, Diagnosis an-&& of Shrimp Diseases - Central Institute of ...

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72 OC for lminute1 cycle of 72 OC 5 minutes3. Place the reaction tubes in thermal cycler, heat to 95 OC and hold at that temperature.4. Add lpl(1 p1 of a 0.5 U solution) Taq polymerase to each sample. Resume program.When program is complete, remove samples, add 5p1 of loading buffer to each samplemix well and spin briefly.Agarose gel electrophoresis1. Assemble large gel electrophoresis box to run a maximum of 20 samples.2. Prepare a 2.0 % agarose gel by adding 1.46 grams of agarose to 73 ml of 1 X TBE buffer*(*1 X TBE buffer is prepared by diluting 5 X TBE. 5 X TBE is prepared by adding54.0 grams Tris base and 27.5 grams boric acid to 980 ml of dHzO. Add 20 ml of 0.5 MEDTA (pH 8.0). Filter solution through a 0.45-micron filter).3. Cover gel with I X TBE and load samples. Load 5 p1 of PCR marker into one lane of thegel.Run the gel at 80 volts for about 1.5 hours, or until the loading dye lane runs off the anodeside of the gel.4. Place gel in a 0.5 pglml ethidium bromide dye solution for 15 to 20 minutes.(A 0.5 pglml ethidium bromide solution is prepared by adding 10 pl of a 1% stock solution to200 in1 of 1 X TBE buffer).5. Remove the gel from ethidium bromide solution and rinse TBE buffer. Place gel on a UVtransilluminator with filter 312 nm to visualize PCR product and photograph.PConclusionMolecular diagnostic technique like PCR provides a fast, sensitive and specific toolfor disease diagnostics in aquaculture. The selective amplification of a small segment ofpathogenic DNA from the mass of unrelated host DNA sequences to the detectable level, andthat too from an extremely small quantity of the starting material, makes PCR a revolutionarytechnology. The PCR has a wide and significant use in checki& the entry of lethal pathogenslike virus to the hatchery and growout system by helping to select healthy broodstock andseeds. This can also be used in the epizootiological study of the pathogen in an effort to drawdisease control measures. In the coming days, DNA based diagnostic tools are going to play amajor role in the newly evolving aquatic animal healthcare.
ANTIOXIDANTS IN MANAGEMENT OF OXIDATIVE STRESS AND HEALTHN. KalaimaniOxidative stress has been implied, in the case of diseases that include immune injury,drug and toxin induced reactions, ischaemia and subsequent reperfusion injury, nutritionaldeficiencies, radiation injury, aging, hemolytic diseases, lung disorders, heart andcardiovascular system, kidney and gastro intestinal disorders and diseases affecting the brain,nervous system, neuromuscular disorders, cataract and retinal damages and a variety of skindiseases. In most diseases and as well as in several conditions of toxicity, increased oxidantand free radical formation is a consequence of the disease. Such pathological conditions havebeen implicated as resulting from damage caused by active oxygen/free radicals (oxy-rad)such as superoxide, hydrogen peroxide, hydroxyl radical, lipid radicals and nitrogen oxide.Free Radicals, Reactive Oxygen Species in Cell and Tissue DamageMolecular oxygen takes up electrons during respiration in the living cells in asequential and orderly manner. Intermediates produced during this process are oxidants like-superoxide and hydroxyl radical and hydrogen peroxide. (Saugstad, 1989).0, +e- 0,- (Superoxide anion)0, + e- + 2~+____,-H202 (Hydrogen Peroxide)Hz02+ e-OH- + OH ' (Hydroxyl radical)OH-+H+ -----+ H20 (Water)Superoxide and hydroxyl free radicals together with H202 and singlet oxygen arecalled the Reactive Oxygen Species (ROS) or peroxidants, and are endogenous in origin.They interact with exogenous free radical inducers and with nitric oxide (NO). Oxygenradicals are formed (albeit in small amounts) during cellular respiration and may leak out ofthe mitochondria and interact with endoplasmic reticulum, plasma, membranes and otherstructures. A second source of ROS is the xanthine oxidase enzyme which also catalyses theunivalent oxidation of purines with concomitant formation of superoxide radical, Hz02 andperhaps singlet oxygen .
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OVERVIEW OF STRATEGIES FOR PREVENTI
Page 7 and 8:
in harmonizing aquatic animal healt
Page 9 and 10:
SI-IRXMP DISEASES: GENEIL4L ASPECTS
Page 11 and 12:
quantitative level of pathogen is i
Page 13 and 14:
presence of prominent eosinophiiic
Page 15 and 16: Disease affected the shrimps of all
Page 19 and 20: BACTERIAL AND FUNGAL DISEASES OF SH
Page 21 and 22: Cause: The epibiotic bacteria stich
Page 23 and 24: PARASiTIC AND NON-INFECTIOUS IIISEA
Page 25 and 26: XOS-INFECTIOUS DISEASESNon-infectio
Page 27 and 28: INVESTIGATING SHRIMP DISEASES: OBSE
Page 29 and 30: lnay be a predisposing factor for b
Page 31 and 32: BACTERIOLOGICAL METHODSS.V. Alavand
Page 34 and 35: In place of ZMA, nutrient agar prep
Page 36 and 37: Interpretation: Positive reaction i
Page 38 and 39: Incubate at 37°C for 1-2 h.Examine
Page 40 and 41: Alkaligens: etc. are resistant. The
Page 42 and 43: Culture Media, Reagents and StainsC
Page 45 and 46: Differential characteristics of Vib
Page 47 and 48: Davidson's Alcohol Formatin Acetic
Page 49 and 50: Paraffin cold impregnationXylene an
Page 51 and 52: ProcedureDe-paraffinise the slid'c
Page 53 and 54: Biochemistry of Nucleic Acids with
Page 55 and 56: DNA e.rl1i6it.v base equivuleizcc!E
Page 57 and 58: hydrolyzed by dilute alkali. The su
Page 59 and 60: Polymerase Chain Reaction (PCR) is
Page 61 and 62: a number of viral, bacterial and pa
Page 63 and 64: from 15-30 bases. Negative controls
Page 65: PCR Primers for Indian WSVTwo sets
Page 69 and 70: Many potent chemical carcinogens ar
Page 71 and 72: cells. It has been shown that possi
Page 73 and 74: AFB, induced ~nutagenesis in Salmon
Page 75 and 76: FIGURE 2. An overview of oxygen rad
Page 77 and 78: were responsible for production los
Page 79 and 80: for Asiatic region. The participati
Page 81 and 82: charge of the DNA fragment. The neg
Page 83 and 84: European countries have in place th
Page 85: irth to a new concept to treatment
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