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

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IntroductionMolecular diagnosis in shrimp disease with special reference to PCR ofIndian white spot virusT.C. Santiago, K.K. Vijayan, S.V. Alavandi and N. KalaimaniIncreasing disease problems mars the growth of promising aquaculture industry bothnationally and internationally. Vulnerability of the aquaculture production system to disease isdue to the co-existence and close interaction of host, pathogen and environment. A small shiftof equilibrium between these three can trigger a disease outbreak leading to mortalityresulting in crop failure. The latest viral disease problem in shrimp farming arena due to thewhite spot virus exposed the vulnerability of aqua-business.Disease problems are inevitable, as aquaculture has to look forward to produce moreanimal protein, more jobs and more revenues for the people. To tackle the menace of diseaseproblems, a scientific health management approach has to be developed emphasizing theconventional wisdom - 'prevention is better than cure'. An integral part of such a program isthe use of diagnostic tests at the strategic point of production cycle to eliminate or control thedisease causing pathogens.Conventional diagnostic methodsThe conventional diagnostic methods practiced in aquaculture are mostly adaptedfrom the field of human health and veterinary sciences. Among the diagnostics mentioned(Table I),visual examination, microscopic, histological examination and bacterialexamination are the most widely used and still form the essential part of disease diagnosis.But these methods often fail to deliver data in time to support a decision making in thehealth management to salvage the crop. This is mainly due to the time consuming andlaborious methodologies and the inability of these tests to detect sub-clinical I latent I carrier,state of infection.DNA - based diagnosticsDevelopments in molecular biology enabled researchers to collect information on thegenetic material that serves as the blueprint for all living organisms. The most recentdevelopment in diagnostics have utilized molecular biology to design new generation ofdiagnostics tools, the Polymerase Chain Reaction (PCR) and Gene Probes. These DNA-baseddiagnostic tools stand out among other conventional diagnostic methods with its speed,sensitivity and simplicity. Polymerase Chain Reaction and Gene probes capable of identifying
a number of viral, bacterial and parasitic pathogens are finding their way into the area ofinfectious disease diagnosis in aquatic species.Table 1: Methods available for disease diagnosis and pathogen detectionMethodHistoryDirect microscopyHistopathologyElectron microscopyCulture andbiochemicalcharacterizationEnhancementBioassaySerological methodsTissue cultureDNA probesPCRTests and data obtainedHistory of disease at facility or region, facility design, source ofseed, type of feed used, environmental conditions etc.Gross, clinical signs, lesions visible, behaviour, abnormalgrowth, feeding or food conversion efficiency, etc.Bright-field, phase contrast, or dark field examination of stainedor unstained tissue smears, whole-mounts, etc. of diseased orabnormal specimensRoutine histological or histochemical analysis of tissue sectionsUltrastructural examination of tissue sections, negatively stainedvirus preparations, or sample surfacesRoutine culture and isolation of bacteria and identification usingbiochemical reactionsRearing samples of the appropriate life stages under controlledconditions to enhance expression of latent or low gradeinfectionsExposure to potential pathogensUse of specific antibodies as diagnostic reagents in immunoblot,agglutination, ELISA, IFAT, or other tests.In vitro culture of pathogens in cell linesDetection of unique portion of a pathogen's nucleic acid using alabelled DNA probeAmplification of unique portion of a pathogen's genome toreadily detectable concentrations using specific primer pairsThe key to the DNA based diagnostics is the generation of unique geneticinformation of the target pathogen through recombinant DNA technology. This is done bypurifying the infectious agent of interest (Fig. 1) and isolating the nucleic acid. Isolated DNAis then subjected to restriction digestion and cloning. From the selected clones, desired DNAfragment has to be sequenced. Once the adequate genetic information (sequence information)is generated, the information can be used in PCR or gene probes.Polymerase Chain Reaction (PCR)PCR is relatively a simple technique by which a DNA or cDNA template is amplifiedmany thousand or a lnillion fold quickly and reliably in a short period of 3-4 hours. So far noother technique has equalled PCR in sensitivity, which is about one DNA target molecule.
Page 5 and 6:
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: Polymerase Chain Reaction (PCR) is
Page 63 and 64: from 15-30 bases. Negative controls
Page 65 and 66: PCR Primers for Indian WSVTwo sets
Page 67 and 68: ANTIOXIDANTS IN MANAGEMENT OF OXIDA
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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