. 0.5% measured amount of seed was ground in mortar and pestle with water. The whole extract was added to the feed ingredients (fishmeal, wheat flour, cod liver oil, vitamin and mineral premix) to prepare the experimental diet. The prepared mixture was then passed through a manual feed maker using 1 mm sieve and the threads formed were air-dried. The dried threads were further broken into smaller pieces manually. Normal diet was prepared using same ingredients, except A. aspera seeds (Table 1). Test groups were fed with experimental diet containing A. aspera, and control groups were fed with normal diet. Feeding was started four weeks prior to the immunization and continued till the end of the experiment. Both groups of fish were fed at 1% of body weight at 9:00 AM.
Fig. 3. Electron micrographs of spleen of control Catla catla on day-14 after immunization (3500, 7100, 8900, 7100).
R. Chakrabarti, R.Y. Vasudeva / International Immunopharmacology 6 (2006) 782–790
2.3. Antigen and immunization After four weeks (28 days) of feeding, fish were anaesthetized with MS-222 and injected intraperitoneally with 500 Al of bovine serum albumin (BSA, Fraction-V, Merck) dissolved (i.e. 10 mg of BSA/fish) in phosphate buffered saline (PBS). 2.4. Sampling Blood was collected from both control and Achyranthes treated test groups, on the 7th, 14th, 21st and 28th day after immunization, allowed to clot at room temperature and serum was obtained by centrifugation. At each sampling time, four fish were sacrificed from each group. After collecting the blood, spleen samples were aseptically dissected out for immuno-electron microscopy. 2.5. Determination of antigen-specific antibody titers by ELISA The wells of the microtiter plate (greiner Bio-One, Germany, ELISA plate, Microlon, 96W, Flat-bott, High binding) were coated with 100 Al of fish serum (2-fold serial dilutions) diluted in phosphate buffered saline (pH
785
7.4). The plates were incubated for 12 h at 4 8C. After incubation the wells were washed three times with PBS containing tween-20 (0.05%). The free binding sites of the wells were blocked by adding 300 Al of 5% gelatin dissolved in PBS per well and incubated for 12 h at 4 8C. The wells were washed with PBS-tween, and 100 Al of bovine serum albumin (BSA) in PBS was added to each well (100 ng BSA/well) and incubated for 2 h at 4 8C. The wells were washed with PBS-tween, and 100 Al of rabbit antiBSA serum diluted to 1 : 500 in PBS was added to each well and incubated for 1 h at 37 8C. The wells were washed with PBS-tween, and goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase, diluted to 1 : 1000 in PBS, was added to all wells (100 Al/well) and incubated for 1 h at 37 8C. The wells were washed with PBS-tween, and 100 Al of substrate (containing 13 mg of o-phenylenediamine dihydrochloride and 10 Al of hydrogen peroxide in 10 ml of citrate–phosphate buffer, pH 5.0) was added to all wells and incubated for 10 min. After the development of color, the reaction was terminated by adding 50 Al of 1M oxalic acid per well. The optical density was measured at 492 nm in an automatic microplate reader (Microscan — MS5605A, Electronic Corporation of India Ltd.). The highest dilution of the serum that gave the OD N 0.1 was taken as the titer.
Fig. 4. Electron micrographs of spleen of control Catla catla on day-21 after immunization (7100, 8900, 4400, 7100).
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2.6. Determination of antigen clearance by immuno-electron microscopy (IEM) Few pieces of spleen (1 mm thickness) of catla were fixed in a solution containing 1% glutaraldehide and 2% paraformaldehide in 0.1 M phosphate buffer for overnight. Then tissues were washed with 0.1 M phosphate buffer and dehydrated with gradient ethanol (30–100%) at 4 8C. The tissues were infiltrated with a mixture of ethanol and LR White (1 : 1) followed by pure LR White (TAAB). Tissues were embedded in beam capsule filled with LR White and polymerized at 55 8C. Ultra thin sections (70 nm) were cut in a microtome (Reichert Jung Ultracut-E) and grids were prepared. The sections on the grid were blocked with 2% fish gelatin in 0.1 M phosphate buffer for 2 h. After blocking, the sections were washed and labeled by incubating with rabbit anti-BSA polyclonal antibodies (ICN Biochemicals, USA) diluted to 1 : 500 in phosphate buffer containing 1% fish gelatin and incubated for 12 h. The grids were washed thoroughly with 1% fish gelatin in phosphate buffer and labeled with secondary antibody, i.e. goat anti-rabbit IgG conjugated with 15 nm gold particles (TAAB), diluted to 1 : 100 in phosphate buffer containing 1% fish gelatin, and incubated for 2 h. The grids were washed with phosphate buffer containing 1% fish gelatin, and finally washed with double distilled water. After
labeling, the sections were stained with uranyl acetate and lead citrate and observed for labeled particles under electron microscope, (Fei-Philips Morgagni 268D Digital TEM) with image analysis system. 2.7. Statistics The data was statistically analyzed using students’ t-test in Microsoft Excel. The level of significance was P b 0.05. 3. Results 3.1. Antibody response In catla, BSA-specific antibody level was peaked on day-7 and then gradually decreased up to day-28. The trend was similar in both control and Achyranthes treated groups. The anti-BSA antibody level was always higher in Achyranthes treated group than the control throughout the study period (Fig. 1). The difference was significant ( P b 0.05) throughout the study period, except on day-28. The titers reduced by 97% in control group and 94% in test group on day-28, compared with their respective titers of day-7. In the control group, the average antibody titers were 896 103, 264 103, 40 103 and 26 103 on days 7, 14, 21 and 28, respectively. In
Fig. 5. Electron micrographs of spleen of control Catla catla on day-28 after immunization (5600, 5600, 5600, 5600).
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Achyranthes treated group, the average titers were 2304 103, 704 103, 288 103 and 128 103 on days 7, 14, 21 and 28, respectively. The anti-BSA antibody titers of Achyranthes treated group was 2.5–7.2 times higher than the control in various days of sampling. 3.2. Antigen clearance The presence of gold particles locates the BSA in the immuno-labeled spleen sections. In control group catla, the amount of antigen (BSA) present in the spleen on day-7 was shown in Fig. 2. It was seen that on day-7 the particles were densely crowded throughout the sections, indicating the abundant presence of BSA. On day-14, the amount of antigen present in the control group catla was shown in Fig. 3. The labeling appeared densely throughout the sections indicating the ample presence of BSA. On day-21, the particles were still densely crowded, indicating the presence of BSA abundantly (Fig. 4). On day-28, the antigen presence in the spleen of control catla was shown in Fig. 5. It was noticed that the density of the particles was lower in spleen sections compared with the earlier days samples of the control catla. Nevertheless the particles were found abundantly throughout the sections, indicating the plentiful presence of BSA. All these results indicate that the injected antigen, i.e. BSA was still present in large quantities in
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control catla even on day-28 after immunization, and the efficiency of antigen clearance was poor in untreated control catla. In Achyranthes treated catla, the amount of antigen (BSA) present in the spleen on day-7 was shown in Fig. 6. Though crowded particles were found on day-7, the density of particles was very low compared with the control one. From the figures it can be clearly seen that the antigen presence was very low even compared to the day-28 control. On day-14, the amount of antigen present in the Achyranthes treated catla was shown in Fig. 7. The labeling was very low and found rarely indicating the presence of low amounts of BSA. On day-21 and day-28, the amount of antigen present in the spleen of Achyranthes treated catla was shown in Figs. 8 and 9, respectively. From day-21 onwards the particles were rarely found throughout the sections, indicating the efficient clearance of injected BSA by the treatment with Achyranthes.
4. Discussion Antibody is the major humoral component of the specific immune system, which plays an adaptive role in neutralizing and killing of invaded pathogens with the help of other humoral and cellular components of the immune system. Enhancement in the production of
Fig. 6. Electron micrographs of spleen of Achyranthes treated Catla catla on day-7 after immunization (5600, 7100, 7100, 5600).
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specific antibodies will help in neutralization and quick removal of antigen that entered into the host. Achyranthes incorporated diets were fed to fish before immunization as a prophylactic treatment. Using BSA as antigen the immunostimulatory property of Achyranthes was determined in catla. It was found that Achyranthes has significantly enhanced the specific antibody response against BSA. Similarly, Achyranthes has enhanced the cRBC-specific hemagglutinating antibody titers and some other components of non-specific immunity in carps C. catla catla, L. rohita rohu and Cyprinus carpio common carp [14–17]. Similarly different plant sources were reported to enhance the antibody response in fishes. Intraperitoneal administration of leaf extract of Acalypha indica, Phyllanthus niruri and seed kernel of Azadirachta indica has enhanced the antibody response in tilapia (Oreochromis mossambicus) against sheep red blood cells (SRBC) [18]. Feeding with Spirulina had significantly increased antibody titers to keyhole limpet hemocyanin (KLH) in channel catfish Ictalurus punctatus [19,20]. When any foreign material enters in to the host, the host’s immune system recognizes and mounts its im-
mune response against it and clears the antigen from the body of the host. As the antigen (BSA) was injected into the fish, the immune system should mount a response and eliminate it from the system of the host as quickly as possible to confer safety of the host and to protect the host from the onset of infection. In earlier experiment it was observed that treatment with Achyranthes significantly protected the fish from a bacterial pathogen. Fingerlings of rohu, fed with either control or Achyranthes incorporated diets, were experimentally infected with A. hydrophila. High survival rate was observed in 0.5% Achyranthes treated group (72%) than the control (23%) [13]. In the present study it was observed that control fish were unable to clear the injected antigen from the system, even 28 days after immunization, as the labeling was seen abundantly throughout the sections (Figs. 2–5). These results indicate that the immune system of control fish is poor in clearing the invaded antigen, hence, high percentage of mortalities were observed in our earlier pathogen challenge experiment. Treatment with Achyranthes has enhanced the efficiency of catla that eliminated the injected BSA from the system. In Achyranthes treated
Fig. 7. Electron micrographs of spleen of Achyranthes treated Catla catla on day-14 after immunization (5600, 7100, 7100, 5600).
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Fig. 8. Electron micrographs of spleen of Achyranthes treated Catla catla on day-21 after immunization (5600, 7100, 7100, 7100).
Fig. 9. Electron micrographs of spleen of Achyranthes treated Catla catla on day-28 after immunization (7100, 5600, 5600, 4400).
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fish very low amount of particles were present on day14, and the particles were scarcely found on days 21 and 28, i.e. BSA was almost cleared from the system even on day-21. Since Achyranthes enhances the specific antibody response and also components of the non-specific immunity such as phagocytic activity, bactericidal activity, anti-proteases [13,15], the injected antigen would have been efficiently cleared. The results of earlier and present studies indicate the potentiality of A. aspera as an immunostimulant. Acknowledgements We are very much thankful to the Dr. T. K. Das and all staff of Electron Microscope Facility, All India Institute of Medical Sciences, New Delhi, for helping in the processing of the tissue samples. References [1] Raa J. The use of immune-stimulants in fish and shellfish feeds. In: Cruz-Sua´rez LE, Ricque-Marie D, Tapia-Salazar M, OlveraNovoa MA, Civera-Cerecedo R, editors. Avances en Nutricio´n Acuı´cola V. Memorias del V Simposium Internacional de Nutricio´n Acuı´cola. 19–22 November 2000. Me´rida, Yucata´n, Mexico, p. 47 – 56. [2] Dey RK, Chandra S. Preliminary studies to raise disease resistant seed (fry) of Indian major carp, Catla catla (Ham.) through herbal treatment of spawn. Fish Chimes March 1995:23 – 5. [3] Thuy NTT, Mukherjee SC, Prasad KP. Studies on the immunostimulatory effect of certain plant extracts on fish. The Sixth Indian Fisheries Forum, CIFE, Mumbai, India; 2002. Abstracts AH-13:153. [4] Edahiro T, Hamoguchi M, Kusuda R. Effect of Glycyrrhizine against Streptococcal infection of young yellowtail, Seriola quinqueradiata. Suisan Zoshoku 1990;38:239 – 43. [5] Girach RD, Khan ASA. Ethnomedicinal uses of Achyranthes aspera leaves in Orissa (India). Int J Pharmacogn 1992;30: 113 – 5. [6] Tang W, Eisenbrand G. Achyranthes bidentata BI. Chinese Drugs of Plant Origin. Berlin7 Springer-Verlag; 1992. p. 13 – 7.
[7] Liersch BKHR. Achyranthes. In: Haensel R, Keller K, Rimpler G, Schneider G, editors. Hagers Handbuch der Pharmazeutischen Praxis, V. Berlin7 Springer-Verlag; 1992. p. 54 – 9. [8] Aziz N, Gilani AH, Rindh MA. Kushta(s): unique herbo-mineral preparations used in South Asian traditional medicine. Med Hypotheses 2002;59:468 – 72. [9] Borthakur SK, Goswami N. Herbal remedies from Dimoria of Kamrup district of Assam in northeastern India. Fitoterapia 1995;66:333 – 40. [10] Akhtar MS, Iqbal J. Evaluation of the hypoglycemic effect of Achyranthes aspera in normal and alloxan diabetic rabbits. J Ethnopharmacol 1991;31:49 – 57. [11] Singh V. Traditional remedies to treat asthma in the North West and Trans-Himalayan region in J. & K. state. Fitoterapia 1995;66:507 – 9. [12] Raman RP, Rahman MK. Herbal therapy in aquaculture. The Sixth Indian Fisheries Forum, CIFE, Mumbai, India; 2002. Abstracts AH-17:157. [13] Vasudeva RY, Das BK, Jyotyrmayee P, Chakrabarti R. Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. Fish Shellfish Immunol 2006;20:263 – 73. [14] Vasudeva RY, Romesh SM, Singh SA, Chakrabarti R. Potentiation of antibody production in Indian major carp Labeo rohita, rohu, by Achyranthes aspera as a herbal feed ingredient. Aquaculture 2004;238:67 – 73. [15] Vasudeva RY, Chakrabarti R. Enhanced anti-proteases in Labeo rohita fed with diet containing herbal ingredients. Indian J Clin Biochem 2004;19:132 – 4. [16] Vasudeva RY, Chakrabarti R. Stimulation of immunity in Indian major carp Catla catla with herbal feed ingredients. Fish Shellfish Immunol 2005;18:327 – 34. [17] Vasudeva RY, Chakrabarti R. Dietary incorporation of Achyranthes aspera seed influences the immunity of common carp Cyprinus carpio. Indian J Anim Sci 2005;75:1097 – 102. [18] Hemapriya VS, Logambal SM, Michael DR. Immunostimulation by leaf extracts of a few south Indian medicinal plants in Oreochromis mossambicus (Peters). Dev Comp Immunol 1997;21:175. [19] Duncan LP, Phillip HK. Dietary immunostimulants enhance non-specific immune responses in channel catfish but not resistance to Edwardsiella ictaluri. J Aquat Anim Health 1996;8:241 – 8. [20] Duncan LP, Phillip HK. Effects of feeding Spirulina on specific and nonspecific immune responses of channel catfish. J Aquat Anim Health 1996;8:308 – 13.