MATERIALS AND METHODSshodhganga.inflibnet.ac.in/bitstream/10603/10437/8/08_chapter 3.pdf · 35 MATERIALS AND METHODS A. Source of Fish: Experiments were performed on the common fresh

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MATERIALS AND METHODS

A. Source of Fish:

Experiments were performed on the common fresh water carp, Labeo rohita (Indian

Major Carp) which is extensively cultured in India and is valued as an important food fish.

B. Collection and maintenance of fish:

Healthy fish with an average weight of 50-60 gm were obtained from Jalipudi fish

farm, Jalipudi Mandal, West Godavari District, Andhra Pradesh and kept in the laboratory's

recirculating units until used. Fish were brought to the laboratory and maintained in cement

tanks. Both the experimental and control fish were acclimated to the laboratory conditions

for about 4-5 days before they were used for experimentation. Dechlorinated ground water

was used during acclimation and experimental period. The water in acclimation tanks were

frequently oxygenated with electrical aerators.

C. Bacterial strain and cultivation:

Aeromonas liquefaciens strain, MTCC 2654 (Virulent Strain) was obtained from

MTCC, Chandigarh, India. From this parent culture, sub cultures of A. liquefaciens were

prepared and doses were made under aseptic conditions.

D. Culture techniques of Aeromonas liquefaciens:

Culture and doses of Aeromonas liquefaciens was done following the method of

Pelczar (1993).

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i). Preparation of Medium:

Nutrient agar medium per 1000 ml is as follows:

Peptone - 5 gm

Beef extract - 3 gm

Nacl - 3 gm

Yeast extract - 2 gm

Agar Agar - 20 gm

Distilled water - 1000 ml

All the above contents were mixed with double distilled water and made to

1000 ml (PH-7.2). PH was checked before adding agar agar. The medium was distributed in

culture tubes (i.e. 5ml agar medium in each tube). The slants of required medium were

prepared by pouring 5ml nutrient agar medium in each and all the tubes were closed with

cotton plugs. The mouths of the tubes were wiped with cotton and autoclaved for 20

minutes. After sterilization, the slants were allowed to solidify in slanting position with

uniform angle with 600 under aseptic conditions using laminar air flow and left them

overnight for proper solidification under aseptic condition.

ii). Inoculation of bacterial strain for the preparation of subcultures:

The inoculation was carried out inside the inoculation chamber (Laminar air

flow) sterilized previously by using germicidal lamp and also by smearing the table surface

as well as sides of the chamber inside with disinfectant solution (surgical spirit). The plugs

of cotton tubes were opened near the flame and gently flamed the mouth of the culture tube.

A loop full of bacterial cell suspension from stock culture was streaked across the surface of



37

a solidified nutrient agar medium with a sterile (previously heated & cooled) inoculation

loop/needle.

After inoculation, the slants were incubated inside the incubator at 300C for 24

hours. Growth was observed in the slants after 24 hours. The same method was followed for

further subcultures.

The Petridishes and pipettes were washed and dried. The nutrient agar medium

was taken in a conical flask and closed with aluminum foil or cotton plug. All the above

were sterilized by autoclaving for 20 minutes. The plates and pipettes were dried in an oven

after sterilization. About 20 ml. of sterilized agar medium was poured into each sterile

petridish or petriplate in the proximity of a flame inside the inoculation chamber, sterilized

previously by cleaning the surface with surgical spirit.

Much care was taken while pouring plates, in opening the lid as little as

possible near the flame to avoid any microbial contamination from the atmosphere. The

dishes were allowed to solidify on uniform surface in the petridish. After solidification of

the medium, the petridishes were inverted and handled them carefully to avoid any

contamination and preserved them for use when required.

iii). Preparation of serial dilutions of bacteria:

100 ml of double distilled water, test tubes with cotton plugs and 10 pipettes

were sterilized by autoclaved for 20 minutes. After sterilization, they were cooled inside the

inoculation chamber. 9 ml of sterilized water was taken in all the test tubes. These were the

dilution blanks. One slant (sub culture) was taken and made it into a suspension with 1 ml

of sterilized water.



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The dilution blanks were labeled as 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 and so on.

§ The initial dilution was prepared by adding 1ml of sample into a 9 ml of dilution

blank labeled as 10-1. Thus the original sample was diluted ten times.

§ From the first dilution 1 ml. of sample was transferred to the second dilution blank

marked as 10-2 with sterile and fresh 1 ml pipette. Thus diluting the sample to 100

times. 1: 100 - 10-2.

§ From 10-2 suspension, 1 ml of suspension was transferred to 10-3 dilution blank.

Thus diluting the original sample to 1000 times 1:1000 -10-3.

§ Dilution of the original sample was repeated up to 10-8 using a fresh pipette.

iv) Plating the sample (Spread plate method):

Serial dilution of the sample was followed by spreading the sample in

petridishes containing medium. The Petridishes containing the sterilized solidified agar

medium was marked as 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 and so on. 0.1 ml of sample from

each dilution was taken i.e. from 10-1 to 10-8 on plates containing agar medium and marked

as 10-1 to 10-8 respectively. The sample was spread uniformly over the surface of the agar

using a sterilized hockey stick shaped glass rod (L-Shaped). The glass rod was sterilized by

being dipping in alcohol and ignited to burn off the alcohol. After spreading, the Petridishes

were incubated for 24 hours at 300C. When suspension was spread over the plate isolated

colonies are developed.



39

The no. of individual colonies was counted under colony counter. Total bacterial count was

estimated by using the following formula.

Total bacterial count:

No. of colonies observed 1_____________________ X ______________ml. of sample (colonies) plated dilution factor

A. Route of bacterial administration:

Aeromonas liquefaciens bacterial suspension was injected to the fish, Labeo robita

intramuscularly near the anal region.

B. Pathogen dose:

Various doses like 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 were injected to the fish

to induce aeromoniasis and observed 10-4 dose as LD50. So 10-5 and 10-6 were selected for

experimentation as optimum doses.

G. Immunostimulants:

i) lmmunex-DS: Manufactured from PVS laboratories Ltd., Vijayawada, Krishna District,

A.P., India was used in the present study. Immunex-DS is a special formulation to induce an

immune response and to protect fish and shell fish against pathogens.

Immunex-DS contains â carotins L-lysine, DL Methionine, Fatty acids, Livamisol

hydrochloride, Vitamins A, D3, E, C, B12, Minerals Zinc, Cobalt, Manganese, Selenium and

probiotics Lactobacillus, Saccharomyces cervisiae. Test dose of immunostimulant was

selected as per the recommended dosage given by PVS lab, ie. 5 gms per kg of pellets.



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ii) H-Treat: Manufactured from PVS laboratories Ltd., Vijayawada, Krishna District, A.P.,

India was used as a herbal treatment in the present study. H-Treat provides additional

strength and immunity by stimulating macrophages, receptors, bacteriods, phagocytosis etc.

It is a herbal mixture of Andrographis paniculate (Arjuna), Withamnia somnifera

(Aswagantha), Vitis vinifera (Grape seed), Terminalia chebula (Harada) and Azadirachta

indica (Neem). These herbal plants are well known for hepatoprotective, antistress,

antioxidant, astringent, expectorant etc.

H. Antibiotics: Ciprofloxacin was selected for the treatment of aeromoniasis. The dosage

was selected as per the recommended dose i.e. 100 gm per 1000 kg fish.

I. Biochemical parameters:

1. Estimation of Total protein:

Total protein content was estimated by the modified method of Lowry et.al (1951).

5% homogenates of gill, intestine, brain and thymus, 2% homogenates of liver and kidney

were prepared in 5% trichloro acetic acid and centrifuged at 3000 rpm for 10 minutes. The

supernatant was discarded. The suspended protein residue was dissolved in I ml of 1N

NaOH. From this 0.2 ml of the extract was taken into the test tube and 5 ml of alkaline

copper solution (50ml of 2% Na2CO3 and 1 ml of 0.5% CuS04 5H20 in 1% sodium

potassium tartrate) was added. The contents were mixed well and allowed to stand for 10

minutes. To this 0.5 ml of 50% Folein phenol reagent diluted with distilled water in 1:1

ratio was added. After 10 minutes, the optical density was measured at 540 nm in a

spectrophotometer against a blank. The standard graph was plotted by the method of Lowry



41

et.al (1951) with bovine serum albumin supplied by Sigma chemical company, USA. The

values were expressed as mg/ml weight of the tissue.

2. Estimation of total carbohydrate:

Total carbohydrate content was estimated by the method of Nicholas et.al (1956).

The homogenates of gill, intestine, brain and thymus were prepared separately in 5 ml of

10% TCA for extraction of carbohydrates. The homogenates were centrifuged at 3000 rpm

for 15 minutes. One ml of clear supernatant, 5 ml of anthrone reagent were added and

thoroughly shaken. Simultaneously 1 ml of TCA was taken for blank preparation and to it 5

ml of anthrone were added. After constant mixing, the tubes were kept in a boiling water

bath for 15 minutes and then cooled to room temperature. The color developed is read at

620nm in a spectrophotometer against a blank. The total carbohydrate value was read from

a standard graph prepared earlier using known glucose standard. The values are expressed

as mg/ml.

3. Estimation of DNA (Diphenyl amine method):

DNA was estimated following the method of Burton (1956).

Preparation of Reagents:

Diphenyl amine reagent: 5 gm of Diphenylamine was dissolved in 500 ml. of glacial acetic

acid. To this 13.75 ml of conc. H2SO4 was added.

DNA standard: 100 mg of DNA in 100 ml of distilled water.

Procedure: 0.1 to 1 ml of standard solutions were taken in different test tubes and made to 1

ml with distilled water. A blank is prepared with 1 ml of distilled water. 0.2 ml test solution

was taken in three test tubes and made to 1 ml with distilled water. 4 ml of freshly prepared



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DPA (Diphenyl amine) solution were added to all the test tubes, mixed well and heated in

water bath for 10 minutes and allowed to cool to develop blue colour. O.D values noted

against blank. The O.D was measured at 595 nm in a spectrophotometer. A standard curve

was plotted by taking the concentration of DNA on X-axis and O.D on Y-axis.

4. Estimation of RNA (orcinol method):

Aim: To estimate the amount of RNA present in the given sample by using orcinol reagent.

Principle: This is a general reaction for pentoses. Acid hydrolysis of RNA releases the

ribose sugar and this in the presence of acid dehydrates to furfural. Orcinol reacts with

furfural in presence of FeC13 as a catalyst gives a green colored complex. To this reaction

purine nucleotides are more reactive than pyramidine nucleotides.

Reagents:

1. Standard RNA Solution (200 mg/ml): 20 mg of yeast RNA was dissolved in 100 ml of

10% TCA and heat the solution at 950C for 15 minutes. The solution is cooled and used as

standard.

2. Orcinol reagent: Dissolve 500 mg of FeCl3, 6H20 in 500 ml of concentrated HCI and to

this add 17.5 ml of 6% orcinol in ethanol.

Procedure: Different volumes of (0.4, 0.8, 1.2, 1.6, 2.0) RNA solution was pipetted out in to

test tubes and 3 ml. of orcinol reagent was added. The contents of the tubes were mixed well

and incubated in boiling water bath for 15 minutes. The O.D was measured at 655 nm in a

spectrophotometer against the blank. A standard curve was plotted by taking the

concentration of RNA on X-axis and O.D on Y-axis.



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J. Blood Parameters:

Blood collection: Blood was collected by caudal cut method and serum was sepatated

following standard procedures.

1. Determination of serum protein by Biuret method:

Principle: The peptide bonds of the protein react with cupric (Cu+2) under alkaline condition

to yield a purple/violet colored complex, which shows an absorption maximum at 540 nm.

Protein reference standard: Casein is used as a reference standard protein (10 mg/ml). 1 gm

of casein was weighed accurately and transferred to a clean and dry 100 ml volumetric

flask. The protein was suspended in 50 ml of distilled water and dissolved by adding few

drops of sodium hydroxide solution and the volume was made to 100 ml.

Reagents:

Biuret reagent: 1.5 gm of cupric sulphate was dissolved in 250 ml of distilled water. 6 gm of

sodium potassium tartrate separately weighed and dissolved in 250 ml of distilled water.

The cupric sulphate and tartrate solutions were mixed in a 1L beaker. To this solution 300

ml of 10% (W/V) sodium hydroxide solution was added with constant stirring and made up

the volume to 1L with distilled water using a volumetric flask and stored in a plastic

container.

Procedure: To one ml of the standard protein solution (containing 1-10 mg of casein) or

appropriately diluted or undiluted unknown protein sample solution, 5ml of biuret reagent

were added and mixed the contents.



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After 30 minutes of incubation at room temperature, the violet color developed was

measured against the reagent blank at 540 nm in spectrophotometer and the absorbance was

recorded. A calibration curve was constructed on a graph paper by plotting the protein

concentration (1-10 mg of protein) on x-axis and absorbance at 540 nm on y-axis and

computed the concentration of the protein in the sample from the calibration curve. While

calculating the protein concentration in the unknown sample, the dilution factor was taken

into account.

2. Assay of total Immunoglobulin M (IgM):

Fish immunoglobulin M (IgM) ELISA KIT (catalog no. CSB-E 12045Fh) was used for the

assay of IgM.

Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with

goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter

plate wells with a Horseradish Peroxidase (HRP)-conjugated IgM and antibody preparation

specific for IgM, and incubated. Then substrate solutions are added to each well. The

enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the

color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The

concentration of IgM in the samples is then determined by comparing the O.D of the

samples to the standard curve.

Detection range: 0.2 µg/ml-50 µg/ml. The standard curve concentrations used for the

ELISA’s were 50 µg/ml, 12.5 µg/ml, 3.12 µg/ml, 0.78 µg/ml, 0.2 µg/ml.

Specificity: This assay recognizes fish IgM. No significant cross-reactivity or interference

was observed.



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Sensitivity: The minimum detectable dose of fish IgM is typically less than 0.125 µg/ml.

The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest

protein concentration that could be differentiated from zero.

Materials provided:

Reagent Quantity

Assay plate 1

Standard 5 x 0.5 ml

HRP-Conjugate 1 x 6ml

Antibody 1 x 6 ml

Wash Buffer 1 x 15 ml

(20×concentrate)

Substrate A 1 x 7 ml

Substrate B 1 x 7 ml

Stop Solution 1 x 7 ml

Standard S1 S2 S3 S4 S5

Concentration

(µg/ml)

0.2 0.78 3.12 12.5 50

Storage:

1. Unopened test kits were stored at 2-8°C upon receipt and the microtiter plate was kept in

a sealed bag. The test kit was used throughout the expiration date of the kit, and stored as

prescribed above.



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2. Opened test plate was stored at 2-8°C in the aluminum foil bag with desiccants to

minimize exposure to damp air. The kits remained remain stable until the expiring date

shown.

3. A microtiter plate reader with a band width of 10 nm or less and an optical density range

of 0-3 OD or greater at 450 nm wavelength is acceptable for use in absorbance

measurement.

Reagent preparation:

1. All reagents were brought to room temperature before use.

2. Wash Buffer - If crystals have formed in the concentrate, warmed up to room temperature

and mixed gently until the crystals have completely dissolved. 15 ml of Wash Buffer

Concentrate was diluted with or distilled water to prepare 300 ml of Wash Buffer.

Other materials used:

- Microplate reader capable of measuring absorbance at 450 nm, with the correction

wavelength set at 540 nm or 570 nm.

- Pipettes and pipette tips.

- Distilled water.

- Squirt bottle, automated microplate washer.

- An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

Sample collection and storage:

Serum: A serum separator tube (SST) was used and allowed the samples to clot for 30

minutes before centrifugation for 15 minutes at 1000 x g. Removed serum and assayed



47

immediately or aliquot and store samples at -20°C. Centrifuged the sample again after

thawing before the assay. Repeated freeze-thaw cycles were avoided.

Assay procedure: All reagents and samples were brought to room temperature before use.

All samples, standards, and controls were assayed in duplicate. All the reagents were added

directly to the liquid level in the well. Care was taken not to contact the inner wall of the

well with pipette.

1. A Blank was set without any solution. Added 50 µl of Standard or Sample per well.

2. Added 50 µl of HRP-Conjugate and 50 µl of Antibody to each well except Blank well.

3. Covered with the adhesive strip. Incubated for 1 hour at 37° C.

4. Aspirated each well and washed, repeating the process three times for a total of three

washes. Washed by filling each well with Wash Buffer (200 µl) using a squirt bottle, multi-

channel pipette, manifold dispenser or autowasher. Care was taken to the complete removal

of liquid at each step. After the last wash, removed any remaining Wash Buffer by

aspirating or decanting. Inverted the plate and blot it against clean paper towels.

5. Added 50 µl of Substrate A and 50 µl of Substrate B to each well. Incubated for 15

minutes at 37°C by keeping the plate away from drafts and other temperature fluctuations in

the dark.

6. Added 50 µl of Stop Solution to each well when the first four wells containing the highest

concentration of standards developed obvious blue color. If color change does not appear

uniform, gently taped the plate to ensure thorough mixing.



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7. Determined the optical density of each well within 30 minutes, using a microplate reader

set to 450 nm.

Calculation results:

A standard curve is made using the professional soft "Curve Exert 1.3".

A standard curve was constructed by plotting the mean absorbance for each standard on the

y-axis against the concentration on the x-axis and a best fit curve was drawn through the

points on the graph. The data was linearized by plotting the log of the IgM concentrations

versus the log of the O.D and the best fit line was determined by regression analysis. This

procedure produced an adequate but less precise fit of the data.

K. Total protein isolation through SDS page:

Materials: Resolving Gel or Separating Gel: (PH 8.8)

Percentage of gel 15%

38: 0.8% w/v acrylamide:bisacrylamide 3.75ml

1.5M Tris-Cl pH 8.8 2.2ml

20% SDS 0.03 ml

dH2O 1.8 ml

Mix together. Add APS and TEMED just before pouring

10% APS 0.03ml

TEMED 7 ul



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STACKING GEL (NOTE PH 6.8)

Staining solution:

Coomassie Blue Stain R-250 2.5g

Methanol 1.0L

Acetic Acid 0.2L

Distilled water 0.8L

Destainer 1 :

48 ml of Methanol

2 ml of Glacial Acetic acid.

50 ml of Distilled water

Destainer 2 :

8 ml of Methanol

2 ml of Glacial Acetic acid.

Percentage of stack 4%

30:0.8% w/v acryl:bisacryl 1ml

1M Tris-Cl pH6.8 630 ul

20% SDS 25 ul

dH2O 3.6 ml

Mix together. Add APS and TEMED just before pouring. 25ul

10% APS 5ul

TEMED 5ml



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90 ml of Distilled water

SDS-Sample Loading Dye (5x):

2 mL 10% SDS

1.2 mL 0.5M Tris-HCl (pH 6.8)

4.8 mL 50% Glycerol

1 mL of 1% Bromophenol Blue

1mL H2O

0.5 mL â-mercaptoethanol

Protein Extraction Buffer:

Urea 8M.

10% SDS.

10 M Tris.

1 mM EDTA.

10x SDS Tank buffer:

1 liter

30 g Tris base

144 g Glycine

5 ml/liter 20% SDS

Make uipto 1 liter to dH2O

Methods:

Total protein extraction Protocol:

Ethanol freeze tissue are retrieved.

50mg of tissue is taken in 1.5ml tube.



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100µl of protein extraction buffer is added and grinded well using micro pestle.

Centrifuged at 10,000 rpm for 5 minutes.

Supernatant transferred into fresh of 1.5ml tube. This sample is used for SDS Analysis.

Preparing sample for SDS PAGE: 50 µl of Extracted proteins sample are mixed with equal

volume of 5X sample loading buffer and heated at 95ºC for 5 minutes.

Cool to room temperature.

20 µl is loaded into 12% SDS PAGE with Lifetech Standard protein Molecular Marker.

Protocol for SDS PAGE:

Procedure:

1. Glass plates assembled the plates without leak proof.

2. Approximately 8 ml of separating gel mix was taken and mixed APS.

3. Poured gel solution immediately in-between glass plates till three fourth volumes of glass

plates. Added 200 to 250 µl of water to make the surface even.

4. Allowed to solidify. It was taken 20-30 minutes. Solidification can be observed as a line

between gel and water. Discarded top water carefully.

5. Taken 3.5 ml of stacking gel and mix with APS (40 µl of APS in case of 4 ml of Stacking

gel, 30 µl of APS for 3 ml of stacking gel) and poured directly onto separating gel. Inserted

gel comb immediately without trapping any bubbles. Comb must be inserted maximum

half-length of staking gel. Allowed stacking gel to solidify.

6. Filled 1X tank buffer in lower tank of SDS PAGE apparatus.



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7. Once stacking gel solidified, carefully removed lower spacer and inserted gel plate into

lower tank. Any bubbles caught between the plates at the bottom of the gel removed by

squirting running buffer through a syringe fitted with a bent needle.

8. Filled running buffer to top reservoir and carefully removed comb.

9. Loaded 10ul of each protein samples along with 10 µl of Molecular weight marker (ready

to use) into wells.

10. When the dye front came to 0.25 cm above the bottom of the gel, turn off the power

pack. Removed the gel plates and gently placed the plates apart. Used a spatula or similar

tool to separate the plates. Cut a corner from the bottom of the gel that is closest to the

number 1 well.

12. Placed a gel in a plastic or glass container with 100ml of Distilled water. Incubated the

gel for 10 minutes. Repeated this step thrice.

13. Discarded water and covered the gel with 30ml of Stain and kept it for 1 hour and the

tray was drained completely and rinsed the gel with distilled water.

14. Poured the destainer 1 solution and agitated for 20 mins and discarded the solution.

Transferred the gel into destainer 2 and destained it completely.

15. Gel photographed at this stage and analyzed.

In M.W.Marker: 7 bands of 97KDa, 66KDa, 45KDa, 35KDa, 25KDa, 20KDa and

14.4KDa.



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L. Histopathology:

Histopathological observations were made from the gill, intestine and thymus of

both experimental and control fish on day 5 of necropsy, pieces of intestine and thymus

were separated, fixed and sectioned at 5µ and stained by H and E method for

histopathological studies.

Statistical Applications:

The observations of different experimets were analysed through student ‘t’ test to

find out the statistical significance of the observed differences in the biochemical

parameters like protein, carbobydrate, RNA, DNA and blood parameters like serum protein

and IgM lvels of different experimetal and control groups of L. rohita (Daniel, 1974).

Student ‘t’ test :

The criterion to caleculate two different samples can be obtained by using the following

formula.

t =

t =

Where

= Mean of the first sample

= Mean of the second sample

S = Combined standard diviation



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= Number of observations of first sample.

= Number of observations of second sample.

S (Combined standard diviation) can be obtained by using the formula.

X = Obtained individual value of first sample.

= Mean of observed values of first sample.

= The value obtained by subtracting each observed value from mean of first sample.

= The sum of the squares of difference between each individual value and mean

of first sample.

n1= Number of observations of first sample.

Y = Obtained individual value of second sample.

= Mean of observed values of second sample.

= The value obtained by subtracting each observed value from mean of second

sample.

= The sum of the squares of difference between each individual value and mean

of second sample.

n2= Number of observations of second sample.



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EXPERIMENTAL DESIGN

Test fish: In the present investigations, Labeo rohita (the Indian Major Carp) was selected

to study the effect of immunostimulants and antibiotics and its immunity to A.

liquefaciens. A series of experiments were conducted to estimate the level of total protein,

carbohydrate, DNA, RNA in tissues of gill, intestine, brain & thymus, serum protein and

antibody like IgM and histopathology of gill, intestine and thymus from the experimental

and control fish and SDS-PAGE analysis of intestinal protein.

Pilot Experiment: The purpose of this experiment was to determine the LD 50 value of L.

rohita experimentally infected with A. liquefaciens. 8 cement tanks 3 ½ feet in width and

4 feet in depth were used. Ninety six L. rohita weighing about 50-60 gm, twelve in each

one placed in 8 cement tanks. After a 4 day acclimation period, fish in 8 cement tanks

were infected intramuscularly with 10-1 CFU/fish, 10-2 CFU/fish, 10-3 CFU/fish,

10-4 CFU/fish, 10-5 CFU/fish, 10-6 CFU/fish, 10-7 CFU/fish and 10-8 CFU/fish. The

infection schedule is shown below:

Determination of LD 50 of A. liquefaciens in L. rohitaDose of bacteria

administered CFU/fish

No.

challengedTotal Mortality Mortality (%)

LD50

CFU/fish

10-8 (group I) 12 2 16.66

10-7 (group II) 12 2 16.66

10-6 (group III) 12 3 25.00

10-5 (group IV) 12 4 33.33

10-4 (group V) 12 6 50.00 10-4

10-3 (group VI) 12 7 58.33

10-2 (group VII) 12 10 83.33

10-1 (group VIII) 12 11 91.66



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Mortalities were recorded on day 1 in the groups V, VI, VII and VIII injected with

10-4/CFU/fish, 10-3 CFU/fish, 10-2 CFU/fish, and 10-1 CFU/fish respectively. LD50 of the

pathogen was determined as 10-4 CFU/fish.

Phase I:

Experiment I:

The purpose of this experiment was to determine the effect of Immunex DS and A.

liquefaciens on L. rohita.

The treatment of Immunex DS and infection schedule adopted is as follows:

GroupsTreatment with

Immunex DSInfection Days of Necropsy

A 40 mg/100 gm of feedfor 4 days --- On day 1,2,3,4 and 5

B --- 10-5 CFU/fish on day 1 On day 1,2,3,4 and 5

C --- --- On day 1,2,3,4 and 5

Three cement tanks 3 ½ feet in width and 4 feet in depth were used. Twenty

L. rohita (group A) weighing approximately 50-60 gm were placed in tank 1 and another

group of twenty fish (group B) of same weight were placed in tank 2.

After a 4 day acclimation period, fish in tank 1 were treated with Immunex DS (40

mg/100 gm of feed) for 4 days. Fish in tank 2 were infected with a bacterial suspension of

10-5 CFU/fish to induce aeromoniasis. Fish in tank 3 were kept as uninfected controls

(group C) for comparison.

Three fish from experimental and control groups were necropsied on day 1, 2, 3, 4

and 5 and studied for biochemical assays, blood parameters, SDS-PAGE analysis of

intestinal protein and histopathological observations.



57

Experiment II:

The purpose of this experiment was to determine the effect of Immunex DS and A.

liquefaciens (10-6 CFU/fish) on L. rohita.

The treatment of Immunex DS and infection schedule adopted is as follows:


Immunex DSInfection Days of Necropsy

D 40 mg/100 gm of feedfor 4 days --- On day 1,2,.3,4 and 5

E --- 10-6 CFU/fish on day 1 On day 1,2,.3,4 and 5

C --- --- On day 1,2,.3,4 and 5

Three cement tanks 3 ½ feet in width and 4 feet in depth were used. Twenty L. rohita

(group D) weighing approximately 50-60 gm were placed in tank 1 and another group of

twenty fish (group E) of same weight were placed in tank 2. After a four day acclimation

period, fish in tank 1 were treated with Immunex DS (40 mg/100 gm of feed) for 4 days.

Fish in tank 2 were infected with a bacterial suspension of 10-6 CFU/fish to induce

aeromoniasis. Fish in tank 3 were kept as untreated and uninfected controls (group F) for

comparison. Three fish from experimental and control groups were necropsied on day 1,

2, 3, 4 and 5 and studied for biochemical assays, blood parameters and histopathological

observations.

Experiment III:

The purpose of this experiment was to determine the effect of Immunex DS, A.

liquefaciens (10-5 CFU/fish) and ciprofloxacin (10 mg/100 gm of fish) on L. rohita.



58

The treatment of Immunex DS and antibiotic and infection schedule adopted is as

follows:

Groups Treatment withImmunex DS Infection Treatment with

antibioticDays of

necropsy

G 40 mg/100 gm of feedfor 4 days

On day 5 - 10-5

CFU/fish _ day 1,2,3,4 & 5after infection

H _ 10-5 CFU/fish onday 1

10 mg/100 gm offish for 2 days

Day 1,2,3,4 & 5after antibiotic

treatment

I 40 mg/100 gm of feedfor 4 days

10-5 CFU/fish onday 5



treatment

J40 mg/100 gm of feed

for 4 days (from day 3 today 6)




treatment

K _ _ _ On day 1,2,.3,4and 5

After a four day acclimation period, fish in tank 1 (group G) were treated with

Immunex DS (40 mg/100 gm of feed) for 4 days; on day 5 the fish were infected with

bacterial suspension of A. liquefaciens 10-5 (CFU/fish) to induce aeromoniasis. Fish in

tank 2 (group H) were infected with bacterial suspension (10-5 CFU/fish) to induce

aeromoniasis. On day 3 the fish were treated with antibiotic, ciprofloxacin (10 mg/100 gm

of fish) as per the recommended dose. Fish in tank 3 (group 1) were treated with Immunex

DS (40 mg/100 gm of feed) for four days. On day 5, the fish were infected with A.

liquefaciens (10-5 CFU/fish) to induce aeromoniasis. On day 7, fish were treated with

antibiotic, ciprofloxacin (10 mg/100 gm of fish) for two days. Fish in tank 4 (group J)

were infected with A. liquefaciens (10-5 CFU/fish). On day 3, the fish were treated with

Immunex DS (40 mg/100 gm feed) for four days. On day 7 fish were treated with



59

antibiotic ciprofloxacin as per the recommended dose. Fish in tank 5 (group K) were kept

as uninfected and untreated controls. Three fish from experimental and control groups

were necropsied on each day for 5 days and studied for biochemical assays, blood

parameters, SDS-PAGE analysis of intestinal protein and histopathology.

Experiment IV:

This experiment was performed to determine the effect of immunostimulant,

Immunex DS (40 mg/100 gm of feed), A. liquefaciens (10-6 CFU/fish) and antibiotic (10

mg/100 gm of fish) on L. rohita.

The treatment of Immunex DS and antibiotic and infection schedule adopted is asfollows:

Groups Treatment withImmunex DS Infection Treatment with

antibioticDays of

necropsy

L 40 mg/100 gm of feed for4 days

10-5 CFU/fish onday 5 _ day 1,2,3,4 & 5

after infection

M _ 10-5 CFU/fish onday 1

10 mg/100gm offish for 2 days


treatment

N 40 mg/100 gm of feed for4 days




treatment

O40 mg/100 gm of feed for4 days (from day 3 to day

6)


10mg/100gm offish for 2 days


treatment

P _ _ _ On day 1,2,.3,4and 5

Separate cement tanks 3 ½ feet in width and 4 feet in depth were used for each

group of fish. Twenty L. rohita in each group weighing approximately 50-60 gm were

placed separately in tanks 1, 2, 3, 4 and 5 and named the groups L, M, N, O & P



60

respectively. After a four day acclimation period, fish in tank 1 (group L) were treated

with Immunex DS (40 mg/100 gm of feed) for four days. On day 5, the IDS treated fish

were infected with bacterial suspension of A. liquefaciens (10-6 CFU/fish) to induce

aeromoniasis. Fish in tank 2 (group M) were infected with A. liquefaciens (10-6 CFU/fish)

to induce aeromoniasis. On day 3 the fish were treated with and antibiotic, ciprofloxacin

(10 mg/100 gm of fish). Fish in tank 3 (group N) were treated with Immunex DS (40

mg/100 gm of feed) for four days; and on day 5, they were infected with A. liquefaciens

(10-6 CFU/fish). On day 7, fish were treated with antibiotic (10 mg/100 gm of fish). Fish

in tank 4 (group O) were infected with A. liquefaciens (10-6 CFU/fish) to induce

aeromoniasis. On day 3, fish were treated with Immunex DS (40 mg/100 gm of feed) for

four days and on day 7, fish were treated with antibiotic (10 mg/100 gm of fish). Fish in

group P were kept as uninfected and untreated controls for comparison. Three fish from

experimental and control groups were necropsied on each day for 5 days and studied for

biochemical assays, blood parameters and histopathology.

Phase II:

Experiment I:

The purpose of this experiment was to determine the effect of H-Treat and A. liquefaciens

on L. rohita.

The treatment of H - Treat and infection schedule adopted is as follows:

Groups Treatment with H-Treat Infection Days of Necropsy

a 40 mg/100 gm of feed for 4 days --- On day 1,2,.3,4 and 5

b --- 10-5 CFU/fish on day 1 On day 1,2,.3,4 and 5

c _ _ On day 1,2,.3,4 and 5



61

Three cement tanks 3 ½feet in width and 4 feet in depth were used. Twenty L.

rohita (group a) weighing approximately 50-60 gm were placed in tank 1 and another

group of twenty fish (group b) of same weight were placed in tank 2. After a 4 day

acclimation period, fish in tank 1 were treated with H-Treat (40 mg/100 gm of feed) for 4

days. Fish in tank 2 were infected with a bacterial suspension of 10-5 CFU/fish to induce

aeromoniasis. Fish in tank 3 were kept as non infected controls (group c) for comparison.

Three fish from experimental and control groups were necropsied on day 1, 2, 3, 4

and 5 and studied for biochemical assays, blood parameters, SDS-PAGE analysis of

intestinal protein and histopathological observations.

Experiment II:

The purpose of this experiment was to determine the effect of H-Treat and A. liquefaciens

(10-6 CFU/fish) on L. rohita.

The treatment of H-Treat and infection schedule adopted is as follows:

Groups Treatment with H-Treat Infection Days of Necropsy

d 40 mg/100 gm of feed for 4 days --- On day 1,2,.3,4 and 5

e --- 10-6 CFU/fish on day 1 On day 1,2,.3,4 and 5

f _ _ On day 1,2,.3,4 and 5

Three cement tanks 3 ½ feet in width and 4 feet in depth were used. Twenty L. rohita

(group d) weighing approximately 50-60 gm were placed in tank 1 and another group of

twenty fish (group e) of same weight were placed in tank 2. After a four day acclimation

period, fish in tank 1 were treated with H-Treat (40 mg/100 gm of feed). Fish in tank 2

were infected with a bacterial suspension of 10-6 CFU/fish to induce aeromoniasis. Fish in

tank 3 were kept as untreated and uninfected controls (group f) for comparison. Three fish



62

from experimental and control groups were necropsied on day 1, 2, 3, 4 and 5 and studied

for biochemical assays, blood parameters and histopathological observations.

Experiment III:

The purpose of this experiment was to determine the effect of H-Treat, A.

liquefaciens (10-5 CFU/fish) and ciprofloxacin (10 mg/100 gm of fish) on L. rohita.

The treatment of H-Treat and antibiotic and infection schedule adopted is as follows:


H-TreatInfection Treatment with

antibioticDays of

necropsy

g 40 mg/100 gm of feed for 4days

10-5 CFU/fishOn day 5 _ day 1,2,3,4 & 5

after infection

h _ 10-5 CFU/fishon day 1



treatment

i 40 mg/100 gm of feed for 4days

10-5 CFU/fishon day 5



treatment

j 40 mg/100 gm of feed for 4days (from day 3 to day 6)




treatment

k _ _ _On day 1,2,.3,4and 5

After a four day acclimation period, fish in tank 1 (group g) were treated with H-

Treat (40 mg/100 gm of feed) for 4 days. On day 5, the fish were infected with bacterial

suspension of A. liquefaciens 10-5 (CFU/fish) to induce aeromoniasis. Fish in tank 2

(group h) were infected with bacterial suspension (10-5 CFU/fish) to induce aeromoniasis.

On day 3 the fish were treated with antibiotic ciprofloxacin (10 mg/100 gm of fish) as per

the recommended dose. Fish in tank 3 (group i) were treated with H-Treat (40 mg/100 gm

of feed) for four days. On day 5, the fish were infected with A. liquefaciens (10-5

CFU/fish) to induce aeromoniasis. On day 7, fish were treated with antibiotic



63

ciprofloxacin (10 mg/100 gm of fish) for two days. Fish in tank 4 (group j) were infected

with A. liquefaciens (10-5 CFU/fish). On day 3, the fish were treated with H-Treat (40

mg/100 gm of feed) for four days; on day 7 fish were treated with antibiotic, ciprofloxacin

as per the recommended dose. Fish in tank 5 (group k) were kept as uninfected and

untreated controls. Three fish from experimental and control groups were necropsied on

each day for 5 days and studied for biochemical assays, blood parameters, SDS-PAGE

analysis of intestinal protein and histopathology.

Experiment IV:

This experiment was performed to determine the effect of immunostimulant, H-Treat

(40 mg/100 gm of feed), A. liquefaciens (10-6 CFU/fish) and ciprofloxacin (10mg/100gms

of fish) L. rohita.

The treatment of H-Treat and antibiotic and infection schedule adopted is as follows:

Groups Treatment withH-Treat Infection Treatment with

antibioticDays of

necropsy

l 40 mg/100gm of feed for 4days

10-6 CFU/fishOn day 5 _ day 1,2,3,4 & 5

after infection

m _ 10-6 CFU/fishon day 1



treatment

n 40 mg/100gms of feed for 4days

10-6CFU/fishon day 5



treatment

o 40 mg/100gms of feed for 4days (from day 3 to day 6)




treatment

p _ _ _ On day 1,2,.3,4and 5

Separate cement tanks 3 ½ feet in width and 4 feet in depth were used for each group of

fish. Twenty L. rohita weighing approximately 50-60 gm were placed separately in tanks



64

1, 2, 3, 4 and 5 and named the groups as l, m, n, o & p respectively. After a four day

acclimation period, fish in tank 1 (group l) were treated with H-Treat (40 mg/100 gm of

feed) for four days and on day 5, the fish were infected with bacterial suspension of A.

liquefaciens (10-6 CFU/fish) to induce aeromoniasis. Fish in tank 2 (group m) were

infected with A. liquefaciens (10-6 CFU/fish) to induce aeromoniasis. On day 3, the fish

were treated with antibiotic, ciprofloxacin (10 mg/100 gm of fish). Fish in tank 3 (group

n) were treated with H-Treat (40 mg/100 gm of feed) for four days and on day 5, the fish

were infected with A. liquefaciens (10-6 CFU/fish) to induce aeromoniasis. On day 7, fish

were treated with antibiotic (10mg/100gm of fish). Fish in tank 4 (group o) were infected

with A. liquefaciens (10-6 CFU/fish) to induce aeromoniasis. On day 3, fish were treated

with H-Treat (40 mg/100 gm of feed) for four days and on day 7; fish were treated with

antibiotic (10 mg/100 gm of fish). Fish in tank 5 (group p) were kept as uninfected and

untreated controls for comparison. Three fish from experimental and control groups were

necropsied on each day for 5 days and studied for biochemical assays, blood parameters

and histopathology.



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MATERIALS AND METHODSshodhganga.inflibnet.ac.in/bitstream/10603/10437/8/08_chapter 3.pdf · 35 MATERIALS AND METHODS A. Source of Fish: Experiments were performed on the common fresh