Materials and Methods - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/10439/10/10_chapter 3.p… · The total ammonia nitrogen was estimated by the phenate method as described

Materials and Methods________

Materials and Methods

59

3.1 Materials and equipment used in the present study

The following equipment, chemicals materials were used in the present

study were provided details of make and supplier information of these materials

has been provided herewith.

3.1.1 Equipments

PCR Thermal cycler, Master cycler (Eppendroff, Germany)

UV Tran’s illuminator (Spectroline, England)

Electrophoresis unit (Broviga, India)

Centrifuge (Remi C-24, India)

Digital pH meter (Environmental Instruments, India, Model no. 112)

Salinity Refractometer (Atago, Japan, model smill -e)

Dissolved oxygen and Temperature meter (Equinox, Taiwan, Model 8404)

Microtome (Leica- USA)

3.1.2 Media

TCBS agar selective, Moller decarboxylase medium, Lysine, Ornithine &

Arginine, Tryptone, Kovac’s Reagent, Cytochrome Oxidase Discs, Saffranin,

Iodine, Glucose, Sucrose, Lactose, Mannitol, Tryptic Soy Agar, Oxidation

Fermentation Medium (Himedia laboratories, mumbai)

3.1.3 Diagnostic Kits used

Shrimple Kit from EnBioTechnology Laboratories, Japan

Rapid Dot Kit from Mangalore Fisheries College


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Monodon baculo virus detection kit for Shrimps from Bangalore Genie catalog

No: 105842

IQ 2000 WSSV detection and prevention kit from Pad lab Biotech – Chennai

Iscreen – Isothermal PCR kit and oven from farming Intelligence Tech. Corp.,

Taiwan

IQ2000 WIT MultiVirTM system -multi viral diagnostic system for marine

shrimp, from Farming Intelligence Tech. Corp.Taiwan.

3.1.4 Other Material

Ethylentdiaminetetra Acidic Acid Disodium salt (Merck-Catalog no. 28025)

The absolute ethyl alcohol acquired from changshu yangyuan chemical, china

Benzalkonium chloride –50 % from Biostadt limited, Mumbai.

B-green from Sujay agrilabs

Neutral from Finar laboratory chemicals, Mumbai

Agarose from Himedia laboratories, Mumbai

Copper sulphate penta hydrate by Merck India limited

Hydrogen peroxide manufactured by Asian peroxides limited

Microscope- Quasmo optics model no pzbc-25

Citric acid, Epsom salt and calcium carbonate are commercial products-

unbranded

100 micron nylon mesh hapa 1 m3 was used for survival estimation of post

larvae stocked

Zooplankton net with graduated test tube was used for quantitative estimation


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Tinospora cordifolia extract supplied by Sri Venkateswara Ayurveda Nilayam,

Kavuru, A.P., India

Feed binder- Probind manufactured by Alpha biologicals, Nellore, A.P., India

Haemocytometer manufactured by Paul Marienfeld GmbH& Co. Germany

Secchi disk for measuring transparency

Mineral mixture-Agrimin manufactured by Virbac India limited

Formaldehyde 37% manufactured by Qualigenes India limited

Iodophores 10%, Mizuphor manufactured by biostadt limited mumbai

Yucca extract manufactured by Alltech, USA.

3.2 Methods

3.2.1 Estimation of alkalinity

The amount of alkalinity was estimated in water sample by using APHA

2320 B titration method.

Reagents

a. Methyl Orange Indicator: 50 mg of methyl orange was dissolved in

100ml of distilled Water.

b. Phenolphthalein: 50 mg of phenolphthalein was dissolved in 95%

ethanol of about 50ml.

c. Standard Sodium Carbonate (0.02N): Dissolved dried sodium carbonate

about 106 mg in 100ml of distilled water. The distilled water was boiled

for 10 –15 min to expel CO2 and cooled before using and it is prepared

freshly.

d. Standard Sulphuric Acid (0.02N): 0.28 ml of H2So4 made up to 500ml in

volumetric flask with distilled water.


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Procedure

To the 25 ml of sample, 2 drops of phenolphthalein indicator was added

and titrated against 0.02N H2So4. The end point is disappearence of pink color

indicates the carbonate alkalinity. In the same sample 2 drops of methyl orange

and 2 drops of bromocresol were added then it was titrated against 0.02 N

H2So4, appearance of orange colour was taken as an end point and measured

as total alkalinity.

Calculation

Total Alkalinity = Vol. Of H2 So4 x Normality of H2 So4 x 50 x 1000Vol of sample

Carbonate Alkalinity = Vol. of H2 So4 x Normality of H2 So4 x 50 x 1000 x 2Vol. of the sample

Bicarbonate Alkalinity = Total Alkalinity – Carbonate Alkalinity

3.2.2 Estimation of hardness, calcium and magnesium

The hardness of the experimental sample was estimated by titration method

as per APHA 2340c.

Reagents

a) Buffer Solution: Dissolved 6.75 g of NH4Cl in 57 ml of concentration

NH4OH and made up to 100 ml with distilled water.

b) Standard Calcium Solution : Weighed 100 mg of CaCo3 and dissolved

with few drops of 1:1 HCl, add 20ml of distilled water, boiled for

10 –15 min, cooled and adjusted the pH to 7 with 3N NH4oH and made up

to 100 ml with distilled water.


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c) Erichrome Black - T: 2.25 g of Hydroxylamine Hydrochloride was added to

0.25 g of erichrome black T indicator and dissolved in 50 ml of 70%

ethnol.

d) Standard EDTA: Dissolved 2 g of EDTA and 50 mg of MgCl2.6H2O in 500

ml of distilled water

Calculation = Normality of CaCO3 (0.02N) x Vol. Of CaCO3

Volume of EDTA

Procedure

5 ml of the sample was taken in a 150 ml conical flask and added 2 ml of

hardness buffer and 2 drops of erichrome black – T indicator and it was titrated

with standared EDTA solution. The endpoint was noted by colour change from

wine red to pure blue. The EDTA rundown was recorded and total hardness was

calculated with the equation

Total Hardness = Vol of EDTA x Normality of EDTA x 50 x 1000(mg/l as CaCO3) Volume of Sample

3.2.3 Estimation of calcium

The amount of calcium was estimated by titration method as per APHA

3500 Ca method. To the 5 ml of sample, 5 ml of 1N NaoH and a pinch of

Muroxide Indicator were added, then it was titrated against standard EDTA, the

change of colour red to violet indicates the end point.

Calcium Hardness = Vol. Of EDTA x Normality of EDTA x 50 x1000Vol. Of sample


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3.2.4 Estimation of magnesium

The magnesium amount was estimated by calculation method as per

APHA 3500 - Mg method and the amount of magnesium was calculated as

follows.

Magnesium Hardness = Total Hardness – Calcium Hardness

3.2.5 Estimation of total ammonia nitrogen

The total ammonia nitrogen was estimated by the phenate method as

described in APHA 4500-NH3 method.

Reagents

a) Phenol Solution: 20 g of Phenol was dissolved in 200 ml of 95% of

ethanol.

b) Sodium Nitroprusside: 1 g of Sodium Nitroprusside was dissolved in 200

ml of distilled water.

c) Alkaline Reagent: 100 g of Sodium Citrate and 5 g of Sodium Hydroxide

were dissolved in 500 ml of distilled water.

d) 1.5 N Sodium Hypochlorite Solution.

e) Oxidizing Solution: 100ml of Alkaline Reagent was mixed with 25ml of

Sodium Hypochlorite and the solution was prepared freshly for each

experiment.

f) Stock Standard: 0.1g of Ammonium Sulphate (NH4SO4) was dissolved in

1000 ml of distilled water and added with 1ml of CHCl3..


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g) Working Standard: From 1 ml of stock standard solution, working stands

was preaperd.

Procedure

To 50 ml of sample, 2 ml of Phenol solution, 2 ml of sodium Nitroprusside,

5 ml of oxidizing solution were added mixed well and allowed to stand for 1 hr at

room temperature, covered with aluminium foil, read the absorbance at 640 nm.

Calculation

Ammonia = Test Optical Density x Concentration of Standeaded Standaderd Optical Denstiy

3.2.6 Estimation of Phenol Oxidase (PO)

The Phenol oxidase activity was assayed as described by Sung et al.,

(1994) by using 3-4-dihydroxyphenyl-alanine (L-DOPA) as a substrate.

Procedure

Phenoloxidase activity (PO) activity was detected spectrophotometrically

(490 nm) by the formation of the DOPA-chrome pigment, after oxidation of the L-

dihydroxyphenylalanine substrate (L-DOPA). Hemolymph was taken from

individual shrimp using a needle inserted into ventral sinus cavity and with drawn

into the syringe containing precooled (40C) anticoagulant (0.45M NaCl, 0.1M

glucose, 30 mM sodium citrate, 26 mM citric acid ,10 mM EDTA, pH 7.0)

(Soderhall and Smith, 1983) using the ratio one volume of hemolymph to two

volumes of anticoagulant. The hemocyte suspension was then centrifuged at 800


66

x g for 10 minutes at 40C. The resulting pellet was resuspended and

homogenized in 10 mM sodium cacodylate buffer (10 mM sodium cacodylate, pH

7.0). After homogenization, the sample was centrifuged at 10,000 x g at 40C for

20 minutes to remove cell debris. The supernatant was collected, referred as

hemocyte lysate supernatant and used for determination of phenoloxidase

activity

Phenol oxidase was determined by incubation of 50 micro liters of

hemocyte lysate suspension with 50 micro liters of L-DOPA (3mg/ml). After

incubation at room temperature for 10 minutes the developed colour was

monitored by its absorbance at 490 nm. The protein content of hemocyte lysate

was determined by folin reaction (Lowry et al., 1951).

Calculation

Prophenol oxidase activity =absorbance after 10 min. –absorbance at 0 min. (Units)

One unit of enzymatic activity = variation of 0.001 in the absorbance /min/ mg of protein (Soderhall, 1984).

3.2.7 Isolation culture and identification of vibrio species

Bacteria was isolated and identified from different parts of the shrimp

according to the procedure given by Lightenr et al. (1996). Bacteria were isolated

during a suspected outbreak from L. vannamei and Penaeus monodon shrimps

were collected for Vibrio culture. The tissues like gills, hepatopancreas and

haemolymph were plated on TCBS agar. Shrimp were disinfected with 70%


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alcohol, gills and hepatopancreas macerated in sterile normal saline solution (1 g

sample to 10 ml normal saline), serially diluted, in order to be scattered on Petri

dishes with Agar TCBS and incubated at 30°C for 24h. Samples of haemolymph

were collected from the ventral sinus, scattered on petri dishes with agar TCBS

and incubated at 30°C for 24 h.

Composition of TCBS agar

Ingredients g/litre

Peptone special 10.00

Yeast extract 5.00

Sodium thiosulphate 10.00

Sodium citrate 10.00

Sodium cholate 3.00

Oxgall 5.00

Sucrose 20.00

Sodium chloride 10.00

Ferric citrate 1.00

Bromo thymol blue 0.04

Thymol blue 0.04

Agar 15.00

3.2.7.1 Identification of bacterial species using Biochemical tests

Bacteria were subjected to various biochemical tests for identification

using the procedures described by Kandler et al. (1986), and Lightner et al.

(1996).


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3.2.7.1.1 Gram staining

Gram staining was done to identify whether the bacaterium isolated is

gram positive or negative.

Reagnets

Crystal Violet and Grams Iodine

95 % Ethyl Alcohol and Saffranin

Procedure

Bateraial culture samples were air dried or heat fixed and flooded the slide

with crystal violet for 1 min and then washed the slide in a gentle and direct

stream of tap water for 2 seconds and again flooded the slide with iodine

mordant for 1 minute. After addition of iodine mordant wash the slide in a gentle

or direct stream of tap water for 2 seconds and blot dry the slide with absorbent

paper, immerse the smear in 95% v/v ethanol for 30 seconds with gentle

agitation and aging blot dry the smear with absorbent paper, immerse the smear

for 2 minutes with counter stain and finally wash the smear in a gentle and

indirect stream of water until no colour appears in the wash water. After staining

the color taken by the bacteria was noted culture appears pink in colour, they are

gram-negative bacteria.

3.2.7.1.2 Motility by hanging drop technique

In this investigation the hanging drop method was used to see motility of a

selected species of bacteria.


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Procedure

Clean depression slide was prepared with cover slip by washing with

warm water and all grease was removed and a very small amount of Vaseline

was placed near each corner of a cover slip for adhesion cover slip to the

depression slide. Shaked the culture tube and transfered two loopfuls of the

culture onto the cover slip. Placed the slide in contact with the cover slip with the

depression over the drop of suspended bacteria. Invert the slide quickly so that

the drop cannot run off to one side.

Microscopic observarion

The slide was first observed with the low-power objective, once the image

is visible under low power, swing the high-dry objective into position and readjust

the lighting. Since most bacteria are drawn to the edge of the drop by surface

tension, focused near the edge of the drop and keep lighting to a minimum.

Living bacteria are almost impossible to see in bright light.

3.2.7.1.3 Estimation of Oxidase

The oxidase test was used to assist in the identification of Vibrio spp.

That produce oxidase enzyme. Oxidase enzyme plays an important role in

electron transport system during aerobic respiration cytochrome oxidase

catalyzes the oxidation of a reduced cytochrome by molecular oxygen resulting in

the formation of hydrogen per oxide the enzyme oxidizes the reagent n n-tetra

methyl-1 paraphenyldiamine dihydrochloride to a coloured product Indophenol.


70

Procedure

Oxidase disc was placed on the bacterial colony.If the organism produces

oxidase enzyme the phenyl amine in the reagent will be oxidized forming deep

purple blue colour and oxidase positive forms deep purple blue colour. Oxidase

negative no colour formation

3.2.7.1.4 Decarboxilase test

Decarboxylases are group of substrate specific enzyme that are capable

of reacting with the carboxyl group (COOH) portion of amino acids forming

alkaline reacting amines, this reaction is known as “decarboxylation” forming CO2

as a second product. Lysine, ornithine and arginine are three amino acids tested

for the identification of Vibrio species.

Moller decarboxylase medium is the base most commonly used for amino

acid decarboxlation test. The amino acids were added to the decarboxylase base

prior to inoculation. A control tube containing only the base without the amino

acid must be kept the tubes incubated anaerobically by overlying with mineral oil.

During initial stages both the tubes turns yellow colour due to fermentation of

small amount of glucose in the medium. If the amino acid is decarboxylated

alkaline amines are formed and the medium turns back to its original purple

color. It removes the CO2 from amino acid in the presence of a specific

decarboxylase enzyme and results in the break down of amino acid with the

formation of corresponding amines. Bromocresol purple is used as a pH indicator

and a positive decarboxylase test yielding excess amino acid results in excess


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amines, which was indicated by purple colour (Bromocresol purple is yellow in

acidic pH).

Procedure

Moller decarboxylase medium was prepared and the test culture was

inoculated. A control tube containing only the base without the amino acid was

kept and inoculated. Overlayed the inoculated broth tubes with sterile mineral oil

to cover 1 cm of the surface. The tubes were incubated at 370C for 3- 4 days.

Conversion of the control tube to yellow colour indicates that the organisms are

viable and the pH of the medium was lowered sufficiently indicative of the

presence of decarboxylase enzyme. The tubes containing amino acid form

purple blue colour indicates the positive test release of amines from

decarboxlation medium. After incubation the tubes containing lysine with test

organisms changed the colour of the medium from purple to yellow and purple

again indicates the positive reaction. If medium remains yellow and never

becomes purple indicates negative reaction.

3.2.7.1.5 Fermentation of Carbohydrates

The test organisms were inoculated in the tubes of tryptone or peptone

medium containing glucose or other carbohydrate with indicator

bromothymolblue. The inoculated medium in one tube is sealed with a layer

liquid paraffin to exclude oxygen. Fermentative organisms utilize the

carbohydrate in both open and sealed tubes and change in the color in the


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medium from green color to yellow was observed. Oxidative organisms are also

able to use the carbohydrate only in the open tube there was no utilization in the

sealed tube. Production of acid may be slow and therefore cultures were

incubated for 7 –14 days.

Procedure

The oxidative fermentation medium was prepared and inoculated in the

test culture aseptically in respective tubes and covered with 10 mm deep layer of

sterile paraffin or molten wax then incubate the tubes at 370C and examined daily

the tubes for carbohydrate utilization. The tubes that turn from green to yellow

are of positive reaction. No colour change indicates negative

3.2.7.1.6 Indole production

Tryptophan is an essential amino acid that undergoes oxidation by

enzymatic activity in bacteria and conversion of tryptophan into metabolic

products mediated by the enzyme tryptophanase. Microorganism’s hydrolysis the

tryptophan and produce indole as an end product. In this experiment tryptone

broth which contains the substrate tryptophan is used. The presence of Indole is

detectable by adding Kovac’s reagent, which produces cheery red colour. This

colour is produced by the reagent, which is composed of p-dimethyl amino

benzaldehyde and hydro chloric acid. Culture producing a red colour ring

following the addition of Kovac’s reagent is Indole positive. The absence of red

colour indicates Indole negative.


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Procedure

Peptone broth was prepared and inoculated with test cultures and tubes

were incubated at 370C for 24 hrs. After incubation, 0.2ml of Kovac’s reagent

was added.

Kovac’s Reagent

Para dimethyl amino benzaldehyde 5.0 g

Amyl alcohol 75.0 ml

Concentratd Hcl 25.0 ml

3.2.8 Bacterial clearance

To determine bacterial clearance rate, shrimp were injected

intramuscularly at the 6th abdominal segment with 10 μl of a suspension of Vibrio

parahemolyticus isolated form an infected shrimp. Three shrimp from each feed-

treatment group were removed immediately after injection and then another 3

each at 30 minutes post-injection to determine the number of bacterial cells in

hemolymph. Hemolymph (50 μl) was collected from the ventral sinus of the first

abdominal segment into a syringe containing 100 μl of anticoagulant solution AC-

1 (0.45 M NaCl, 0.1 M glucose, 30 mM Na-citrate, 26 mM citric acid, 10 mM

EDTA, pH 7.0) (Söderhäll and Smith, 1983) and added immediately to a tube

containing 350 μl of TSB. This was serially diluted 10 fold in Tris-buffered saline

(TBS, 50 mM Tris-HCl pH 7.3, 100 mM NaCl) before 20 μl quadruplicate aliquots

were dropped onto plates of thiosulphate, citrate, bile-salt (TCBS) and agar to


74

obtain bacterial counts (CFU) after incubation at 30°C for 10 h. Only serial

dilution plates giving individual drop counts in the range of 20 to 40 colonies were

used. Counts were calculated according to dilution and recorded as the mean

CFU count for the quadruplicate counts (Söderhäll and Smith, 1983)

3.2.9 Hemolymph clotting time

Haemolymph clotting time was estimated as per the procedure described

by Lightner (1996). After, surface disinfection of the shrimp with alcohol,

tuberculin syringe with 27 gauge needles were used to obtain blood sample from

Ventral sinus using no anticoagulant. Transfered drop of hemolymph to slide (at

room temperature) as quickly as possible after extraction from a shrimp

Noted when hemolymph drop gels and elapsed time = clotting time.

3.2.10 Total Haemocyte Count (THC) and Differential Haemocyte Count (DHC)

Haemolymph was collected in a syringe containing fixative (10% formalin

in 0.45 M NaCl) with ratio of one volume of haemolymph to one volume of

fixative. The amount of 20 l of fixed haemocyte was mixed with 20 l of Bengal

Rose solution (1.2% Bengal rose in 50% ethanol). Total haemocyte count was

determined by using Haemocytometer. The smear of fixed haemocyte was

prepared and stained with Mayer’s hematoxylin solution by the process below


75

Dipped the smeared slide in hematoxylin solution for 5 minutes. Then

washed with running water and then rinsed with 80% alcohol. After that serial

dilution was performed in 95% ethanol, 2 times with absolute ethanol) and 2

times with clear in xylene for 3 times and mounted with DPX. Numbers of

granular cells were recorded in 200 cells of total haemocytes (Söderhäll and

Smith, 1983).

3.2.11 DNA extraction lysis buffer, phenol chloroform and spin column

3.2.11.1 Preservation of RNA and DNA in tissues

For routine diagnostic testing by PCR, RT-PCR, samples were prepared

to preserve the pathogen’s nucleic acid. Preservation of samples in alcohol (70–

100%) was the most preferred method for subsequent molecular tests (Oie,

2009).

3.2.11.2 DNA Extraction by Phenol Chloroform Method

Sample was prepared by the proteinase K/SDS method, from 30 to 50 mg

minced tissue, according to current protocols in molecular biology (Sambrook

and Russel, 2001). The purified shrimp DNA was resuspended in TE buffer (10

mM Tris-HCl, pH 7.5, 1 mM EDTA) and stored at 4°C. DNA quality and integrity

were confirmed by agarose gel electrophoresis.


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Materials

The materials used were as follows: TE Buffer, 20 mg/ml proteinase K,

Phenol/Chloroform/Isoamylalcohol (25/24/1), Isopropanol, Extraction Buffer and

Ethanol 70%.

Procedure

To 20mg of sample tissue 500 µl lysis buffer was added, homogenated the

tissue and after homogenation added 5 µl of proteinase K to the tissue, mixed

well and incubated for 1 h at 370C. After incubation added equal volumes of

phenol/chloroform/isoamyalalcohol (25:24:1) and mixed well and centrifuged at

13000 RPM for 15 minutes. Collected the aqueous layer and transferred into a

new tube. Aqueous layer was precipitated with 0.6 ml of Isopropanol. Centrifuged

at 13,000 rpm for 15 minutes. Discorded the supernatant and pellet present at

bottom was dried. To the pellet 100 µl of TE buffer was added to dissolve the

DNA and stored at –200 C for longer time (or) DNA dissolved in 100 µl of water to

use for further use.

3.2.11.3 DNA Extraction by Spin Column Method (QIAGEN)

After ttissue lysis and added proteinase K, which breaks down proteins.

Protein free sample was loaded on column and nucleic acids bind to membrane

and column washing was done for removing any contaminants. DNA was

released from column and ready for use in downstream application.


77

Materials

The following materials were used for the experiment: ERC Buffer, PE

Buffer, EB Buffer, Spincolumns, and Micropipette (0.5 to20ul- 200ul), Tissue

griders, and Disposable gloves.

Procedure

To 20 mg of tissue added with 300µl of ERC buffer, grinded the tissue and

checked the colour of the mixture was yellow. Placed a spin column in a 2 ml

collection tube in a suitable rack. To bind DNA, applied the sample to the mini

elute column and centrifuged at 12,000rpm for 1 minute. Discarded the fiow-

through and placed the column back into the same tube. The samples were

washed with, 750 µl Buffer PE to the column and centrifuged for 1 minute.

Discarded the flow-through and place the column back in the same tube.

Centrifuged the column for an additional 1 minute at maximum speed. Placed the

column in a clean 1.5 ml microcentrifuge tube. To elute DNA, added with 10 µl

Buffer EB to the center of the membrane, allowed the column to stand for 1

minute, and then centrifuged for 1 minute.the DAN that was collected in the

microcentrifuge tube was used MBV PCR, DNA quality and quantity estimation.

3.2.12 Quality and Quantity estimation of DNA

After isolation of DNA, quantification and analysis of quality was estimated

to ascertain the approximate quantity of DNA obtained and the suitability of DNA


78

sample for further analysis. The used methods for quantifying the amount of

nucleic acid in a preparation were spectrophotometric analysis (Sambrook and

Russel, 2001). Analysis of UV absorption by the nucleotides provides a simple

and accurate estimation of the concentration of nucleic acids in a sample.

Purines and pyrmidines in nucleic acid showed absorption maxima around 260

nm (eg. dATP: 259 nm; dCTP: 272 nm; dTTP: 247 nm), if the DNA sample is

pure without significant contamination from proteins or organic solvents. The ratio

of OD260/OD280 should be determined to assess the purity of the sample.

Procedure

To 5 l of DNA sample, 995 l TE (Tris-EDTA buffer) was added and

mixed well. Using TE buffer blank was set in spectrophotometer and reading was

noted at OD260 and OD280. Calculated the OD260/OD280 ratio at a ratio of 1.8-2.0

denotes that the absorption in the UV range is due to nucleic acids. Ratio lower

than 1.8 indicates the presence of proteins and/or other and higher than 2.0

indicates that the samples may be contaminated with chloroform or phenol. The

amount of DNA can be quantified using the following formula:

DNA concentration (g/ml) = OD260 x 200 (dilution factor) x 50g1000


79

Spectrophotomteric Conversions for Nucleic Acids:

1 unit of Absorbance at 260nm of ds DNA = 50 g/ml

1 unit of Absorbance at 260nm ss oligonucleotides = 33 g/ml

1 unit of Absorbance at 260nm ss RNA = 40 g/ml

3.2.13 PCR for WSSV, MBV, TSV and IHHNV

PCR methods for WSSV and IHHNV were followed as per the procedures

of IQ 2000 KIT method. Isothermal PCR for WSSV was performed as per the

procedure of Iscreen kit manufactured by Framing Intelligence Technology

Corporation Taiwan. PCR method for MBV was followed the procedures of

Bangalore Genie Kit. And the PCR method for TSV was followed using WIT

MULTIVIRTM system

3.2.14 Microscopic detection of Monodon Baculo Virus

The diagnosis of Monodon Baculo Virus was made by the demonstration

of single or multiple spherical occlusion bodies in squash preparations of

hepatopancreas, midgut, or feces examined by phase or bright field microscopy.

MBV occlusion bodies were shown to be intranuclear and to range in diameter

from less than 0.1 µm to nearly 20 µm. Even in fresh feces, MBV occlusions

often remain in clusters, held together by the nuclear membrane. Staining the

tissue squash with a stain like 0.05% aqueous malachite green, which aids in

demonstration of the occlusion bodies by staining them more intensely than

similar sized normal host cell nuclei, nucleoli, secretory granules or

phagolysosomes and lipid droplets (Oie, 2009)


80

3.2.15 Rapid Field Stain Method

The moribund shrimp suspected of having WSSV, excised gills,

appendages or stomach were minced and then squashed, dabbed, or smeared

onto a slide. Fixed the smear in methanol for 6 minutes or fixed by heating the

slide carefully. And then smear was flooded with an appropriate stain such as

Giemsa or other blood smear stains. Stain for ~ 1 to 5 minutes. After placing the

coverslip, the smear was examined with 10, 20 and 40x objectives. Tissue

squashes or impression were prepared from gills or cuticular epithelium of shrimp

with clinical signs, which have displayed the cells with hypertrophied nuclei with

diagnostic inclusion bodies. Occlusion bodies were absent in WSSV infected

nuclei. Normal cell nuclei are 4 to 10 µm in diameter and display chromatin

threads and a nucleolus. Infected nuclei are hypertrophied and contain usually a

single eosinophilic to bluish inclusion body in severely affected shrimp (Lightner

et al., 1996).

3.2.16 Histology

a) Fixation of the samples

For the histological studies, the diseased shrimp were collected with a

minimum handling stress. The shrimp were transported to the laboratory in a

well oxygenated water-filled utensil. Adequate aeration was supplied to the

container. Ten volumes of Davidson’s fixative was used for one volume of tissue

sample .The fixative reduces autolytic changes in decapod crustaceans, and its

acidic content decalcifies the cuticle.


81

Reagents

The formulation for Davidson’s AFA Fixative (for 1 litre): 330 ml 95% ethyl

alcohol, 220 ml 100% formalin (a saturated 37–39% aqueous solution of

formaldehyde gas), 115 ml glacial acetic acid and 335 ml tap water .

Procedure

For larvae and post larvae that were too small to be easily injected with

fixative using a tuberculin syringe were fixed by immersing directly in the fixative

for 12–24 hours in fixative and then transferred to 70% ethyl alcohol for further

processing. For larger post larvae, juveniles, and adults injected the fixative into

the live shrimp. The hepatopancreas (HP) was injected first and at two or more

sites, with a volume of fixative sufficient to change the HP to a white to orange

colour, and then injected the fixative into adjacent regions of the cephalothorax,

into the anterior abdominal region, and into the posterior abdominal region. The

fixative was divided between the different regions, with the cephalothoracic

region, specifically the HP, receiving a larger share than the abdominal region.

Immediately following the injection, the cuticle was cut with dissecting scissors,

from the sixth abdominal segment to the base of the rostrum, care has been

taken that not to cut deeply into the underlying tissue. The incision in the

cephalothoracic region was just lateral to the dorsal midline, while that in the

abdominal region was approximately mid-lateral. For shrimp larger than ~12 g,

after injection of fixative, the body was transversely bisected, at least once, just

posterior to the abdomen/ cephalothorax junction, and again mid-abdominally.

For large crustaceans the organs of interest were excised after injection of


82

fixative. Following injection, incisions and bisection/trisection, or excision of key

organs, immersed the specimen in the fixative (use 10:1 fixative: tissue ratio).

Allowed fixation to proceed at room temperature for 24–72 hours depending on

the size of the shrimp being preserved. Following fixation, the specimens were

transferred to 70% ethyl alcohol, where they can be stored for an indefinite

period.

b) Preparation for Embedding

The following procedure was used for embedding the tissues in paraffin,

following a standard method, "gut-gill panorama".

Procedure

Removed preserved shrimp from 50% ethyl alcohol and placed on a

cutting surface.with a single edge razor blade, bisected shrimp transversely (for

shrimp greater in length than 3.0 cm) just posterior to the junction of the

cephalothorax and abdomen. Longitudinally bisected the cephalothorax just

lateral of the mid-line from the half of the cephalothorax without the mid-line,

removed, with a diagonal cut starting at the distal surface, the branchiostegal

region containing the gills. Utilizing a razor blade, separated abdominal segments

#1, 3 and 6 from the remainder of the abdomen, removed distal ends of the

uropods.

Shrimp tissues were processed in the following solutions:

a. 70% ethyl alcohol - two separate 1 hr baths.

b. 80% ethyl alcohol - two separate 1 hr baths.


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c. 95% ethyl alcohol - two separate 1 hr baths.

d. 100% ethyl alcohol - two separate 1 hr baths.

e. Clearing agent [xylene] - two separate 1 hr baths.

f. Paraffin -two separate 1 hr baths.

Tissues successfully embedded, as accomplished above and were then

placed in embedding molds to form blocks ready to be sectioned for further

observation.

c) Sectioning

The embedded samples were used for sectioning. The sections of 5 µm

were taken and placed on the water in water bath to spread.

d)Staining

The sections were stained with Mayer-Bennett Hematoxylin and

Phloxine/Eosin (H&E):

Mayer-Bennett Hematoxylin and Phloxine/Eosin (H&E)

Reagents

Mayer-Bennett Hematoxylin was prepared by using the following chemicals

Hot distilled water 2000 ml, Hematoxylin 2.0 g, Sodium iodate 0.4 g,

Potassium aluminum potassium sulfate 180 g, Citric acid 2.0 g, Chloral

hydrate 100 g, was mixed in order listed

Eosin-Phlocine

Stock eosin (1% aqueous eosin Y) 100 ml, Stock phloxine (1% aqueous


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phloxine B) 10 ml, 95% ethanol 780 ml, Glacial acetic acid 4 ml, were

mixed in order listed

Staining Method

The sections were processed in the foloowing sequence

Xylene -5 minutes

Xylene - 5 minutes

100% EtOH - 10 dips

100% EtOH - 10 dips

95% EtOH - 10 dips

95% EtOH - 10 dips

80% EtOH - 10 dips

80% EtOH - 10 dips

50% EtOH - 10 dips

Distilled water - 6 rinses (change water for each rinse)

Hematoxylin - 4-6 minutes.

Running tap water - 4-6 minutes.

Phloxine/eosin - 2 minutes

95% EtOH - 10 dips

95% EtOH – 10 dips

100% EtOH - 10 dips

100% EtOH - 10 dips

Xylene - 10 dips

Xylene - 10 dips

Xylene - 10 dips

Xylene - 10 dips

Coverslip with dpx.

Labelled


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3.2.17 Direct Microscopy

Samples for microscopic examination were processed as quickly as

possible as per the procedures described by Lightner et al., 1996 and Oie, 2009.

The following aspects were studied using bright field microscopy.

a) Observation of exoskeleton for morphology of white spots on shrimp

and prawns

b) Morphological characters of post larva

c) Observation of consumption of micro diets by the post larvae of

Penaeus monodon and its effect of feed texture on gut wall

d) Wet mount of pleopod for Chromatophore stage and protozoan

infestation, gills ,exoskeleton in case of unusual fouling, white fecal

matter

e) Slides of histology

3.2.18 Wet-Mount Diagnostic Procedures for gregarines

a) Removed mid gut.

b) Stripped mid gut contents onto a clean glass slide using the edge of a

cover slip and forceps.

c) Spread the gut contents and prepared a wet-mount.

d) Examined wet-mounts by direct microscopy with 10x and 20x objectives

for gregarine sporozoites, trophozoites, or gametocytes


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3.2.19 Immunodot/Rapid Dot

WSSV outbreaks were confirmed by rapid dot kit supplied by Mangalore

Fisheries College. The following procedure was followed as per the protocol

mentioned in the kit.

Step 1: Sterilized the mortar by washing with by burning with spirit (Sol. A). after

cooling the mortar added 40 drops of Sol. B to it.

Step 2: Gently plucked and taken 5-gills from a shrimp or required number of PL

by a sterilized forceps. Macerated the sample thoroughly (5 minutes) in

the mortar, transferred it to an eppendorff tube and kept for 10 minutes at

room temperature to settle down the bigger particles.

Step 3: Using a micro tip take 1.0 μl of supernatant sample from the eppendorff

tube and dot it on nitrocellulose paper (NC) kept in the reaction tray (Petri

plate). Also dot 1.0 μl of control +ve and -ve (provided in the kit box)

starting from notched end of the NC paper. Used separate new tip for

each sample. Air-dry the NC paper for 5 minutes.

Step 4: Added 1ml (25 drops) of Sol. C on the NC paper and shaked vigorously

for 2 minutes and incubated for 11 minutes at room temp (RT).

Step 5: Shaked vigorously for 2 minutes and discarded Sol. C. Washed the NC

paper thrice gently with Sol. D (2 ml) and removed it. Tap the reaction tray

against paper towel to remove the rest of the solution.

Step 6: Added 1ml of Sol. E (25 drops) or until NC paper was completely

immersed and shaked vigorously for 2 minutes and incubated for 41

minutes at RT.


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Step 7: Shaked vigorously for 2 minutes and poured off Sol. E. Washed NC

paper gently thrice with Sol. D. Tap the reaction tray against paper towel

to remove the rest of the solution.

Step 8: Added 1 ml of Sol. F (25 drops) or until NC paper was completely

immersed, shaked vigorously for 2 minutes and incubated for 11minutes

at RT.

Step 9: Shaked vigorously for 2 minutes and poured off Sol. F. Washed NC

gently thrice with sol. D. Tap the reaction tray against paper towel to

remove the rest of the solution.

Step 10: Added Sol. G (20 drops) on NC paper and kept shaking the tray (10

min) for development of a dark blue colour dot in positive control, without

any colour development in negative control dot. Development of blue

colour on sample dot indicates presence of White Spot Virus in the

sample.

Step 11: Washed the paper with tap water and noted the results.

Details of the solutions

Solution A - Spirit ; Solution B - Sample preparation buffer; Solution C -

Blocking buffer ; Solution D - Wash buffer; Solution E - 1st Antibody;

Solution F - 2nd Antibody; Solution G - Developer

3.2.20 Shrimple Kit Procedure

Shrimple kit supplied by EnBioTec Laboratories., Co. LTD, Japan was

used as rapid kit for the detection of WSSV. Shrimp that look sick and unhealthy


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were cleaned with water. Collected the pleopods at the bottom and put them into

a microtube. Care was taken not to put other portions than the swimming legs.

Homogenated the pleopods and gills in the microtube with pistile provided in the

kit. Allowed to settle for five minutes and took the supernatant with the dropper

and made four droppings into the round hole of the shrimple main equipment.

The appearance of a red band at the C point of the SHRIMPLE main equipment

indicates it is working properly. When a red band appears at the point T right

before the red band at the point C within 15 minutes after the dropping was

made, shows a positive result (WSSV infection). When it does not appear, it

shows a negative result (no WSSV infection).

3.2.21 Temperature

Water temperature was measured using a built in temperature sensor with

dissolved oxygen meter.

3.2.22 pH

pH of water was collected at different depths and was measured by using

the pH meter (EI 112) at the sampling sites from where the water was collected.

The value of the pH was then recorded from the meter reading.

3.2.23 Disease diagnosis using clinical signs and symptoms

The following gross signs and symptoms were observed on site as per the

guidelines prescribed by OIE (2009). The signs and symptoms like, colour of the


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shrimp, Antennae necrosis, Blisters, Tail rot, Gill colour and fouling, Body cramp,

White spots on exoskeleton, Muscle opaqueness, White faecal matter, Morbidity

and mortality were studied and reported.

3.2.24 Determination of growth rate

Growth sampling was done on site every week. For each sampling, 100

shrimps were collected and weighed. Average body weight was calculated by

dividing the total weight by number of shrimp.

3.2.25 Hapa survival and bioassay

The shrimp larvae (100 nos.) were stocked in a mesh hapa (1x1x1.2 M)

tied in the same pond. The survival was estimated after 24, 48 and 72 hours. The

same hapa was used for bioassay by stocking the healthy juveniles in a

problematic pond.

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