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80

MATERIAL AND METHODS:

Site of collection: Chhawni slaughter house,

Cantonment area,

Aurangabad (M.S.), India.

Hosts:

1) Goat (Capra hircus.Linnaeus,1758).

2) Sheep (Ovis bharal.Linnaeus,1758).

The areas from where the goats and sheep are being supplied to the slaughter

house, the locations are given below. These places also can be seen to an

advantage in the accompanying map.

I. Tisgaon.

II. Padegaon.

III. Vaijapur

81

Map of Aurangabad

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To collect the nematode parasites of goat and sheep the author used to daily

visit to the slaughter house in the morning where about 50- 60 goats and sheep are

slaughtered in everyday. Viscera of sheep and goats were obtained for examination

after external observation was made for nodules bleedings etc. these viscera were

exposed and parasites collected. Sometime the squeezing of intestine with little

pressure helps in passage of parasites along with faecal matter. The collection of

roundworm was done within hour of slaughtering of the host and pulled them in

Tyroide’s solution (Nacl- 8.00 gm ; potassium chloride- 0.20 gm; magnesium

chloride – 0.20 gm; sodium dihydrogen phosphate- 0.60 gm; calcium chloride-

0.30 gm; sodium bicarbonate- 1.00 gm and glass distilled water- 1000cc) in flask

and were brought to the lab for analytical work. The attempts to collect these

parasites at different concentration of Tyroide’s at various temperature revealed

that at 1.5 dilution and 370c temperature the activity of the parasite was at optimum

condition.

82

If the parasite in the same faecal matter with little water yielded better result

as the parasite were found to be in motile and active condition for longer time.

Nematode parasites in this condition were used for ionic studies. After bringing the

material to the lab the parasites were isolated and washed them.

I) ESTIMATION OF GLYCOGEN:

Glycogen was estimated by the method of Kemp and Kito Van Heijingen, (1954).

Kemp Method:

The nematode parasites were washed well with Tyrode’s solution and

weighed, they were separated into different species sexwise and were grind in 5ml

of 80% (v/w) methanol and the suspension was centrifuged the supernatant

containing glucose was decanted. To the precipitate 5 ml of depeproteinising

solution ( 5% TCA containing 100 mg of silver sulphate) was added and glycogen

was extracted heating in a boiling water bath for 15 minutes. The volume was made

upto 5 ml to compensate the evaporation during heating and it is centrifuged for 15

minutes at 3000 rpm.

1 ml of the clear supernatant was added in a wide test tube to 3ml of

concentrated sulphuric acid, mixing with vigorous shaking. The mixture was kept

in a boiling water bath for 4- 6 minutes and subsequently cooled under running

water the intensity of pink colour formed was read at 520 Mµ in spectrophotometer

( Carlzins Jena Spectrophotometer) using silica cuvetts of 1 cm light path against a

blank. The glycogen content was expressed as mg/gm wet weight of the tissue-

standard graph was prepared with analytical grade pure glucose and assays were

compared. The methanol supernatant containing glucose was taken into a

centrifuge tubes and 10mg of powdered charcoal was added in order to absorb the

organic substance which interferes in the colour development but not hexoses. The

methanol was removed completely under reduced pressure by keeping in the tube

in warm water. Then deproteinising solution was added and its volume was made

to 5 ml. After through mixing was centrifuged. 1 ml of supernatant was analyzed

for glucose in GOD period method (Werner W.H.G. and H. Wielinger, 1970 ).

83

GLUCOSE ESTIMATION BY GOD PERIOD METHOD:

Calorimetric Method:

Werner W. H.G.Ray and H.wielinger, (1970).

(Boehringer Mannheim GMbH kit was used for estimation)

Test Principle:

Glucose + O2 + H2O GOD

Gluconate + H2O2

H2O2 + ABTS POD

Colour complex + H2O

The sample solutions were prepared by deprotenizing 0.10ml of tissue

homogenate in 1 ml of uranyl acetate solution ,thoroughly mixed and centrifuged.

Out of which 0.10 ml of test solution was used for estimation of glucose.

Reagents:

1.Standrad glucose-9.1mg/100ml(0.505mmol/l)

2.Buffer/Enzyme/Chromogene

Phosphate Buffer-100mmol/l,7.0 pH

POD—0.8U/ml. Diammonium 2,2,azinotris(3-ethylbenzothiazoline 6-

sulphonate)

GOD—10U/ml

ABTS—1.0mg/ml

Test: pipette into the test tubes.

84

Observation Table

Mixed,incubated at 250c for 30 minutes and the optical density was measured at

578 nm with photometer with 1 cm.light path cuvette having against a

blank,module photometer 120/Boehringer Mannheim GMbH.

Calculation:

C = Concentration of glucose.

C = 100 X A sample

(Mg/100 ml)

A stand

Blank. Stand. Sample.

Distlled water

Solution -1

Deprotenized supernatant

Solution-2

0.2ml

-----

-----

5.0ml

------

0.2ml

-----

5.0ml

------

----

0.2ml

5.0ml

85

DETERMINATION OF PYRUVIC ACID (CH3COCOOH):

The determination of pyruvic acid was done by friademann and Hangen(1943).

The nematode parasites were collected, separated sexwise,washed and

weighed.The weighed parasites were homogenized in distilled water with a mortar

and pestel and made into a known quantity.2ml of the homogenate was ejected

rapidly through a needle into 10 ml of 10% trichloroacetic acid solution in a

centrifuge tube,stoppered and mix well and centrifuged.3ml of the clear cold

supernatant was pipetted out into a wide test tube and contents were warmed up to

25oc to that 1ml of dinitrophenylehydrozone was added and allowed to react at

room temperature for 5 minutes.The same procedure was carried out with dilute

pyruvate standard solution preparing with dilute trichliro acetic acid and mixing

with 2,4- dinitrophenylehydrozone .To each tube 3ml of tolune was added and

stream of air was passed through a narrow tube into the mixture for 2 minutes.

After the mixture settled, the lower layer was removed with a fine tipped

droper and discarded. To the remaining solution 6 ml of 10 % sodium carbonate

was added and mixed by passing bubbling air for 2 minutes. The mixture was

allowed to settle .5ml of the aqueous layer (lower) was placed in a small tube and 5

ml of 1.5N sodium hydroxide solution was added and mixed .It was allowed to

stand for 10 minutes. The colure developed was read at 520mM(Carlzein’s Jena

Spectrophotometer) keeping the mixture of 5 ml of 10% sodium carbonate and 5

ml of 1.5 N sodium hydroxide solution as blank.

Calculation: Density of unknown � 1

Density of standard

= Mg of pyruvic acid per 100 ml of the homogenate.

The pyruvate concentration was expressed in Mg of pyruvate per gram wet weight

of the tissue of parasite.

86

DETERMINATION OF LACTIC ACID:

Calorimetric determination of lactic acid by Barker and Summerson (1941).

Principle:

In the tissue homogenate glucose and other interfering material of protein

free filtrate were removed by the Van-Slyke-Salkowski method of treatment with

copper sulphate and Calcium hydroxide .An Aliquot of the resulting solution was

heated with concentrated sulphuric acid to convert lactic acid to acetaldehyde

which in turn reacts with p-hydroxy-diphenyl in presence of copper ion.

Procedure : -

The nematode parasites were collected, washed in Tyrode’s solution,

weighed and homogenized in distilled water. The homogenized was deprotinized

with trichloroacetic acid at 1:10 dilution. Three gradated centrifuged tubes were

taken and labeled test, standard and blanks. In the test centrifuged tube 2ml of

protein filtrate is taken. In the standard tube 5ml of standard lactic acid solution

was taken containing 0.01 mg. of lactic acid per 1 ml .In the third tube water taken

which acts as blank as well as control. To each tube 1ml of 20% copper sulphate

solution was added and diluted to the marked up to 10 ml with water. To that 1gm

of powdered calcium hydroxide was added to each tube Stopperd and shaken

vigorously until the solids are uniformly dispersed.

The tubes were allowed to stand for half an hour with a repeated shaking

then the tubes were centrifuged.1ml clear supernatant was transferred into a clean

dry test tube having wide internal diameter. To each tube 0.05ml of 4% copper

sulphate was added and 6ml of concentrated sulphuric acid was added. While

adding sulphuric acid, care was taken to run the acid drop wise initially and the

contents were thoroughly mixed, then the remaining acid was added. The tubes

were cooled under running water and 0.1ml of P-hydroxyl diphenyl reagent was

added dropwise. While adding reagent precipitated on coming in contact with acid,

so it was dispersed through out the solution quickly and uniformly by lateral

shakings. Then the tubes were placed in water at 300c allowed to stand for 30

minutes. The precipitated reagent was redispersed and the tubes were placed in

vigorously boiling water for 90 seconds. They were removed and cooled to room

87

temperature. The colors developed was read at 560nM (Carlzin’s Jena) photometer

using water as blank setting apparatus at zero density.

Calculations:

: Density of unknown � 0.005

����50

����100

Density of standard

= Mg of lactic acid 100ml of the homogenate.

So the amount of lactic acid was expressed in mg. of lactic acid per 1gram

wet weight of the parasites.

LACTATE DEHYDROGENASE:

Lactate dehydrogenase (LDH) L-Lactate NAD Oxidoreductase

(EC1.1.1.27) is a glycolytic oxidizing enzyme. This enzyme acts where the

pyruvate is reduced to lactate or the lactate is oxidized to pyruvate.

Pyruvate + NADH + H LDH

L-Lactate +NAD

The lactate dehydrogenase activity was estimated by the modified

calorimetric method of Nachlas et. al. (1960) using Lithium Lactate as substrate.

Reagents:

1) 0.25M sucrose solution.

2) 0.2M Disodium phosphate solution.

3) 0.2M Monopotassium phosphate solution.

4) Phosphate buffer at 7.4 pH by mixing above 2nd

and 3rd

solution and

pH was adjusted.

5) 0.2 mole solution of INT.

6) 0.2 Lithium lactate solution.

7) 0.002 M NAD solution.

88

Procedure:

The nematode parasites were collected from the sheep and goats and

washed with Tyrode’s solution-blotted and weighed. They were homogenized into

(10W/V) with 0.25 Mcold sucrose solution. The homogenate was centrifuged at

2500 RPM for 15 minutes. The supernatant of the homogenate was used for

estimation of enzyme activity. The reaction mixture in final volume contained

0.5ml of the homogenate supernatant 0.4ml of lithium lactate 0.5ml of phosphate

buffer 0.5ml of INT (2-P-idophenyl, 3-P-Nitrophenyl-5-phenyl tetrazolium

chloride) and 0.1ml of NAD solution.

The incubation was carried out for 30 minutes at 370c .After incubation the

reaction was stopped by the addition of 5ml of glacial acetic acid. The formazone

formed was its extracted over night in 5 ml. of toluene at 50c.The intensity of the

colour developed was read at 495mM.After setting the Carlzein’s Jena

spectrophotometer at zero with blank, the standard graph was prepared as per the

procedure and test results were compared. LDH activity was expressed in M moles

of formazone formed per mg of protein per hour.

SUCCINATE DEHYDROGENASE:

Succinate dehydrogenase (SDH) succinate accepter Oxidoreductase (EC

1.3.99.1) is mitochondrial enzyme responsible for dehydrogenation of succinate to

fumarate. It was estimated by the procedure of Nachlas et.al. (1960).

Principle:

SDH removes hydrogen atom from succinic acid in Kreb’s cycles and

transfers it to electron transport chain through FAD.Here tetrazolium chloride is

used as an electron acceptor from succinate and due to this the tetrazolium chloride

is converted into water insoluble coloured formazone(red).The intensity of colour

developed had proportional relation to that of enzyme activity.

89

Procedure:

The nematode parasites were collected from the intestine of goat and sheep

separated and washed thoroughly in Tyrode’s solution, wiped and weighed. The

weighed parasites were homogenized in 0.25M cold sucrose solution (10W/V) and

it was centrifuged at 2000rpm for 15 minutes and supernatant was used for the

estimation of enzyme activity which was kept in cold condition. The reaction

mixture in final volume of 2.0ml contains 75M moles of sodium succinate 100M

moles of potassium phosphate buffer (pH 7.0)4M moles of INT (2-P-indophenyle-

3-p Nitro phenyle-5-phenyl tetrazolium chloride) and 0.5 ml of the homogenate

supernatant. The incubation was carried out at 370c for one hour and the reaction

was stopped by adding 5ml of glacial acetic acid. The idoformazone formed was

extracted over night in 5ml of tolune at 50c.The optical density of the colour was

measured at 495nm in the U. V. spectrophotometer (Carlzein’s Jena) against blank.

The standard graph was prepared by taking different concentration of INT as per

the procedure and the test results were compared .The SDH activity was expressed

as M moles of formazone formed per mg protein per hour.

MALIC DEHYDROGENASE:

The malic dehydrogenase activity was estimated by Nachlas method

(1960).The roundworms were homogenised in sodium phosphate buffer (0.1M at

pH 7) and made (10W/V) homogenate at 50c.

Procedure:

The incubation mixture contain 1ml of 10 % homogenate ,1ml of sodium

phosphate buffer (0.1M at pH 7.0)0.5ml of sodium malate solution (0.1M)and

0.5ml of INT solution (0.5W/V).The mixture was incubated for 30 minutes at 370c

in water bath. At the end of incubation 6ml of glacial acetic acid was added to stop

the reaction. The formazone formed due to enzyme activity was extracted in 6ml of

tolune kept over night in refrigerator for complete extraction. The colour in the

tolune, the formazone level of the extract was measured at 495nM in Carlzein’s

Jena spectrophotometer with 10mm.width silica Cuvette. The MDH activity was

expressed in M moles of formazone per mg protein per hour.

90

PHOSPHOENOL PYRUVATE CARBOXY KINASE (EC4.1.1.32)(PEPCK):

Phosphoenol pyruvate carboxy kinase activity was estimated by modified

(Utter and Kurahashi, 1954) W. Korting and J. Barrett (1977) was followed with

slight modification.

Principle:

PEP + CO2 +ADP PEPCK OAA +ATP

OAA + NADH++

MDH Malate + NAD

In the present study, the reaction mixture instead of Immidazole buffer,

phosphate buffer was used. The phosphate ion acceptor which was IDP (inosin

diphosphate) as per Utter and Kurahashi (1954) and according to Castro et. al.

(1969), GDP (guanine diphosphate) and as per T. W. Moon et. al. (1977) relative

reactions is GDP >IDP > >ADP. The author had to restrict to ADP in the present

estimation because of non availability of other two chemicals.

Reagents:

Na phosphate buffer (pH adjusted to 7.4) 150 mol/ml.

NADH 6 M Mole/lit.

PEP 32 M Mol/Lit.

ADP 0.1M mol/Lit.

NaHCO3 10 M Mol/Lit.

(MDH) 5 M gr/ml.

Procedure:

91

The reactions was carried out by adding 1ml of phosphate buffer 0.2ml of

NaHCO3, 0.05ml of NADH, 0.05ml of tissue homogenate into a small test tube and

incubate for 5 minutes at 270c .Afterwards 0.04ml of MDH, 0.1ml of ADP was

added and mixed well. Immediately it was transferred into a silicon cuvette and

NADH extension was measured at 1minute intervals with time adjusted,

computerized (modulab) UV spectrophotometer at 366 nM. The enzyme activity

was calculated as per the procedure of PK given below and compared. In order to

have a comparative idea all the parameters while estimated PK and PEPCK were

kept constant.

PYRUVATE KINASE (EC.2.7.1.40):

Pyruvate kinase was estimated as per method of Beisenherz. G. et.al.

(1953).

Principle:

ADP + PEP PK

ATP + Pyruvate

Pyruvate + NADH LDH

L. Lactate + NAD+

Procedure:

The tissue homogenate of the parasites was done with 0.25 M sucrose

solution and the homogenate was centrifuged at 3000 rpm per 15 minutes. The

supernatant was used for for enzymatic estimations.

Reagents:

Buffer

Triethonalamine 0.16 mol/lit.

Kcl 0.12 mol/lit.

MgSO4 21 mol/lit.

EDTA 1.3 mol/ lit.

NADH 6 mol/ lit.

PEP 32 mol/ lit.

LDH 240 mol/ lit.

ADP 0.1 mol/lit.

Procedure:

92

In the reaction mixture in the volume of 3.25 ml the reaction was carried out

by adding 2 ml of buffer solution, 0.9 ml of water , 0.1 ml of PEP, 0.1 ml of NADH

, 0.1 ml of tissue homogenate, 0.05ml of LDH and incubated for 5 minutes at 27oc .

Then the reaction mixture was transferred into a temperature controlled cuvette and

0.1 ml of ADP was added and mixed well. The optical density extension was noted

at 366 nM and reading were recorded for 5 minutes. The experiment was repeated

and enzymatic activity was calculated for NADH disintegration rate. the calculation

was followed as per the procedure given by Boehringer Mannheim, GMbH test

procedure.

DETERMINATION OF ALKALINE PHOPHATASE :

1) Method of King and Armstrong 1934; King et al. 1937, 1942.

Reagents:

i) Disodium phenyl phosphate, 0.01M- Dissolve 1.09 gm in water and make

up to 500 ml. Bring quickly to the boil cool, add a little chloroform and

keep in the refrigerator.

ii) Sodium carbonate- Sodium bicarbonate buffer, 0.1M- Dissolve 3.18 gm of

unhydrous sodium carbonate and 1.68 gm of sodium bicarbonate in

water and make upto 500 ml.

iii) Buffered substrate for use- Prepare by mixing equal volumes of solutions

(i) and (ii). This has a pH of 10.

iv) 20% sodium carbonate solution- Dissolve 20 gm of unhydrous sodium

carbonate in water and make up to 100 ml. This solution is almost

saturated at room temperature and so should be kept in a warm place to

prevent the salt from crystallizing out.

v) Standard phenol solution- Stock solution 100 mg of phenol per 100 ml of

solution -Dissolve 1 gm of pure crystalline phenol in 0.1 N hydrochloric

acid and make upto 1 lit. with the acid. This can be standardized as

follows -

93

a) Place 20ml into 250 ml. flask. solution add 50 ml of 0.1 N Sodium

hydroxide and heat to 65oc .

b) To the hot solution add 25 ml of 0.1 N iodine.

c) Stopper and allow to stand for 30- 40 minutes. Add 5 ml of

concentrated hydrochloric acid and titrate the excess with 0.1 N

sodium thiosulphate.

d) Each ml of 0.1 N iodine is equivalent to 1.57 mg of phenol.

vi) Diluted phenol standard for use- Dilute the stock standard 1in 10 to obtain

a standard solution containing 10 mg phenol per 100 ml of solution.

vii) Standard phenol solution and reagent- containing 0.5 mg phenol per 100 ml.

Take 2.5 ml of the dilute standard add 15 ml of the diluted phenol

reagent and make upto 50 ml with water. This is best prepared daily.

Homogenetic preparation :

The nematode parasites were collected from the intestine of Capra

hircus and Ovis bharal , washed and weighed and homogenation was done in

distilled water and centrifuged for 15 minutes at 3000 rpm. The supernatant was

kept in refrigerator and was used for alkaline phosphatase enzyme estimation.

Procedure:

Pipette 6 ml of the buffer substrate into a test tube and placed in the water

bath at 37oc for a few minutes. Add 0.3 ml of homogenate, preferably without

removing from the bath. Mix and cork add allow to remain in the bath exactly 15

minutes. Then remove and immediately add 207 ml of the diluted phenol reagent.

At the same time set up a tube for the control containing 6 ml of substrate and 0.3

ml of homogenate, which is added immediately to 2.7 ml of diluted phenol

reagents. Mix well in both cases and centrifuge. Take 4 ml of supernatant fluid

from each and 1 ml of 20% sodium carbonate. Put up a standard prepared by

adding 1 ml of sodium carbonate solution to 4 ml of the standard phenol and

reagent. Place the 3 tubes in the 37oc water bath for 15 minutes and read in the

Colorimeter. As blank take 2.8 ml of water and add 1.2 ml of diluted phenol

94

reagent and 1 ml of 20% sodium carbonate. A red filter is used, with transmission

at 680 mM (millimicron).

DETERMINATION OF ACID PHOSPHATASE :

1) Method of Gutman and Gutman (1938, 1940):

The King Armstrong method for alkaline phosphatase was adapted for

estimation of acid phosphatase by Gutman and Gutman, who substituted a

different buffer so that the reaction could be carried out at pH 4.9.

Reagents:

i) Disodium phenyl phosphate, 0.01 M – Dissolve 1.09 gm in water and make upto

500 ml.

ii) Citric acid, sodium citrare buffer- Dissolve 21 gm of crystalline citric acid in

water and

add 188 ml of normal sodium hydroxide and make upto 500 ml with water.

Adjust to

pH 4.9 by adding sodium hydroxide or hydrochloric acid. Alternatively dissolve

29.41

gm of Sodium citrate C6H5O7 Na3, 2H2O, in 0.2 N hydrochloric acid and make

up to 500

ml with the acid fix solution (i) and (ii) in the refrigerator.

iii) Buffer substrate- Prepare freshly for use by mixing equal quantities of the two

solutions

given above.

The solutions required are the same as for the estimation of homogenate

alkaline phosphatase.

Homogenate preparation:

The parasites were collected washed and weighed and homogenation was

done in distilled water and centrifuged for 15 minutes at 3000 rpm. The supernatant

was kept in refrigerator and was used for alkaline phoaphatse for enzyme

estimation.

95

Procedure:

The method is the almost same as that given for homogenate alkaline

phosphatase. Use 6 ml of the buffer substrate with 0.3ml of homogenate and

incubate for 1 hour at 37oc. Subsequent procedure is identical with that given

previously.

Calculation:

The result in this case is expressed in terms of phenol liberated by 100 ml

homogenate in 1 hour at 37oc; each unit corresponding to 1 mg of phenol thus

formed. The calculation is thus exactly the same as that given for alkaline

phopsphatase. In the original method the time of incubation was 3 hours. It is

however, more convenient to use 1 hour.

The result is expressed in mg of phenol liberated by 100 ml of homogenate

in 15 minutes at 37oc; each unit corresponding to the liberation of 1 mg of phenol

per 100 ml of homogenate so :

Units phosphatase per 100 ml homogenate

= Reading of Unknown X 15 – Reading of Blank X 15

Reading of Standard Reading of Standard

96

INFLUENCE OF GAS ON NEMATODE PARASITES ACTIVITY:

(Fig.of Gas phase)

Fig.—2

Activity and survival studies under different gas phase i,e.Oxygen,Carbon

dioxide and Nitrogen of Oesophagostomum columbianum,O.asperum and

Bunostomum trigonocephalum .The experiment indicate that the parasites are more

active and survival for longer periods in CO2 than to the Oxygen and Nitrogen.

97

EXPERIMENT WITH THE GAS PHASE OF PURE OXYGEN,

CARBOHYDRATE AND NITROGEN TO STUDY THEIR INFLUENCE ON

PARASITES ACTIVITY:

To understand the influence of the various gases on the parasite and their

behavior the other devised and experiment by selected 100 ml capacity saline bottle

which were filled with cooled boiled Tyroide’s solution. Freshly collected

nematode parasites were brought to the laboratory in the above solution and

washed thoroughly. They were introduced into the bottle and filled with Tyroide’s

solution. After inserting hypodermic needle into the rubber cork it was fixed

carefully by allowing excess lipid to come out slowly without leaving an air gap,

and without exerting much pressure on parasites.

Now the close bottle is connected to the pure oxygen, carbon dioxide and

nitrogen cylinders with tubing having another hypodermic needle at it as end

inserted into the rubber cap of the bottle. When the respective gases were allow to

flow in, an equal amount of solution was displaced. The bottles were filled with the

above gases for more than half of the bottle, after establishing the saturated

conditions both the needles were removed and the bottles were made air tight buy

applying petroleum jelly. The parasite motility and longevity was observed and

recorded.

DEMONSTRATION OF RESPIRATION WITH THE HELP OF BLOOD

GAS ANALYSER MODEL CORNING PH/ BLOOD/ 165/2:

The respiration and also the rate of oxygen consumption and release of CO2

in the parasites were demonstrated by using automatic corning blood gas analyzer.

The instrument automatically records the pH, PO2, PCO2 ,HCO3 , total co2 and

mean base excess within 0.5 ml of solution injected into the sample chamber where

the respective oxygen, carbon dioxide, pH, microelectrode are positioned. By

taking relative above values the instrument automatically calculates and expresses

the above parameters.

The device is usually used to calibrate the PO2 tension in the blood at the

time of cardiac operation or in alkalosis, and acidosis etc. in hospitals. The author

98

devised an experiment by selecting a special type of polyethylene syringe gas kit to

make it completely air tight. The outlet was closed with high grade rubber copings

supplied by corning company. The line parasites were collected and washed

thoroughly Tyrode’s solution, wiped and weighed. The weighed parasites were

introduced into the air tight syringe along with 5 ml of Tyrode’s solution (1:5)

dilution. Here utmost care was taken to prevent the entry of live parasites into the

sampling chamber of the instrument through inlet port by keeping a glass wool plug

at the tip of the syringe. The initial readings of the sample were taken and the outlet

was closed immediately with rubber copings. The readings were taken at hourly

intervals of four hours.

The same procedure was repeated for all parasites sexwise, except

Bunostomum male, due to no availability of sufficient parasites in required

quantity. The experiment was conducted at 4 pH concentration, in order to

understand the effect of H ion concentration on rate of oxygen consumption. At the

same time the experiment was repeated with Tyrode’s solution with glucose and

without glucose in order to understand the effect of the glucose in the medium on

the rate of oxygen uptake in the gas dynamics. Te experiment was conducted 0.001

M Kcl in the medium to understand, the inhibitory effect.

HYDROGEN ION CONCENTRATION OF HOST INTESTINAL REGIONS:

The information regarding the Hydrogen ion concentration of the host

intestine (capra hircus and Ovis bharal) was not readily available for reference

work. In order to overcome this problem the author attempted to take pH reading of

the different intestinal regions with systronics portable battery operated pH meter

with monomicro electrode. Soon after the slaughter of the host, the intestine which

was almost warm and fresh was punctured with the sharp blade and electrode was

inserted at different regions of the entire length of the host intestine and pH

readings were noted. About 25 samples of each sheep and goats male and female

species were studied and average pH readings were recorded and given in the table

. This gives a fair idea about the intestinal environment of the host at the time of

nematode parasites collection.

99

ESTIMATTION OF PROTEIN:

Sample preparation:

The roundworms were washed in Tyrode’s solution several times and

weighed in living condition after blotting with filter paper. Then parasites were

homogenized in a hand driven mortar and pestle in a known quantity of glass

distilled water and it is made upto a known volume. The homogenate was allowed

for few minutes and centrifuged at 3000 rpm for 15 minutes; the supernatant was

used for the estimation of soluble proteins. The precipitate was dissolved in 0.1 M

NaoH keeping overnight at 60oc and made upto the known volume and the

insoluble proteins were estimated from the precipitate dissolved solution.

Method:

The proteins were estimated by Lowry et al. (1951), Lowry et al.(1964)

which is very sensitive 100- 200 mg when compared to other methods.

Principle:

Protein reacts with Folin – Ciocalteacu reagent give a coloured complex.

This colour is due to the action of alkali copper with protein as in the case of buret

test and the reduction of phospho molybedate by rosin and trytophane present in the

protein.

Material:

Alkaline sodium carbonate solution (2% Na2CO3 in 0.1 N NaOH)

Copper sulphate- sodium potassium tartarate solution

(0.5% CuSo4 in 1% sodium potassium tartarate solution)

(Prepared by mixing stock solution which were kept separately)

Alkaline solution:

Prepared before the estimation by mixing 50ml of solution 1 and 1 ml of solution 2.

Folin- Ciocalteacu reagent – The reagent was diluted with equal volume of water

before estimation.

Standard protein – Albumin solution having 0.2 mg / ml (standard bovine albumine

used for standard graph)

100

Procedure:

To 5 ml of alkaline solution 0.1 ml of test samples added, mixed thoroughly

and allows standing this mixture for 10 minutes to the above solution. Folin

Ciocalteacu reagent solution was added with immediate mixing and allow stand.

The colour extinction read at 750nm with (Carlzein’s Jena) Spectrophotometer

against the appropriate blank after 30 minutes. By using standard albumin standard

graph was prepared and tabulated.

GLUTAMATE PYRUVATE TRANSAMINASE (G.P.T.):

METHOD:

Reitman and Frankel (1957) method.

Principle:

Glutamate pyruvate transaminase (G.P.T.) catalyses the reaction between

aniline and ketoglutarate to form glutamate and pyruvate. The pyruvate reacts with

2, 4-dinitrophenyl hydrazines (DNPH) to form hydrazone which in alkali condition

gives brown colour.

Reagents:

1) Alkaline kertoglutarate substrate (7.4 pH)

2) DNPH- solution

3) Pyruvate standard (2 mM)

4) Sodium hydroxide (4 N)

Procedure:

Into a test tube which was labeled as test 1 ml of alkaline ketoglutarate

substrate solution is taken and it was placed in a water bath at 37oc at 5 minutes.

To the above test tube 0.2 ml of homogenate was added mixed and allow

incubating at 37oc for 30 minutes. Then 1 ml of DNPH colour reagent was

added and mixed well and then allowed to stand at room temperature for 20

minutes. To the test mixture 10 ml of working sodium hydroxide 0.4 N was

added and mixed and allow to stand at room temperature for 10 minutes. The

101

optical density was read at 504 nM using a green filter against distilled water as

blank which was set to zero.

The standard graph was prepared by using different concentration of

pyruvate standard ranging from -

1) 0.1ml of pyruvate standard 0.9 ml of solution 1,

2) 0.2 ml of pyruvate and 0.8 ml of solution 1,

3) 0.3+ 0.7

4) 0.4+ 0.6 which will represent 28, 57, 97, 150 SGPT units, and graph was

ploted and test volume were compared.

GLUTAMATE OXALOACETATE TRANSAMINASE (G.O.T.):

Method:

Reitmen and Frankel (1957) method.

Principle:

Glutamate oxaloacetate Transaminase catalyses the reaction between

aspartic acid and �- Keto glutarate to form glutamate and oxaloacetate.

Oxaloacetate reacts with 2- 4 DNPH (Dinitro phenyl hydrazine) to form

hydrazones which in alkaline media gives brown colour.

Reagents:

1) Asparate- Ketoglutarate substrate (pH 7.4)

2) DNPH colour reagents

3) Pyruvate standard (2 nM )

4) Sodium Hydroxide (4N)

Working reagents was prepared by diluted 1: 10 with distilled water. The

parasites were collected weighed, washed and homogenized in sucrose

solution.

Procedure:

1 ml of aspartate ketoglutarate substrate solution was taken in a test tube

and placed in a water bath at 37oc for 5 minutes. To the above 0.2 ml of nematode

parasite homogenate was added and mixed well and kept at 37oc water bath for

1hour. Then the test tubes were removed and 1 ml of DNPH colouring reagent was

added mixed well and allow staying at room temperature for 20 minutes,

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To that 10 ml of 0.4N working sodium hydroxide solution was added mixed

with and allow standing for 10 minutes at room temperature. Then optical density

was measured at 505nM CZ Colorimeter against distilled water blank set as zero

and then standard graph was prepared as in the same manner except changing the

substrate.

ESTIMATION OF FREE AMINO ACID:

Total free amino acid level was estimated by the method of Moore and

Stein (1954).

After washing the parasites weighed and roughly 5% homogenate in 10% (W/V)

trichloroacetic acid (TCA) and centrifuged at 3000rpm. To 0.5ml of the above

supernatant 2.0 ml of ninhydrine reagent was added and kept in boiling water bath

for exactly 6.6 minutes and immediately cooled. It was diluted to 10 or 15 ml

depending on the intensity of the colour and bluish pink colour was read at 570 nM

in UV Spectrophotometer standard graph was prepared using analar grade tyrosine

and the amino acid content of the tissue is expressed as mg of amino acid per wet

weight of the parasite.

DETERMINATION OF PROTEASE ACTIVITY:

Protease activity was estimated by using the method of Moore and Stein

(1954) considering the amount of free amino acid liberated from protein substrate

as a measure proteolytic activity. A 70% parasite homogenate was prepared in cold

distilled water in a glass grinder jacketed with ice the homogenate was centrifuged

at 1000 rpm for 15 minutes and supernatant was used for enzyme assay.

Procedure:

The assay mixture in final volume of 1.0ml contained 100M moles of

suitable for required pH (citrate buffer was used to cover pH to 2.5 – 5.5, phosphate

buffer for 6.0 – 8.0 pH, tris buffer for pH range of 8.5- 9, carbonate – carbonate

buffer for 9.5- 10 pH range ). 12 mg heat denatured haemoglobin protein and 0.5

ml of homogenate supernatant was used. The reaction was stopped by the addition

of 2 ml of 10% TCA. Un-incubated sample was also treated with 2 ml of 10%

TCA, prior to the addition of homogenate supernatant. The content of both tubes

were incubated for 1 hour and were filtered. The amino acid level was determined

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in the filtrate. To the filtrate ninhydrine reagent was added, kept in a boiling water

bath for 6.5 minutes and immediately cooled. It was made upto 10 or 50 ml and

colour was read at 570 mµ in U.V. Spectrophotometer.

PROCEDURE FOR ESTIMATION OF IONIC COMPOSITION OF

NEMATODE PARASITES:

Routinely the parasites were collected from the slaughter house of the

Chhavni Cantonment area Aurangabad Maharshtra without any ringer solution or

water. They were brought to the laboratory , the wet weight was noted sex wise and

dried in hot air over at 100oc for 24 hours . Then the dry weights of the parasites

were noted then they were digested into the solution by using mixed acid digestion

and separated sex wise as well as specisewise.

Reagents:

1) Perchloric acid 60%

2) Nitric acid concentrated

3) Sulphuric acid concentrated.

Procedure:

Round about 0.50 gm of dried nematode parasites were placed in 100 ml

flask , 5 ml of 60% Hclo4 (Perchloric acid), 5ml of concentrated HNo3 and 0.5 ml

of H2So4 were added. The contents were swirled gently first at moderate heat, then

heat increased latter. It is digested for about 30 minutes, until the disappearance of

brown fumes. After the appearance of white fumes it was digested for 10 minutes

cooled and diluted with D.D. glass distilled water.

In the same manner blank digestion was carried out, the clear digested

solution was used for estimation of Na, K, Ca, Cu, Fe, PO4 except sulphate where

the number of blanks of digestion was carried out and estimation was repeated to

get concurrent values. The estimation Na, K, Ca, Cu, Fe were done on atomic

absorption spectrophotometer where the accuracy is more, sensitivity is 0.001 ppm.

Observation Table of Elements:

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Elements Wave length Flame gases

Na 585.0 nm Air acetyline

K 766.3 nm Air acetyline

Ca 421.5 nm Air acetyline

Cu 323.4 nm Air acetyline

Fe 244.1 nm Air acetyline

Atomic absorption spectrophotometer- PERKIN- ELMER 2380 with varied wave

length and computerized system emission spectra.

(Na- K) ATPase:

The parasites were washed with distilled water, then homogenized in

phosphate buffer 0.05M an immediately cooled and kept it in deep freeze. The total

(Na+ - K

+) ATPase was estimated by the procedure of Narayan Reddy K. and

Kaplay S. (1983).

Reaction mixture for 50 assays:

12.5 ml of reaction mixture was prepared (0.5 M NaCl, 0.5 M KCl, 0.5 M

MgCl2, 0.1 M EDTA, 0.25 M Tri-HCl buffer at 7.5 pH and glass distilled water

0.75 ml ) which is sufficient for 50 assays.

Reagents:

1) Reaction mixture: 0.25 ml per assay in a final volume of 0.50 ml gives 140

mM Nacl, 14 mM KCl, 3 mM MgCl2 , 0.2 mM EDTA, 20 mM Tri HCl, pH

7.6.

2) Ouabine : 2 mM molecular weight 728.6, 1.46 mg / ml water.

3) 30 mM ATP sigma vanedium free 181.8 mg in 6- 7 ml , 1.0 ml of 0.5 tris –

buffer at 7.0- 7.2 made upto 10 ml.

105

Observation Table:

Assay - Ouabine +Ouabine Blank

R.M.1 0.24 0.24 0.24

2 mM Ouab ine 2 Tissue

homogenate supernatant

-

0.10

0.04

0.10

-

-

water 0.10 0.10 0.10

Per incubate for 10 minutes at 37oc, add 30 mM ATP 0.05 ml each to above. Again

incubated for 2 hours at 37oc.The reaction was terminated by adding 0.5ml of 10%

ice cold TCA. Each tube was mixed and kept at cold temperature. At this stage 0.10

ml of tissue homogenate supernatant was added to the blank.

Then the tubes were centrifuged after keeping 10 minutes in the ice. The

clear supernatant was decanted. The inorganic phosphate was determined in the

supernatant by Fisk and Subbarow method.

Protein in the tissue homogenate supernatant was determined by Folin’s

method (Lowery et al. 1951).

DETERMINATION OF INORGANIC PHOSPHOROUS (BY FISK AND

SUBBAROW, (1925) :

The proteins of the homogenate are precipitated with trichloroacetic acid.

The protein free filtrate is treated with an acid molybdic acid, which form phosphor

molybdic acid is reduced by the addition of 1, 2, 4- aminonapthol sulphonic acid

reagent to produce a blue colour and the colour intensity is proportional to the

amount of phosphate present.

Procedure:

To 8ml of 10% trichloroacetic acid solution in a small flask, 2ml of

homogenate solution was added and mixed well. It is filtered through an ashless

filter paper. 5 ml of filtrate was transferred into a graduated cylinder and 1 ml of

106

molybdate solution was added and mix, to the above solution 0.4 ml of amino

naphtho sulphonic acid reagent solution was added and mixed well. Then it was

diluted to the mark of 10ml with water mixed well and allows standing for 10

minutes. The colour intensity was read with a spectrophotometer (C.Z.Spect.) at

680 nm against a blank. The standard graph was prepared with standard phosphate

and values of inorganic phosphate were expressed in mg/gm wet weight of the

nematode parasites.

VALIDITY OF EXPERIMENTAL PROCEDURES:

Aliquots for assay:

Aliquots were selected for the assay such as initial levels were

approximately as near as possible. Yet providing sufficient product to fall in a

convenient range for spectrophotometric measurement. All substrates and

metabolites are expressed as mg/gm. All ions were expressed in millimoles per

gram five replicate values were taken for each parameter.

Enzyme units:

Enzyme activity was expressed as standered units i.e. millimoles of product

formed or substrate cleared per milligram protein per hour.

Substrate requirements:

All enzyme activity levels were determined at saturating substrate

concentration.

Lamberts- Beer’s Laws:

Almost all the products of the reaction were measured by Colorimetric

procedure in which optical density is proportional to concentration of reaction

product.

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