MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS

III. MATERIALS AND METHODS

3.1 Glassware and plasticware

Scrupulously cleaned, grease free glassware of Corning / Borosil / Scott Durham

brand were used. Molecular / Analar / GR / Excellar grade of chemicals were used to

prepare buffers and all other reagents in triple glass distilled water.

Plasticware including centrifuge tubes, microcentrifuge tubes, cryo vials, tissue

culture Petri plates, real time PCR plates, real time PCR plate sealing film, FACS tubes,

adjustable volumetric micropippets, microtips were procured from Nunc Denmark, BD

Biosciences USA, Eppendorf Germany and Axygen India.

3.2 Marek's Disease virus (MDV)

The Marek's disease virus (MDV-1) with 4x103TCID50/ml (code MDCVD1C1)

used for challenge studies was procured from Indovax private limited, Gurgaon, India.

3.3 SPF eggs and chicks

One hundred numbers of Specific Pathogen Free (SPF) embryonated chicken eggs

were procured from Indovax Pvt. Ltd. Gurgaon for CEF culture, safety test studies and

confirmation of virulence of MDV-1 virus. The virulence of the virus was confirmed by

inoculating 500 PFU [assuming 1TCID50 = 0.69 PFU (Luria et al., 1978)] intra

abdominally into ten SPF chicks. Virus was tested for the presence of Meq gene, a

principal oncogene (Lupiani, 2004) by processing the feather tips of above chicks by

PCR, using specific primers as per the method described by Raja et al. (2009).

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3.4 Vaccines

a) Commercial Marek’s Disease Freeze dried HVT Vaccine, living BP (vet). 50

ampoules, stored at 4°C until use.

Upon reconstitution with the diluent supplied along with the vaccine

containing HVT FC 126 live virus vaccine strain, each dose of 0.2ml would give

1,000 PFU.

b) HVT+SB-1 cell associated vaccine - 50 ampoules. The vaccine was stored in a

liquid nitrogen container until use.

c) Other vaccines: Live attenuated Newcastle Disease (ND) vaccine, La Sota strain,

vial containing 500 doses, provided with 15 ml of diluent, procured from the

Institute of Animal Health and Veterinary Biologicals, Bangalore. Each dose

contained freeze dried vaccine virus more than 106 EID50 virus suspended in a

suitable stabilizer.

3.5 Chicks and feed

Two hundred fifty numbers of day old chicks (BV 300) were procured from the

hatchery facility of M/s Venkateshwara Hatcheries Pvt. Ltd. Hosur, Tamil Nadu. All the

chicks were reared in a contained environment in the animal experimentation facility of

Veterinary College, Bangalore. Necessary permission was obtained from the Institutional

Ethics Committee of Veterinary College, Bangalore. Required feed for the birds was

procured from Poultry feed plant, Department of Poultry Science, Veterinary College

43

Bangalore. Chicks were reared under standard managemental conditions by providing

adlibitum clean water and feed.

3.6 Titration of Cell Associated Marek’s Disease HVT+SB-1 vaccine virus

The titration was carried out as per the protocol, Supplemental assay methods 406

(2005) of Center for Veterinary Biologicals of USDA.

3.6.1 Materials

3.6.1.1Equipment/instrumentation

i) Centrifuge (MPW 350R, Poland)

ii) Humidified, rotating egg incubator (Cirugia deluxe Pvt. Ltd. Mumbai)

iii) Water-jacketed incubator with a humidified 5 ± 1% CO2 atmosphere and

temperature set at 37°± 1°C (Stericult, Thermo Scientific, USA)

iv) Magnetic stir plate

v) 250-ml trypsinizing flask with a sterile magnetic stir bar

vi) Electronic water bath

vii) Erlenmeyer flask with a sterile magnetic stirring bar

viii) Neubauer hemocytometer

ix) Sterile scissors, curved tip forceps etc.

3.6.1.2 Other supplies

i) Tissue culture Petri dish, 150 mm (Borosil)

ii) Plastic funnel covered with 4 layers of fine gauze

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iii) Collagen treated 60-mm gridded cell culture dish (Nunc Denmark)

iv) Specific pathogen-free (SPF) chick embryos, 9 to 11days old

vi) Fetal Bovine Serum (FBS) (Hi-media)

3.6.1.3 Media and Reagents

i. Calcium magnesium free Phosphate buffered saline (0.1M pH 7.4)

(CMF-PBS)

Sodium chloride : 8.00 g

Disodium hydrogen phosphate 2H2O : 1.44 g

Potassium chloride : 0.20 g

Potassium dihydrogen phosphate : 0.20 g

The above salts were dissolved in triple glass distilled water; the volume was

made up to 1000 ml and sterilized by autoclaving at 121o C for 20 min at 15lb pressure

ii. Sodium bicarbonate solution (4.4%)

Sodium bicarbonate : 4.4 g

Triple glass distilled water : 100.0 ml

The solution was sterilized by autoclaving at 121°C for 20 min at 15lb pressure and

stored in one ml aliquots at 4°C

iii. Tryptose phosphate broth

Tryptose phosphate powder : 2.98 g

Triple glass distilled water : 100.0 ml

45

The broth was distributed in 10 ml of aliquots in screw cap tubes and sterilized by

autoclaving at 121°C for 20 min at 15lb pressure and stored at 4°C.

iv. Vitamin supplements

Choline chloride : 0.5 g

Folic acid : 0.5 g

Nicotinamide : 0.5 g

Pantothenic acid : 0.5 g

Pyridoxyl Hydrochloride : 0.5 g

Thiamine : 0.5 g

Riboflavin : 0.5 g

Triple glass distilled water : 100 ml

The solution was sterilized by filtration using a membrane filter of APD 0.22 µ

(Millipore, India) and stored in eight ml aliquots at 4° C.

v. Growth medium (10X stock solution).

Dulbecco's modified minimum essential medium (MEM) procured from M/s Hi-

Media Laboratories, Mumbai and the stock solution was prepared as follows.

Dehydrated medium (1 Unit) : 0.92 g

Amino acid concentrate : 0.032 g

Vitamin supplements : 8.00 ml

Triple glass distilled water up to : 100 ml

46

The ingredients were mixed and stirred on a magnetic stirrer for 30 minutes. The

solution was sterilized by filtration using a membrane filter of APD 0.22 µ (Millipore

India) and stored in 10 ml aliquots at 4°C.

vi. Growth medium (working strength)

Dulbecco's modified MEM (10X) : 10.0 ml

Tryptose phosphate broth : 10.0 ml

Foetal bovine serum : 6.0 ml

Sterile triple glass distilled water : 73.0 ml

Streptomycin / Penicillin stock solution : 1.0 ml

(Streptomycin 10 mg and Benzyl Penicillin 10,000 IU /ml)

Fungizone (2.5 mg/ml) : 0.1ml

pH was adjusted to 7.4 with 4.4 per cent sodium bicarbonate solution.

vii. Maintenance medium

Dulbecco's modified MEM (10X) : 10.0 ml

Tryptose phosphate broth : 10.0 ml

Foetal bovine serum : 2.0 ml

Sterile triple glass distilled water : 77.0 ml

Streptomycin and Penicillin stock solution : 1.0 ml

(Streptomycin 10 mg and Benzyl Penicillin 10,000 IU /ml)

Fungizone (2.5 mg/ml) : 0.1 ml

The ingredients were mixed under sterile conditions just before the use and the

pH was adjusted to 7.4 with 4.4 per cent sodium bicarbonate solution.

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3.7 Cell culture

3.7.1 Preparation of primary Chick Embryo Fibroblast (CEF)

Primary CEF cultures are prepared from specific-pathogen-free (SPF) chick

embryos of 9 to 11 days old as per the protocol of supplemental assay methods (2005),

Centre for Veterinary Biologicals, USDA.

Air cell ends of the required numbers of embryonated eggs were disinfected with

70 per cent ethanol and the shells were broke opened with sterile forceps. The

membranes were opened with forceps and each of the embryos was lifted out and placed

in a sterile disposable 150 x 10 mm Petri dish. Heads and viscera of the embryos were

removed with sterile forceps and discarded. The embryo was washed several times with

PBS to remove excess blood. The washed embryos were placed in a sterile dry 100 x 10

mm Petri dish and were minced thoroughly using sharp sterile scissors.

The minced tissue was put into a trypsinizing flask containing a magnetic stir bar

and 50 ml growth medium without FBS. The flask was placed on a magnetic stir plate

and was stirred with a moderate vortex for five min until the tissue was completely

homogenized; care must be taken to avoid any froth formation. The cells were allowed

to settle and the supernatant was decanted.

Before proceeding for trypsinization any residual media was rinsed out from the

cells by adding 10 ml of the 0.25 per cent trypsin solution and then the trypsin solution

was completely decanted. Actual trypsinization was carried out by adding 40 ml of 0.25

per cent trypsin and mixing thoroughly. The whole suspension was transferred onto a

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sterile gauze wrapped glass funnel placed into the opening of a 250 ml conical flask. Two

ml of FBS was added to stop the trypsin activity. The total volume was brought to

approximately 100 ml with growth media, centrifuged for 10 minutes at 250 x g at 10°C.

Supernatant was discarded and the cells were resuspended in 100 ml growth

media. The cells were washed once again by centrifugation and finally re-suspended in

fresh growth medium. A cell suspension of 0.5ml was diluted with an equal amount of

trypan blue solution and the cells were counted in haemocytometer.

Concentration of the cells was adjusted to one million cells / ml of growth

medium and dispensed into tissue culture flasks and incubated for three days at 37 C

in an incubator with five per cent CO2 and 75 per cent humidity to obtain confluent

monolayer.

3.7.2 Secondary CEF preparation

Secondary CEF culture was prepared by removing the medium from the primary

culture flask and adding 5 ml of 0.25 per cent trypsin solution to each flask. The trypsin

solution remained in contact with the cell sheet for 90 sec, before it was removed.

Primary culture flask was placed in a horizontal position with the cell sheet down and

incubated at 37 C for an additional 10 min.

To each of the flasks, 15 ml fresh growth medium was added and shook to loosen

and break up the cell clumps. A cell suspension was poured into an Erlenmeyer flask with

a stirring bar. After a thorough mixing, cell count was made with a hemocytometer. The

volume was adjusted to obtain a cell concentration of approximately 3, 75,000 cells per ml.

49

Four ml of secondary cell suspension was placed into 60-mm plastic tissue

culture dishes and incubated in a humidified incubator at 37°C, with five per cent CO2 for

24 hr prior to inoculation.

3.8 Determination of optimum thawing temperature of vaccine

3.8.1 Reconstitution of vaccine

One ampoule of HVT+SB-1 vaccine was taken out from the liquid nitrogen

storage container and thawed at 45 and 60 seconds by immersing in a electronic water

bath containing distilled water at different temperatures ranging from 20°C to 45°C

and different holding intervals given in Table 1. Immediately the vaccine was diluted

with the diluent, supplied by manufacturer, warmed to RT. It was done gently by

aspirating the vaccine into a 10-ml syringe through an 18-gauge needle and then five ml

of the diluent was collected into the same syringe and mixed gently. The contents of the

syringe were offloaded into the diluent bottle. Two ml from the diluted vaccine was

aspirated and used to rinse the ampoule once, then added back to the diluted vaccine.

Diluted vaccine was mixed gently by slowly inverting the bottle. This mixture constituted

“field strength” of the vaccine equivalent to one dose per 0.2 ml.

3.8.2 Holding period

The diluted vaccine was tested for virus titer at different holding temperatures

(Table 1) and at intervals of 30, 90 and 120 min on ice cubes. The vaccine was gently

mixed at 30 min before proceeding to virus titration. Shortly before the end of the

holding period, a two ml of diluted virus was mixed with 8.0 ml of growth medium to

give an initial dilution of 1:5; further dilutions were carried out to get 1:25, 1:250 and

1:500.

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Table 1. Determination of optimum thawing temperature: different temperatures,

duration of thawing and holding period employed

Sl. No. Thawing temperature Duration of thawing Holding period

1 20°C 60 sec 30, 90 & 120 min on ice

2 26°C 45 sec 30, 90 & 120 min on ice

3 35°C 45 sec 30, 90 & 120 min on ice

4 35°C 45 sec 30, 90 & 120 min at RT

5 40°C 45 sec 30, 90 & 120 min on ice

6 45°C 45 sec 30, 90 & 120 min on ice

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3.8.3 Plaque assay

After a gentle but thorough mixing one ml of each of the final dilutions were

seeded into 60 mm Petri dishes containing CEF culture as per 3.7.2. Five replicates were

maintained for each dilution. A thorough and gentle mixing was done by taking

adequate care to avoid any damage to the cells. The entire process of dilution and

seeding was completed within two min to prevent cells from attaching to the surface of

the dilution tubes. Further, the seeded plates were swirled gently to ensure uniform

distribution of virus. The plates were incubated in a 75 per cent humidity atmosphere at

37ºC containing five per cent CO2.

Twenty-four hours post-inoculation, the medium from the plates was removed

and replaced with 5 ml maintenance medium. The same was replaced on day three. Petri

plates with cells alone without seeding the vaccine virus were served as controls to

monitor the integrity of monolayer.

The plaques were counted on day five post inoculation using an inverted

microscope. The cell controls were used to confirm the validity of the assay. The test is

considered to be valid only if the monolayer in the control plates remained healthy.

Following formula was used to determine the titer (PFU/ml) of the vaccine per

dose of 0.2 ml

No. of plaques x V d x 5

= PFU/dose d=dilution factor

V = volume of diluted virus added to the plate

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3.9 Experimental design to asses potency and to evaluate the immune response.

To assess potency and to evaluate the immune response of bivalent HVT+SB-1

MD vaccine against Monovalent HVT vaccine, 210 numbers of one day old unvaccinated

layer chicks were randomly divided into three groups as shown in Table 2.

The indicators chosen to assess potency and to evaluate the immune response are;

i) T helper and T cytotoxic cells count by flow cytometry, ii) quantification of challenge

virulent MDV load against the vaccine virus load in feather pulp by Real time PCR and

iii) analyzing the histopathological changes in selected visceral organs of vaccinates

(HVT alone and bivalent HVT+SB-1) against unvaccinated control chicken.

Vaccines were administered subcutaneously at hatch at a dose of 1,000

PFU/0.2ml to Groups 1 and 2. The Group 3 was sham vaccinated to serve as controls. On

day 9 pv, chicks in all the groups were challenged with MDV-1, an oncogenic virus, by

Subcutaneous route, at a dose of 500 PFU/0.18ml. Blood samples were collected

randomly from six birds in each group on days 8th dpv and 7th, 14th, 28th, 43rd and 61st

dpc, for estimation of T helper and T cytotoxic cell population by flow cytometry. Six

birds from each group were sacrificed on 7th, 14th, 28th, 43rd and 61st dpc. Spleen, liver,

bursa, kidney, proventriculus, thymus, heart and sciatic nerves were collected to study

histopathological changes. Whole feather tips collected from these birds just before the

sacrifice served as source of DNA to estimate MDV-1, the challenge virus load, as a

means to evaluate the efficacy of vaccine.

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Table 2. Experimental design to study the immune response and histopathology

to evaluate HVT and HVT+SB-1 MD vaccines

Particulars Group 1 Group 2 Group 3

Number of SPF Chicks 60 60 90

Vaccination on ‘0’ day HVT HVT+SB-1 Sham vaccinated

Dose of vaccine 1,000 PFU /0.2 ml

Challenge virus Virulent MDV-1 500 PFU/0.18ml

Day of Challenge 9th dpv

Collection of Feather pulp to estimate the virus load and visceral organs for histopathology.

7th, 14th , 28th , 43rd and 61st dpc; 6 birds from each group

Collection of Blood for Flow cytometric analysis 8th dpv and 7th, 14th , 28th , 43rd and 61st dpc; 6 birds from each group

Period of observation 70 days

54

Vaccinated chicks in Groups 1 and 2 and controls in Groups 3 were raised

separately throughout the experiment. All the chicks were vaccinated on day one except

group 3 as shown in Table 2.

3.10 Determination of safety and potency of HVT+SB-1 cell associated vaccine and

HVT cell free vaccine

Safety and potency were determined as per the methods prescribed in the OIE

Terrestrial Manual 2010 adopted by the World Assembly of Delegates of the OIE in

May 2010.

3.10.1 Safety test

Twenty five, one day old SPF chicks received 10,000 PFU of HVT+SB-1 and

HVT vaccine. The chicks were observed for 21 days. The test is not valid if more than

five birds showed abnormal clinical signs or died from causes not attributable to the

vaccine. The vaccine complied with the test if no chicken showed notable clinical signs

of disease or died from causes attributable to the vaccine.

3.10.2 Potency test

3.10.2.1 Materials

a) 30, one day old chicks in group 1 and group 2 were identified and marked separately

for potency testing.

b) Vaccines: HVT+SB-1cell associated bivalent and HVT cell free freeze dried vaccine.

55

3.10.2.2 Method

Thirty, one day old chicks received 1,000 PFU of dose of the HVT vaccine and

HVT+SB-1 vaccine; 30 chicks were used as unvaccinated challenged controls with each

vaccine. On day nine after immunization, each chick was challenged by intra abdominal

route with 500 PFU of virulent MDV. Each of the birds was observed daily for the

development of clinical signs and death up to 70 dpv. Number of birds dying post

vaccination was recorded and per cent mortality and protective index (PI) was calculated

as per the formula given by I.P.VET (2000). At the termination of experimental period all

the survivors were sacrificed, gross lesions, if any, were scored and organs were collected

for histopathological examination. The PI was calculated.

% MD in unvaccinated and challenged - % MD in vaccinated and birds challenged birds

PI = % MD in unvaccinated challenged birds

3.11 Estimation of CD4 and CD8 cell counts by Flow cytometry

Estimation of CD4 and CD8 cells were carried out at the Center for Cellular and

Molecular Patterns, National Centre for Biological Sciences, Bangalore. The protocol

supplied along with the Monoclonal antibodies by the Manufacturer, AbD Serotech

USA, was followed.

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3.11.1 Materials

3.11.1.1 Phosphate buffered saline with bovine serum albumin (PBS-azide-BSA)

Phosphate buffered saline : 1000 ml

Sodium azide : 5.0 g

BSA (sigma) : 10.0 g

The solution was freshly prepared and sterilized by filtration.

3.11.1.2 Wash buffer (PBS-Azide)

Sodium chloride : 8.00 g

Disodium hydrogen phosphate : 1.21 g

Potassium chloride : 0.20 g

Potassium dihydrogen phosphate : 0.20 g

Distilled water (DW) : 1000 ml

Sodium azide : 0.5 g

The solution was sterilized by autoclaving at 121oC for 15 min at 15 lb pressure

and stored at 4oC in aliquots of 100 ml.

3.11.1.3 Cell fixing buffer (Para Formaldehyde in PBS-Azide)

Phosphate buffered saline : 1000 ml

Sodium azide : 0.5 g

Para Formaldehyde (Sigma) : 20.0 g

3.11.1.4 FACS Tubes

Round bottom polypropylene tubes (5 ml) with caps were used (M/s Becton

Dickinson, UK).

57

3.11.1.5 Antibodies

Monoclonal anti chicken CD4 mouse antibody tagged with FITC and anti chicken

CD8a mouse antibody tagged with PE were procured from M/s AbD Serotech, USA.

3.11.2 Optimization of anti chicken CD4 and CD8 antibodies

An end point titration was carried out to determine the optimum concentration of

anti chicken CD4 and CD8 monoclonal antibodies to be used to bind lymphocytes. It

was performed by using the different concentrations 2.5, 5.0, 7.5 and 10µl of the anti

chicken CD4 and CD8 monoclonal antibodies (1µg/µl) to bind to the lymphocytes.

3.11.3 Protocol for measurement of CD4 and CD8 T cells by Flow cytometry

3.11.3.1 Isolation of lymphocytes from EDTA treated blood from chicken

3.11.3.1.1 Materials

i) HiSep ™ LSM 1084 medium used for the isolation of mononuclear cells from EDTA

treated, defibrinated chicken blood was procured from M/s Hi media Mumbai.

ii) Two ml tubes treated with EDTA

iii) Sterile glass Pasteur pipettes

iv) Centrifuge (MPW 350R, Poland)

v) Five ml FACS tubes

vi) Ice cooler

vii) Trypan blue stain, 0.4%

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3.11.3.1.2 Method

1. 3.0 ml of HiSep LSM 1084 was aseptically transferred to clean centrifuge tubes of 15

ml capacity

2. 3.0 ml whole blood was carefully overlaid on HiSep LSM 1084

3. Erythrocytes and granulocytes were sedimented out by centrifuging the tubes at 400

x g for 30 min at RT. Lymphocytes formed a band above HiSep LSM 1084.

4. Supernatant containing plasma and platelet above the interface band was aspirated

and discarded.

5. Lymphocyte cell band was collected using a clean glass Pasteur pipette.

6. Lymphocyte fraction was added to 10 ml PBS in 15 ml centrifuge tube and

centrifuged for 10 minutes at 250 x g and the supernatant was discarded.

8. The cell pellet was re-suspended with one ml PBS in 5ml FACS tube and mixed

gently by inverting several times.

9. The contents were centrifuged at 250 x g for 10 min and the supernatant was

discarded.

10. Steps 8 and 9 were repeated again to remove any traces of HiSep LSM 1084 from the

lymphocytes.

11. Cells after the final wash were dissolved with one per cent BSA and 0.9 per cent

Sodium azide in 100µl of PBS.

12. Cells were counted in Neubauer slide and optimized to one million cells/ml

13. Anti chicken mouse monoclonal antibodies, 7.5 µl of CD4 and 2.5 µl of CD8 were

added and incubated on ice for 30 min in dark room with intermittent mixing.

14. One ml of PBS was added to each tube and washed at 250x g for 5 min.

59

15. Supernatant was completely decanted and one per cent BSA and 0.9 per cent Sodium

azide in 500 µl of PBS was added to the pellet. FACS tubes were gently tapped to

dissolve the pellet.

16. Finally, the contents were subjected to FACS analysis for cell differentiation and

data acquisition.

3.12 DNA extraction from feather tips

3.12.1 Materials

i) Proteinase K (MBI Fermentas)

ii) Proteinase K buffer

EDTA : 0.930g

SDS : 2.500g

Tris HCl : 0.605g

Deionized water : 50ml

iii) Sodium acetate (5M)

Sodium acetate : 8.2g

Deionized water : 20ml

iv) Proteinase K mixture

Proteinase K : 4mg

Proteinase K buffer : 10ml

60

v) Phenol: Chloroform: Isoamyl alcohol

Phenol : 25ml

Chloroform : 24ml

Isoamyl alcohol : 1ml

vi) Isopropanol

vii) Ethanol (70%)

3.12.2 Method

Extraction of DNA from feather follicles was carried out as per the procedure

described by Handberg et al. (2001). Briefly, the procedure includes;

a) Proximal shaft (about one cm) of five feather tips of different sizes collected from

each chicken were shaken over night at 55ºC with 200 µl of proteinase K mixture

(400µg of proteinase K / ml of proteinase K buffer).

b) To this, 20µl of 5 M sodium acetate and 200µl of Phenol: chloroform: Isoamyl

alcohol was added and centrifuged at 13,000g for 15 min at 4°C.

c) The aqueous phase was transferred to 200µl isopropanol and was centrifuged again at

13,000g for 15 min at 4°C.

d) Supernatant was removed and the DNA pellet was washed twice with cold (-20oC),

70 per cent ethanol for 10 min by vortexing and subsequent centrifugation at 13,000g

for 15 min at 4°C.

e) After decanting the ethanol, the traces were removed by air drying the DNA pellet

under laminar flow hood for 15 to 30 min.

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f) DNA pellet was dissolved in 100 µl of sterile nuclease free water and left at RT for at

least 30 min before storing at -20ºC till further use.

3.12.3 Assessment of Quality and Quantity of DNA

The quantity of DNA was calculated by spectrophotometric method. The optical

density (OD) at 260 and 280 nm was taken in UV spectrophotometer with distilled water

as reference. Purity of DNA was estimated on the basis of OD ratio at 260:280 nm. The

samples with acceptable purity (i.e., ratio between 1.6-1.8) were quantified using the

following formula and used for PCR. Concentration of DNA (µg/ml) = OD at 260 x

dilution factor x 50 Where, 50 is concentration of dsDNA expressed in µg/ml at OD of

one.

The quality of DNA was further checked by submarine agarose gel

electrophoresis using 0.8 per cent agarose in 0.5X TBE (pH 8.0) buffer (Sambrook and

Russel, 2001). Ethidium bromide (1 %) was added @ 0.5µl /100ml. The wells were

charged with 5µl of DNA preparations mixed with IX Bromophenol Blue dye.

Electrophoresis was carried out at 5V/cm for 20 min at RT and then the DNA was

visualized under UV transilluminator.

3.13 Polymerase chain reaction

The conventional PCR was carried out to ensure the specificity of the primers in

amplifying the gene of interest. The PCR product was subjected for gel electrophoresis,

to check the size of the amplicon. The Real Time PCR was carried out to quantify MDV-

1 DNA in vaccinated-challenged and unvaccinated challenged control birds.

62

3.13.1 Materials

3.13.1.1 Clinical samples

Feathers were collected from the six birds in each group at different intervals as

mentioned in Table 2. A minimum of ten feathers from each bird were collected into a

sterile polythene zip lock bag on days 7, 14, 28, 43 and 61 post challenge. The feathers

were stored at -20°C until use.

3.13.1.2 Reference MD viruses

a) Cell free HVT vaccine virus commercially available from Ventri Biologicals Ltd.,

Pune, India.

b) Cell associated HVT+SB-1 vaccine virus commercially available from Ventri

Biologicals Ltd., Pune, India.

3.13.1.3 Equipments / reagents (End point PCR)

a) Palm cycler, CGI 960 (M/s. Corbett Research, Australia)

b) Micropipettes (Thermo scientific, Finland)

c) Thin walled PCR tubes of 200 µl capacity (Axygen)

d) Red Taq PCR Master Mix (Bangalore genie) containing

i) 0.05U/µl TaqDNA polymerase (recombinant) in reaction buffer,

ii) MgCl2 (4mM) and

iii) dNTPS (0.4 mM each).

e) Agarose gel (1.8%)

f) Tris - EDTA (TE) buffer

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3.13.1.4 Primers

Three sets of primers for Meq (MDV-1), SORF1 (HVT) and α2 (VI) collagen

(chicken) genes as reported by Islam et al. (2004) was made use in real-time PCR. α2

(VI) collagen (chicken) gene was used as an internal standard to determine the relative

expression of Meq and SORF1 (HVT) genes. The specificity of the primers was checked

with the Basic Local Alignment Search Tool (http: //www.ncbi. nlm.nih.gov/BLAST). All

the three sets of HPLC purified primers (Table 3) were synthesized at Bioserve Pvt. Ltd.,

Hyderabad, supplied in lyophilized form. The primers were reconstituted prior to use in

TE buffer to get a concentration of 20 pmol/µl.

3.13.1.5 Template DNA

The DNA concentration was determined and samples were diluted upto the final

concentration of 100 ng/µl with sterile MiliQ water and stored at - 20°C. DNA used for

PCR was one µl per reaction.

3.13.2 Method (End point PCR)

PCR was carried out in a final reaction volume of 25 µl using 200 µl capacity thin

walled PCR tubes. A reaction mixture was prepared as per the details given in Table 4.

PCR tubes containing the mixture were tapped gently and spun briefly. The PCR tubes

with all the components were transferred to thermal cycler (Palm cycler, CGI 960 (M/s.

Corbett Research, Australia). The PCR conditions used for all the three sets of primers is

shown in table 5. All PCR runs included three non-template DNA samples as negative

controls.

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Table 3. Details of the primers used in PCR and Real Time PCR

Sl. No. Primers* Primer sequence Targeted gene

location Amplicon Size (bp).

Accession No.

1 Meq (MDV-1) [F] 5'-GGA TCG CCC ACC ACG ATT ACT ACC-3'

147386-147705 ~ 400 NC_002229.3 [R] 5'-ACT GCC TCA CAC AAC CTC ATC TCC-3'

2 Meq (MDV-1) [F] 5’-GGA GCC GGA GAG GCT TTA TG-3’

1239-1306 68 M89471 [R] 5’-ATC TGG CCC GAA TAC AAG GAA-3’

3 SORF1 (HVT) [F] 5’-GGC AGA CAC CGC GTT GTA T-3’

130934-131010 77 NC_002577 [R] 5’-TGT CCA CGC TCG AGA CTA TCC-3’

4 α2(VI) Collagen (chicken)

[F] 5’-GGG AAC TGG AGA ACC CAA TTT T-3’ 2360-2435 75 X56595

[R] 5’-CGT GCC GCT GTC TCT ACC AT-3’

* Meq gene unique for MDV-1, absent in MDV-2 and HVT

SORF1 is a unique putative gene encoded within the HVT genome

α2 (VI) collagen (chicken) gene was used as an internal control gene

65

Table 4. Composition of PCR mixture

Components Quantity

2X PCR Master Mix 12.5 µl

Forward Primer (20 pmole/µl) 0.5 µl

Reverse Primer (20 pmole/µl) 0.5 µl

DNase-RNase free water 9.5 µl

DNA Template 2.0 µl

TOTAL 25.0 µl

Table 5. Thermal cycling conditions

Steps Temp Time

Initial denaturation 95°C 5 min

Denaturation 95°C 15 sec

35 cycles Annealing 60°C 1 min

Extension 72°C 2 min

Final extension 72°C 10 min

66

3.13.3 Visualization of PCR product

To confirm the targeted PCR amplification, ten µl of the PCR product from each

tube was mixed with two µl of 6X gel loading buffer and electrophoresed along side 50bp

DNA molecular weight marker (Bangalore Genei) in 1.8 per cent agarose gel containing

ethidium bromide (0.5µg/ml) at constant 80V for 30 min in 0.5X TE buffer. The

amplified product was visualized under UV light and documented by gel documentation

system (Alpha Imager, Alpha Infotech, USA).

3.14 Real Time PCR

3.14.1 Equipments / reagents

a) Applied Biosystems 7300 real time thermal cycler

b) 96 well real time PCR plates (Axygen)

c) PCR film-self adhesive (Eppendorf Germany)

d) Micropipettes

e) 1.5 ml microcentrifuge tubes for preparing Master Mix

f) MaximaTM SYBR Green 2× Master Mix (Catlog # K0221 Fermentas Inc, USA)

containing

i) MaximaTM Hot start Taq DNA Polymerase

ii) MaximaTM SYBR Green qPCR Buffer

iii) SYBR Green I

iv) ROX passive reference dye

v) Primers –mentioned in Table 3

vi) Nuclease free water

vii) DNA extracted from feather tips

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3.14.2 Real Time PCR protocol

3.14.2.1 Plate preparation

The reaction was prepared in a 96-well plate (96-Well Optical Reaction Plate,

Barcoded, serial No. 4306737, Applied Biosystems, USA), using SYBR green qPCR

Master Mix (Fermentas Inc. USA; Catlog No. K0221). The final volume per well was

25 µl.

3.14.2.2 Determination of primer efficiencies

For the comparative CT method of relative quantitation to be valid, the variation of

the efficiency of amplification between the target and that of the active reference (House

keeping gene) must not exceed five per cent (Real time PCR Handbook, University of

Illinois, 2003).

Primer efficiency was determined as per the protocol given by Fraga et al. (2008).

A tenfold dilution (1, 1:10, 1:100, 1:1000 and 1:10,000) of 100 ng of DNA from feather

tip sample that was detected as positive for Meq, SORF1 and α2 (VI) collagen (chicken)

gene was tested using real-time PCR to obtain a standard curve. Threshold Cycle (CT)

value was plotted as a function of the dilution to draw a linear regression line through

the plot. The slope of the regression line was calculated using Graph Pad Prism software

version 5.

1. Ten fold Serial dilution of a 100 ng of DNA (100 to 10-4) representing five data points

was prepared in triplicate.

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2. A separate PCR Master Mix for each of Meq, SORF1 and α2 (VI) collagen gene was

prepared in 1.5 ml micro centrifuge tubes as mentioned in table 6. The Master Mix

(24 µl) was dispensed into each of the wells of the real time PCR plate in triplicates.

3. One µl of the serially diluted DNA template was added to each reaction well in PCR

plate.

4. PCR plates were placed in AB 7300 Thermal Cycler and PCR reaction was run to

determine CT values for each dilution.

5. CT values versus the logarithm of the concentration of DNA were plotted.

6. Slope of the line was used to determine the efficiency of the PCR using equation.

E = (10 –1/slope –1) × 100

7. Amplification efficiency values for each primer set were recorded.

3.14.2.3 Determination of relative primer efficiencies

The CT values obtained for each of the primers at different dilutions of template

DNA was used to calculate relative primer efficiencies. The CT values obtained were

compared by subtracting the CT values of primer set for target genes [Meq and SORF1

(HVT)] from primer set of house keeping gene (α2 (VI) Collagen) and the difference

was plotted against the logarithm of the template amount (a tenfold serial dilution). If the

slope of the resulting line is <0.1, the amplification efficiencies are comparable.

3.14.2.4 Real-time polymerase chain reaction

AB 7300 Real Time Thermal Cycler was used to carry out real-time PCR

amplification reactions as per the protocol given by Fraga et al. (2008).

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Table 6. Various components used in SYBR Green based Real Time PCR

Sl. No. Components Quantity

1 Fermentas SYBR Green PCR Master Mix (2X) 12.5µl

2 Forward Primer (20 pmol/µl) 0.3 µl

3 Reverse Primer (20 pmol/µl) 0.3 µl

4 Template DNA 1.0 µl

5 Nuclease Free water 10.9 µl

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i. SYBR Green 2× Master Mix, primers, template DNA, nuclease free water and 1.5 ml

microcentrifuge tubes were kept on ice.

ii. PCR mix was prepared in 1.5 ml microcentrifuge tubes separately for each primer as

shown in Table 6. Master Mix was mixed thoroughly and 24 µl of it was dispensed to

each wells of Real time PCR 96 well plate.

iii. Template DNA was added to each reaction well and the plate was sealed with

adhesive tape.

iv. AB 7300 Real Time Thermal Cycler was set up and programmed to the conditions

mentioned in Table 7.

v. Reaction plate was placed in the Thermal Cycler and PCR reaction was run to

determine CT values for each sample.

vi. Once the reaction cycles were completed, the dissociation curve analysis was

obtained and the results were analyzed using ∆∆CT method (Livak and Schmittgen,

2001).

3.14.2.5 Analysis of real-time data

Relative quantification of the Meq and SORF1 gene was carried out using the

comparative CT (∆∆CT) method. The raw CT values for the Meq (target) gene and the α2

(VI) collagen (chicken) (house keeping gene) gene for the three groups (Control,

HVT+SB-1 and HVT) and CT values of SORF1 gene for the HVT and HVT+SB-1 group

of birds were exported from the ABI 7300 SDS program into Excel (Microsoft Windows

XP). The relative fold change in the expression of a particular gene, as reflected in DNA

level, between two different groups (either HVT+SB-1 v/s controls or HVT v/s controls

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Table 7. Thermal cycling conditions of SYBR Green based Real Time PCR

Steps Temp Time

Initial denaturation 95°C 5 min

Denaturation 94°C 15 sec 40 cycles

Annealing and Extension 60°C 1 min

72

or HVT+SB-1 v/s HVT ) was calculated for each interval (7th, 14th , 28th, 43rd and 61st

day pc) as per the steps mentioned below.

i. The target gene was normalized to the Housekeeping gene (endogenous control):

∆CT = CT target gene – CT endogenous control.

ii. The sample was normalized to the calibrator sample. In HVT+SB-1 v/s control and

HVT v/s control comparison, the control was taken as calibrator whereas HVT+SB-1

and HVT were considered as samples. In HVT v/s HVT+SB-1 comparison, the

calibrator was HVT and the sample was HVT+SB-1. 

∆∆CT = ∆CT sample – ∆CT calibrator

iii. The amount of target, normalized to an endogenous reference and relative to a

calibrator, is given by:

Fold change = 2 –∆∆CT

3.15 Histopathology

3.15.1 Collection of tissue specimen

Tissues like liver, spleen, kidney, proventriculus, bursa, thymus and sciatic nerves

were collected into 10 per cent neutral buffer formalin, from randomly selected six birds

from each of the three groups; 1, 2 and 3 (Table 2) on 7th, 14th, 28th, 43rd and 61st dpv.

3.15.2 Histopathological examination

The formalin fixed tissues were processed by paraffin wax embedding method of

tissue sectioning. The sections were cut at 6-8 microns thickness with automatic section

cutting machine (SLEE-MAINZ, Germany) and were stained with haematoxyline and

eosin (H & E) stain (Luna, 1968). The H & E stained slides were read under microscope

and histopathological changes were recorded.