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Dhanalakshmi

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EFFECT OF MEDICINAL PLANTS BHRINGARAJ (ECLIPTA ALBA) AND ALOE VERA (ALOE BARBADENSIS MILLER) METHANOL EXTRACT SUPPLEMENTED DIETS ON IMMUNITY AND DISEASE RESISTANCE AGAINST BACTERIUM AEROMONAS HYDROPHILA IN CIRRHINUS MRIGALA

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Dhanalakshmi

  1. 1. FOURTH INTERNATIONAL CONFERENCE ON AGRICULTURE, ANIMAL SCIENCE AQUACULTURE & FISHERIES- 2016 Oral presentation by Mrs. P. Dhanalakshmi
  2. 2. EFFECT OF MEDICINAL PLANTS BHRINGARAJ (ECLIPTA ALBA) AND ALOE VERA (ALOE BARBADENSIS MILLER) METHANOL EXTRACT SUPPLEMENTED DIETS ON IMMUNITY AND DISEASE RESISTANCE AGAINST BACTERIUM AEROMONAS HYDROPHILA IN CIRRHINUS MRIGALA Under the supervision of Dr. V. RAMASUBRAMANIAN., M.Sc., M. Phil., Ph.D. Mrs. P. DHANALAKSHMI. UNIT OF AQUATIC BIOTECHNOLOGY & LIVE FEED CULTURE, DEPARTMENT OF ZOOLOGY BHARATHIAR UNIVERSITY
  3. 3. INTRODUCTION 1. World aquaculture has grown tremendously during the past 20 years becoming an economically important field. 2. Aquaculture is one of the fastest growing food- producing sector in the World. The increased intensification of aquaculture has led to a increased number of disease outbreaks with an high range of pathogens causing them (Kirubakaran et al., 2010).
  4. 4. 3. Hence in recent years, increasing attention has been given to the use of immunostimulants in the aquaculture industry. Improvement of eco-friendly alternatives to antibiotics that may keep fish healthy such as plant based immunostimulants and probiotics has increased, indigenous technological knowledge for controling diseases is enjoying attention in disease control and fish health (Sahu et al., 2007). 4. The immunostimulation effects of plant medicines in various fish species has been reported (Pugh et al., 2001).
  5. 5. OBJECTIVES  The main objective of the study is to control the disease out breaks in aquaculture farms using plant extracts.  To determine the effect of plant extracts (E. alba and A. vera) on the non-specific immune parameters of C. mrigala.
  6. 6.  To determine the efficacy of the plant extracts (E. alba and A. vera) against the challenge pathogenic bacteria of A. hydrophila -gram negative bacteria.  To evaluate the effect of plant extracts on the immune response and survival of C. mrigala fingerlings after the challenge of A. hydrophila- gram negative bacteria.
  7. 7. MATERIALS AND METHODS In the present study, fresh water fish C. mrigala was used as an experimental organism to study the effect of plant extracts against A. hydrophila. FIGURE-1. CIRRHINUS MRIGALA (MRIGAL CARP)
  8. 8. CLASSIFICATION Classification of Mrigal carp • Kingdom - Animalia • Phylum - Chordata • Class - Actinopterigii • Order - Cypriniformes • Family - Cyprinidae • Genus - Cirrhinus • Species - mrigala • Binomial name - Cirrhinus mrigala • Bloch(1795) • Comman name – mrigal carp A) Eclipta alba (Bhringaraj) • Kingdom - Plantae • Division - Magnoliophyta • Class - Magnoliopsida • Order - Asterales • Family - Asteraceae • Genus - Eclipta L. • Species – alba(L) • Botanical name - Eclipta alba • Popular name - Karisha-langanni, Karisirang-kanni, Babri, and Kesuri.
  9. 9. B) Aloe vera • Kingdom - Plantae • Clade-Angiosperms • Clade-Monocots • Order - Asparagalessz • Family - Xanthorrhoeaceae • Sub family - Asphodeloidae • Genus - Aloe • Species - vera • Binomial name - Aloe vera(L) (Burn. F.) B) Aeromonas hydrophila Domain - Bacteria Kingdom – Proteobacteria Phylum - Gamma proteobacteria Class - Aeromonadales Genus – Aeromonas Species – hydrophila Binomial name – Aeromonas hydrophila Chester (1901) Stanier (1943)
  10. 10. PREPARATION OF PLANT EXTRACTS Plant extract A: (E. alba) The powdered aerial parts (15g) of E. alba were extracted separately to exhaustion in a soxhlet apparatus using 99% methanol (150 ml). The extract were filtered through a cotton plug followed by what man filter paper and then concentrated by using a rotary evaporator at low temperature (40o-50oC). The extract were preserved in airtight containers and kept at 4o c until further use.
  11. 11. FIGURE-2. A VIEW OF ECLIPTA ALBA FIGURE- 3. CLEAN ECLIPTAALBA PLANTS & LEAVES
  12. 12. FIGURE- 4. AERIAL PARTS OF ECLIPTA FIGURE-5. ECLIPTAALBA POWDER ALBA IN DRIED FORM FIGURE- 6. SOXHLET APPARATUS WITH EXTRACT FIGURE- 7. ECLIPTA ALBA PLANT EXTRACT MIXED FEED
  13. 13. Plant extract B: (A. vera) The A. vera leaves were sterilized properly. Fresh A. vera leaves with gel was dried in the oven at 40oc for 48 hours and then powdered. In the process of maceration, 15g of crushed plant part was dissolved in 150ml of organic solvent i.e. methanol. The conical flask was covered by cotton plugs to avoid solvent evaporation. The extract was placed in shaking incubator at 250 rpm for 48 hours. After shaking, it was filtered with muslin cloth. The filtered extract was centrifuged at 8000 rpm for 20 minutes. The supernatant was collected in sterile flask. Then, it was stored at 40c.
  14. 14. FIGURE- 8. A VIEW OF ALOE VERA PLANT FIGURE- 9. A CLEAN ALOE VERA & ALOE VERA PIECES
  15. 15. FIGURE-10. ALOE VERA PIECES IN DRIED FORM FIGURE-11. ALOE VERA POWDER
  16. 16. FIGURE-12. ALOE VERA PLANT EXTRACT FIGURE-13. ALOE VERA PLANT EXTRACT MIXED FEED
  17. 17. EXPERIMENTAL FOOD PREPARATIONFor group 1 Normal balanced feed composed of 42% fish meal, 20% groundnut oil cake powder, 15% tapioca powder, 15% wheat flour, 5% egg white and 3% mineral- Vitamin mixture was used as control diet (protein: 39%, carbohydrate: 24% lipid: 11% and ash: 9%). Group 2 The diet used in the experiment group 2, were prepared by mixing commercial carp food with the methanol extract of E. alba in ratio 5g of E. alba per kilogram of food. (i.e, 0.5 % E. alba). Group 3 The diet used in the experiment group 3 were prepared by mixing commercial carp food with the methanol extract of A. vera in ratio 5g of A. vera per kilogram of food. i.e, 0.5% A. vera.
  18. 18. EXPERIMENTAL SETUP Group 1: fishes were fed with 200 mg of control feed. Group 2: fishes were fed with 200 mg of E. alba mixed feed Group 3: fishes were fed with 200 mg of A. vera mixed feed
  19. 19. SAMPLING SCHEDULE 0’day - 1st blood sample collection. 15thday - 1st infectious challenge with A. hydrophila. 30thday - 2nd blood sample collection. 35th day - 2nd infectious challenge with A. hydrophila. 45thday - 3rd blood sample collection.
  20. 20. LYSOZYME ACTIVITY Serum lysozyme activity was measured by adapting the method described by (Parry et al., 1965). SERUM BACTERICIDALACTIVITY The method used for serum bactericidal activity was followed a modified version of that adopted by (Kajita et al., 1990). TOTAL SERUM PROTEIN Samples were analyzed for total protein using the method outlined by (Lowry et al. 1951). SERUM GLOBULIN Serum albumin content was measured using a standard albumin estimation kit and the globulin content was estimated by subtracting albumin from total protein.
  21. 21. HEMATOLOGY LEUKOCYTE COUNT Leukocytes (WBC) were counted by the method of (Rusia and Sood 1992) using haemocytometer. RED BLOOD CELL COUNT Red blood cell (RBC) were determined as described by (Schaperclaus et al., 1991). PACKED CORPUSCULAR VOLUME (PCV): The packed cell volume (PCV) was determined by centrifugation at 2000 rpm for 20 min. STATISTICAL ANALYSIS The data’s were statistically evaluated by One-Way analysis of variance (ANOVA) followed by Post Hoc multiple comparison test using SPSS software. The levels of significance P < 0.05 was considered as statistically significant and P < 0.01 as highly significant.
  22. 22. RESULTS Fig: 14. Serum lysozyme (U/ml-1) count for C. mrigala fed same dosages (200mg) of E. alba and A. vera at different days. Data are represented as mean ± SD, n=4 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  23. 23. Fig.15. Serum bactericidal activity for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n=4. 0 20 40 60 80 100 120 140 160 180 200 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  24. 24. Fig.16. Serum protein (g/dl) level for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n = 4. 0 0.5 1 1.5 2 2.5 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  25. 25. Fig.17. Serum globulin (g/dl) level for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n = 4. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  26. 26. Fig.18. Leukocyte count (/mm3) for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n = 4. 0 5 10 15 20 25 30 35 40 45 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  27. 27. Fig.19. Red blood cell count (×106 cells /mm3) for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n = 4. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  28. 28. Fig.20. The packed cell volume (PCV - %) for C. mrigala fed same dosages (200mg) of E. alba, A. vera at different days. Data are represented as mean ± SD, n = 4 23 23.5 24 24.5 25 25.5 26 26.5 27 27.5 28 28.5 Day-0 Day-15 Day-30 Day-45 control Eclipta alba Aloe vera
  29. 29. Fig.21 . Rate of mortality (%) 0% 5% 10% 15% 20% 25% 30% Day-4 Day-8 Day-12 Day-14 percentageofmortality Mortality rate control Eclipta alba Aloe vera
  30. 30. FIGURE-22. NUCLEATED FISH RBC
  31. 31. FIGURE-23. A) CULTURE OF THE PATHOGENIC BACTERIA AEROMONAS HYDROPHILA B) MICROSCOPIC VIEW OF AEROMONAS HYDROPHILA
  32. 32. DISCUSSION  Application of immunostimulators, particularly plant immunostimulants in the fish culture industry, can be considered a remarkable advantage because of their safety and the fact that they are considered environmentally friendly.  In the present study, oral administration of A. vera and E. alba increased serum lysozyme activity compared with control groups .  In this study, the serum bactericidal activity increased significantly (p < 0.05) in A. vera and E. alba treated groups when compared with control group However, the presence of anti A. hydrophila antibody in the treated fish could be the cause for the increased bactericidal activity.
  33. 33. In this study in C. mrigala, serum protein and globulin content were significantly (P< 0.05) increased after oral administration of E. alba and A. vera, when compared with control group. The present study indicates that there was in increase in TLC in the E. alba and A. vera supplemented groups while control group showed depressed level of leukocyte count. After the fingerlings were intra peritoneally challenged with A. hydrophila. This is mainly due to the immune response of the fish immune system against the bacterial invasion. The gradual reversion of the leukocyte count back to the normal may be indicative of recovery from systemic damage.
  34. 34. In this study, administration of A. vera and E. alba in treated groups enhanced the survival rate after a challenge with live A. hydrophila. The highest survival rate in A. vera (80%), and E. alba (70%) were observed respectively. In this study, significant (P <0.05) differences were observed in RBC count and PCV-values of the E. alba and A. vera treated groups compared with control group after challenge with A. hydrophila in mrigal carp.
  35. 35. CONCLUSION The present results have demonstrated that the oral administration of E. alba and A. vera can enhance the specific and non- specific immune responses. The results reported that a 0.5% E. alba and A. vera supplementation can increase the resistance against A. hydrophila in C. mrigala.
  36. 36. REFERENCES 1. Christybapita, D., Divyagnaneswari, M. and Michael, R. D., (2007). Oral administration of Eclipta alba leaf aqueous extract enhances the nonspecific immune responses and disease resistance of Oreochromis mossambicus, Fish and Shellfish Immunol. 23, p. 840-852. 2. Daniel, M., (2006). Medicinal plants, Chemistry and properties, Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi, 148-149. 3. Devasagayam, T.P.A. and Sainis, K. B., (2002). Immune system and antioxidants, especially those derived from Indian medicinal plants. Ind. J. Exp Biol. 40: 639-655.
  37. 37. 4. Dugenci, S. K., Arda, N. and Candan, A., (2003). Some medicinal plants as immunostimulants for fish. Journal of Ethnopharmacology. 88: 99-106. 5. Eshun, K. and He, Q., (2004). Aloe vera; a valuable ingredient for the food, pharmaceutical and cosmetic industries – A review. Critical Reviews in Food Science and Nutrition, 44(2), 91-96. 6. Gopalakannan, A. and Arul, V., (2006). Immunomodulatory effect of dietary intake of Chitin, Chitosan and Levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture, 255: 179-187.
  38. 38. 7. Guz, L. and Kozinska, E., (2004). Antibiotic susceptibility of Aeromonas hydrophila and A. sobria isolated from farmed carp (Cyprinus carpio L). Bull Vet Inst Pulawy, 48, 391-395. 8. Indian Herbal Pharmacopoeia., (1998). Vol- I A Joint publication of IDMA and RRL Jammu-Tavi .81-85. 9. Jian, J. and Wu, Z., ((2004). Influence of traditional Chinese medicine on nonspecific immunity of Jian carp. (Cyprinus carpio var. Jian). Fish and Shellfish Immunology. 16: 185-191. 10. Joshi, P. K., Bose, M. and Harish, D., (2002). Hematological changes in the blood of clariasbatrachus exposed to mercuric chloride, Ecotoxicalogical Environment Monitoring, 12(2): 119- 122).
  39. 39. Acknowledgements I am thankful to my respected guide Dr. V. Ramasubramanian M.Sc., M. Phil., Ph.D. Associate professor, Department of Zoology, Bharathiar University for his valuable guidance, continuous encouragement and constant support in my research work. I express my sincere thanks to Department of Microbiology, PSG-IMSR, Coimbatore, Tamil Nadu, India, for providing the microorganisms.

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