Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)10153. Negative Staining : The result <strong>of</strong> negativestaining <strong>of</strong> the pellet from the filtrate <strong>of</strong> diseasedPenaeus monodon epidermis is shown in fig. 4.Viral particles with rod shaped morphology canbe seen.margination <strong>and</strong> chromatin condensation were alsoevident at lower level (Fig. 5d).Fig. 4 shows rod shaped viral particles by negativestaining in epidermal homogenate.4. Histopathology : Histo-pathological changesin foregut, gills, hepatopancreas <strong>and</strong> muscle fromtiger shrimp naturally infected with white spotsyndrome were observed. The most pronouncedchanges were observed in gut epithelia. The cellsexhibited hypertrophied nuclei with eosinophilicto basophilic inclusion bodies. Large number <strong>of</strong>nuclei demonstrated central chromatin diminution<strong>and</strong> margination. Complete condensation <strong>of</strong> nucleiwas also observed (Fig. 5b). Pyknosis <strong>and</strong>karyorrhexis were evident with the necrosis <strong>of</strong>t<strong>issue</strong> leading to changes in the normalarchitecture <strong>of</strong> the t<strong>issue</strong>s. The WSSV infectionin gills was indicated by hypertrophied nuclei,basophilic inclusion bodies <strong>and</strong> completecondensation <strong>of</strong> nuclei. Chromatin marginationwas detected at lower level (Fig. 5a). Pyknosis<strong>and</strong> karyorrhexis were more pronounced inconnective t<strong>issue</strong> compartment <strong>of</strong>hepatopancreas; though damage to the tubularepithelial cells <strong>of</strong> hepatopancreas was not clearlyevident (Fig. 5c). The muscle t<strong>issue</strong> showedsevere necrosis, fragmentation <strong>and</strong> haemocyticinfiltration. Nuclear hypertrophy, chromatinFig. 5a to 5d t<strong>issue</strong>s <strong>of</strong> P. monodon infected with WSSV.The histopathological changes like hypertrophied nucleus(HY), pyknosis (P), chromatin margination (CM),karyorrhexis (K) <strong>and</strong> necrosis (N)-in gills (a); gut (b);hepetopancreas (c); <strong>and</strong> muscle (d) (Fig. 5a to d scalebare=50µm).5. SDS PAGE : A clear change in expression <strong>of</strong>proteins in muscles <strong>and</strong> hepatopancreas has beendetected in infected shrimps as compared touninfected ones during SDS-PAGE analysis. Ininfected muscle t<strong>issue</strong>, four newly expressedproteins were detected (Fig. 6b). Two newlyexpressed proteins are <strong>of</strong> higher molecularFig. 6a In infected hepatopancreas, lane 1, 2, 3 showsexpression <strong>of</strong> three proteins 17kDa, 20kDa <strong>and</strong> 46kDa;lane 5, 6, 7 shows expression <strong>of</strong> protein from controlsamples <strong>and</strong> lane 4 is molecular weight marker b<strong>and</strong>.Figure 6b In muscle, Lane 3, 4, 5, 6, 7 shows fournewly express proteins 79kDa, 58kDa, 36kDa <strong>and</strong>17kDa; Lane 1 is molecular weight marker b<strong>and</strong> <strong>and</strong>lane 2 is for control sample.Protein expression in WSSV
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1016weight, 79kDa <strong>and</strong> 58kDa; whereas two newlyexpressed proteins are <strong>of</strong> low molecular weight,36kDa <strong>and</strong> 17kDa. Hepatopancreas <strong>of</strong> infectedshrimps exhibited two new protein b<strong>and</strong>s with lowmolecular weight, 17kDa <strong>and</strong> 20kDa, as well asa protein b<strong>and</strong> with high molecular weight, 46kDa(Fig. 6a). In both the infected t<strong>issue</strong>s, a newlyexpressed protein with molecular weight 17kDahas been detected. In hepatopancreas, a 62kDaprotein b<strong>and</strong> has not been detected which hasbeen expressed in uninfected shrimps.DiscussionThe outbreak <strong>of</strong> white spot syndrome incultured penaeid shrimps have been observed inIndia since 1983 (5,17). In Gujarat (India), shrimpculture is being practiced in the coastal farms inthe southern region; but the outbreak <strong>of</strong> thediseases in these cultured ponds appears not tohave been analyzed systematically. During themonitoring <strong>of</strong> hygienic condition <strong>of</strong> theseaquaculture units, P. monodon with themorphological symptoms <strong>of</strong> WSSV have beendetected. Monitoring <strong>of</strong> WSSV in the growoutponds has been suggested as one <strong>of</strong> the healthmonitoring methods in the farming <strong>of</strong> shrimps (18).Infected shrimps exhibited lethargy, anoxia,opaque musculature, red body colouration <strong>and</strong>white spots on carapace as well as appendages.White spot syndrome virus is the extremelyvirulent, contagious causative agent <strong>of</strong> white spotsyndrome <strong>of</strong> shrimp; <strong>and</strong> it causes high mortality<strong>and</strong> affects commercially cultured marine shrimps(2).Under the SEM, the white spots wereobserved as spheres on both inside <strong>and</strong> outsidesurfaces <strong>of</strong> the carapace cuticle. White spots <strong>of</strong>inside surface were quite larger (25µm to 135µm)as compared to outside surface, where very smallwhite spots (25 to 32µm) have been detected (Fig.2a-2b). The formation <strong>of</strong> white spot is acharacteristic clinical sign <strong>of</strong> WSSV infection (1).The crustacean cuticle is secreted by underlyingepidermal cells <strong>and</strong> mainly made up <strong>of</strong> chitin,calcium carbonate as well as protein (19).Cuticular epithelial cells are one <strong>of</strong> the mostpreferred sites <strong>of</strong> the WSSV <strong>and</strong> are amongstthe first sites to be infected (10). White spots arereported to derive from the abnormalities <strong>of</strong>cuticular epidermis. Blockage <strong>of</strong> secretaryproducts from cuticular epithelial cells possiblyby WSSV viral infection might have affectedcuticle with coloured spots. Presently observedmorphological analysis <strong>of</strong> white spots, analyzedby SEM is found to be similar to the morphologicalanalysis <strong>of</strong> white spots during WSSV infectionreported by Wang et al. (1).The chemical analysis <strong>of</strong> white spot byEDX analysis with SEM indicates changes in thelevels <strong>of</strong> certain elements like C, O, P <strong>and</strong> Ca ascompared to healthy shrimps. Though Wang etal. (1) has reported that the chemical composition<strong>of</strong> the white spot is similar to that <strong>of</strong> normalcuticle; in the present investigation, a clear increasein Ca <strong>and</strong> O as well as decrease in C in whitespot region has been detected in comparison withhealthy shrimps. Damage to the cuticularepithelium by WSSV infection seems to beresponsible for the change in the chemicalcomposition <strong>of</strong> white spots.Evidence <strong>of</strong> histopathologicalmanifestation in the target t<strong>issue</strong>s is one <strong>of</strong> thecriteria used in the diagnosis <strong>of</strong> WSSV infection(9). This paper describes the histopathologicalchanges in foregut, gills, muscles <strong>and</strong>hepatopancreas <strong>of</strong> WSSV infected P. monodon.The WSSV target t<strong>issue</strong>s are reported to begenerally <strong>of</strong> ectodermal <strong>and</strong> mesodermal in originincluding connective <strong>and</strong> epithelial t<strong>issue</strong>s,hematopoietic nodules, haemocytes, gills,epidermis, foregut, striated muscles <strong>and</strong> nerves(2,20). In the present investigation, the cells <strong>of</strong>infected gut <strong>and</strong> gills exhibited nuclearhypertrophy, chromatin diminution <strong>and</strong>margination as well as dense basophilicBhatt et al