full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 4 (5) 1011-1020 January 2011. ISSN 0973-8916in few drops of sterilized brackish water prior tonegative staining. For negative staining, one dropof suspension was mixed with 4 drops of themixture of 0.1% bovine serum albumin and 2%phosphotungstic acid (1:2, pH 7.0). The mixturewas placed on a grid for 30 to 60 s and excesssuspension was removed with filter paper. Thepreparation was allowed to dry before beingexamined. The results were observed under TEM(Technai, 20; Philips series) (15).4. Histopathology : The histopathology of gills,hepatopancreas, muscle and gut were performedfrom WSSV infected and healthy shrimps. Tissueswere blotted free of heymolymph, fixed in Bouin’sfluid, sections were cut at 5-8µm and stained withH & E. The photographs were taken withAxioplan image analyzer at 40X and 100X.5. SDS PAGE Analysis : Muscles andhepatopancreas, 0.5gm each, from infected andhealthy shrimps were homogenized in 10ml TNbuffer (20 mM Tris-HCl, 400 mM NaCl, pH 7.4),centrifuged at 8510×g for 10min at -4ºC andsupernatant was subjected to polyacrylamide gelelectrophoresis using 10% polyacrylamideresolving gel and 5% stacking gel (16). Theresolving gel (30ml) was polymerized by 300µl of10% ammonium peroxodisulphate and 70µl of N,N, N, N-Tetramethylenediamine (TEMED).Stacking gel (10ml) was polymerized by adding50µl APS and 20µl TEMED. The samples weremixed with sample buffer, boiled for 5 min and allgel were run using a dual midi vertical gelelectrophoresis system at constant 100V in 1Xbuffer (25mM Tris, 25mM glycine, pH 8.3, 0.1%SDS). Proteins were visualized by silver stainingkit (Genie, Bangalore).Results1. Morphological symptoms : The infectedshrimps exhibited lethargy, anoxia, opaquemusculature and were detected near pond edges.They were detected with white spots on the1013carapace, on appendages and on other body partsas well as with pink to red body colouration (Fig.1). The other symptoms of infected P. monodonwere loose binding of the epidermis with cuticleand enlarged hepatopancreas with yellowishwhite colouration.Fig. 1 White spots of P. monodon. Arrow indicates the whitespots on the cuticle and other body parts.2. Scanning electron microscopy : The scanningelectron micrographs show white spots on bothupper and lower surfaces of carapace of infectedshrimps. On the inner surface of the carapace,spots were larger in size, usually rounded in shapemeasuring 25µm to 135µm in diameter. On theouter surface of carapace, white spots were verysmall in size and measuring between 25 to 32µmin size (Fig. 2a and 2b).Figure 2a-2b Scanning electron micrograph of whitespots of WSSV infected shrimps. Flattened White spotcovering the cuticle of carapace (a, b).EDX quantification of elements like C, O,Ca and P in carapace from both inner and outersurface with SEM in infected and control shrimpindicated that composition of inner and outersurface of white spots have not been changedand were measured as 38%; 32 to 33 %; 0.8 toProtein expression in WSSV