NEWCASTLE DISEASE

THERMOSTABLE VACCINE AGAINST 

NEWCASTLE DISEASE 

Dr. J. John Kirubaharan 

Fulbright Visiting Scholar, CVM, MSU, US 

Associate Professor (Veterinary Microbiology) 

Madras Veterinary College, TANUVAS, 

Chennai – INDIA

Organization of the talk 

• How did we develop the vaccine? 

• How in house validation was carried out? 

• How the demands of the industry was satisfied? 

• What are the utilities of the vaccine? 

• What we are doing now and where we go from here?

Stages in development 

• Carried out in 7 Stages 

• Stage 1 – Selection of a suitable strain / isolate 

• Stage 2 – Studies on thermostabilization 

• Stage 3 – Production & Standardization of the vaccine 

• Stage 4 – Laboratory trials - post-vaccinal responses 

• Stage 5 – Genetic characterization of the seed virus 

• Stage 6 – Field trials 

• Stage 7 – Technology transfer

Stage 1 – Selection of isolate 

• Isolation of ND from apparently normal birds, 

which were not vaccinated 

• From 160 cloacal swabs - 16 isolates 

• All isolates characterized 

• ICPI values ranged from 0.14 to 1.75 

• ICPI values correlated with the sequence of FPCS 

region 

• Selection criteria for identification of seed virus 

• Based on OIE (2000), EEC norms – with lowest ICPI 

• D58 – ICPI 0.14 (Lower than F and LaSota)

Stage 2 - Thermostabilization 

• Thermostability studies 

• Protocol – Aidni Idris et al, 1991 

• Exposure of virus to temperature ranging from 25- 

56 o C and passaging in eggs after every temperature 

treatment 

• Duration of temperature treatment 

• At 25 o C – 36 days 

• At 37 o C – 29 days 

• At 56 o C – 5 hrs 

• Virus presence confirmed by HA and EID 50 estimation 

at regular intervals 

• Thermostable clones were finally amplified in ECE 

• Virus titration - EID 50

Stage 2 – Thermostabilization 

HA TITRE - Log 10 values 

25 o C 37 o C 56 o C 

2.4082 3.0103 2.7093 

EID 50 / 0/1ML 

INTITIAL 

THERMOSTABILIZATION 

25 o C 37 o C 56 o C 

FINAL 

10 8.5 10 4.5 10 5.5 10 2.5 10 7.5

Stage 2 – Thermostabilization 

• Checking the thermostability 

• Checked by stability of haemagglutinin at 

various temperature 

• No variations in HA titre at 4 o C, 25 o C and 37 o C 

• HA activity dropped by one well at 56 o C 

• Was also validated by a Government vaccine 

manufacturing unit

Stage 3 – Production & Standardization 

• Virus purified by plaque purification method 

• Master seed virus (MSV) prepared as per OIE 2000 - 

titre of MSV EID 50 10 8.0 /0.1 ml 

• Working seed virus (WSV) produced as and when 

required 

• Vaccine production 

• Produced in such a manner that every dose 

contains EID 50 10 6.5 /0.1 ml

Stage 3 – Production & Standardization 

• Followed the standards specified in OIE 2000 

• Standardized for safety, purity and potency 

• Safety – 

• 10 doses given to unprimed birds 

• No harmful effects, symptoms or death 

• Purity – Bacterial, fungal and viral (IB) 

• Free from aerobic, anaerobic, fungal and negative for 

IB 

• Potency – No Challenge based potency 

• 1/10th of dose given to birds 

• Birds developed serum antibody titre of > 16 at 21 days 

(Group average)

Stage 4 – Post vaccination trials 

• Laboratory trials # 1 

• Unprimed and mat. Ab negative chicks - 120 

• Primed with D58 

• Sero-conversion studies 

• HI and ELISA 

• Serum collection – 0,7,14,21 PV and 7,14 PC 

• Four fold increase in serum antibody titre 

• Challenged with virulent NDV (obtained from IVPM) – 10 6 

EID 50 

• 100 % protection in vaccinated and varying mortality in 

control birds with exhibition of symptoms

Log 

HI titre 

Stage 5 – Post vaccination trials (Lab) 

Immune response in unprimed mat.Ab negative chicks 

3 

2.5 

2 

1.9115 

2.3995 

1.5 

1 

1 

0.5 

0 

0 7 21 

Days post vaccination 

Mean HI titre


• Laboratory Trial # 2 

• Vaccination of chickens with thermostable D58 vaccine 

• Assessment of CMI by under agarose leucocyte 

migration inhibition assay and colorimetric 

blastogenesis assay (in the absence of humoral 

response). 

• Assessment of mucosal immune responses by HI and 

IgA ELISA. 

• Assessment of humoral immune responses by HI and 

IgG ELISA. 

• Challenge of experimental and control chickens with 

virulent virus.


Laboratory Trial # 2 

• Laboratory trial 2 

• Scope – To assess mucosal, CMI and humoral responses 

• Vaccination by intra ocular and intranasal route 

• Number of birds : 36 

• Unvaccinated control : 18 

• B cell ablated and vaccinated : 9 

• B cell non-ablated and vaccinated : 9 

‣ Slaughter days : 7, 14, 21 

‣ Samples collected 

‣ Harderian gland, lachrymal fluid, tracheal swab, intestinal 

wash, bile, spleen and serum 

‣ Challenge : 28 th day

Numerous lymphoid 

follicles



‣ Colorimetric blastogenesis assay (CBA) 

• The Stimulation index (SI) values started to increase from 7 th 

day post vaccination in both ablated and non ablated groups 

• No SI values could be recorded in unvaccinated controls 

• Statistically significant difference was observed between SI 

values on days 0 and 7 indicating the setting of CMI response 

after vaccination 

‣ Under agarose leucocyte migration inhibition technique (LMIT) 

• The pattern of percentage migration inhibition values were in 

similar pattern as CBA

Proliferation Index 

Percentage Migration inhibition 



Cell Mediated Immune Response - CBA - LNDV 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

-0.05 

-0.1 

0 7 14 21 

Days Post-Vaccination 

Ablated-Vaccinated 

Non-ablated-Vaccinated 

Ablated_Not Vaccinated 

Non-ablated-Not vaccinated 

Cell Mediated Immune Response - LMI - LNDV 

30 

25 

20 

15 

10 

5 

0 

0 7 14 21 

Days Post-vaccination 

Ablated - Vaccinated 

Non-ablated - Vaccinated 

Ablated - Not vaccinated 

Non-ablated - Not vaccinated



‣ Mucosal immune response - IgA ELISA 

• Statistically significant difference was observed in OD 

values between vaccinated (both ablated and non ablated 

groups) and unvaccinated control birds on day 21 

• Indicates the mucosal immune response in vaccinated 

group 

‣ Humoral immune response 

‣ IgG ELISA and HI test 

• Statistically significant difference observed in OD values 

between vaccinated and unvaccinated birds and also 

between ablated and non ablated birds of vaccinated groups 

indicates the presence of humoral immune response in 

vaccinated group

Mean OD Values 




Mucosal Immune Response - IgA OD values - LNDV 

(ABLATED & VACCINATED) 

0.300 

0.250 

0.200 

0.150 

0.100 

0.050 

0.000 

7 14 21 

Days Post Vaccination 

Hardarien Lachrymal Trachea Intestine Bile Serum 


(NON-ABLATED & VACCINATED) 

0.300 

0.250 

0.200 

0.150 

0.100 

0.050 

0.000 

7 14 21 


Hardarien Lachrymal Trachea Intestine Bile Serum






(ABLATED & NOT-VACCINATED) 

0.300 

0.250 

0.200 

0.150 

0.100 

0.050 

0.000 

7 14 21 


Hardarien Lachrymal Trachea Intestine Bile Serum 


(NON-ABLATED & NOT VACCINATED) 

0.300 

0.250 

0.200 

0.150 

0.100 

0.050 

0.000 

7 14 21 


Hardarien Lachrymal Trachea Intestine Bile Serum

Stage 5 – Genomic characterization 

• Gene sequences of D58 strain available at GenBank 

• All the six genes (both CDS and UTR) have been sequenced 

• NP gene 

• DQ839549; EF442116 

• P gene 

• EU419882 

• M gene 

• GQ 184565 

• F gene 

• EU330230 

• HN gene 

• EU305607 

• L gene 

• GQ 2220701

Stage 5 – Genetic characterization of vaccine seed - 

Phylogenetic tree (NP gene)


Phylogenetic tree (P gene)


Phylogenetic tree (M gene) 

79 

98 

55 

99 

D58 

Lasota 

JL1 

Clone 30 

B1 

B1 - Takaaki 

100 95 

VG-GA 

63 

96 

66 

Roakin 

BeaudetteC 

Texas-GB 

DE-R49 

99 

100 

PHY-LMV42 

100 

NDV-08-04 

53 

PHY-LM1 

V4HB92 

89 

90 

89 

100 

I2 

I2 progenitor 

96 

ULSTER67 

Mukteswar 

90 

83 

99 

Australian V4 

Herts 33 

Italien 

Anhinga 

90 

99 

ZJ1 

NA1 

Sterna 

Malay sian 

0.01


Phylogenetic tree (F gene) 

LaSota 

JL-1 

HB92 isolate V4 

D58 

clone 30. 

B1 Takaaki 

B1 

ITA/45 

Beaudette C 

AQI-ND026 

Tex/48 

chicken/USA/Roakin 

Que/66 

D26/76 

I-2 

I-2progenitor 

Ulster/67 

F48E9 

Muktesw ar 

Texas) 

Herts/33 

PHY-LMV42 

Italien 

0.01


Phylogenetic tree (HN gene) 

LaSota 

Clone 30 

JL-1 

HB92-V4 

D58 

B1 

B1 Takaaki 

Beaudette C 

Tex/48 

AQI-ND026 

Roakin 

Texas 

ITA/45 

Italien 

HERTS/33 

Muktesw ar 

F48E9 

ULSTER/67 

PHY-LMV42 

D26.76 

QUE/66 

I-2 

I-2 PROGENITOR 

0.01


Phylogenetic tree (L gene) 

99 

81 

100 

B1 

D58 

Lasota 

Lentogenic 

96 

55 

B1 TAKAII 

Beaudette C 

Lentogenic 

Velogenic 

VG-GA 

Others 

100 

100 

88 

CLONE30 Lentogenic 

KBNP Others 

JL1 PPM 

83 

TEXAS GB 

Velogenic 

61 

100 

PHY-LMV42 

PHY-LM1 

Others 

87 

ULSTER67 

HB92-V4 

Apathogenic 

80 

100 

I2 

I2 PROGENITOR 

Apathogenic 

100 

ANHINGA Mesogenic 

STERNA Velogenic 

75 

96 

100 

ZJ1 

NA1 

Others 

46 

100 

Herts33 

Italien 

Velogenic 

53 

40 

MUKTESWAR Mesogenic 

F48E9 Others 

AUSTRALIAN VICTORIANApathogenic 

100 

DE-R49 

NDV08-CHINA 

Others 

0.01

Stage 6 – Field trials 

• Use of vaccine seed D58 as priming vaccine for broilers instead of F 

strain 

• 8500 doses 

• Vaccine given through nostrils 

• 10 th day primed with D58 

• 21 st day serology 

• HI titre - 2 4 to 2 7 

• ELISA titre – More than log 2.7 

• Titre maintained till date of lift 45 th day of age (35 days after 

vaccination) 

• One dose provided sufficient immunity

Log 

Titre 

Stage 6 – Field trials 

3.5 

3 

3.0066 

2.5 

2 

1.9605 

1.5 

1 

0.5 

0 

1 1 

0 7 21 

Days 

1.4 

Vaccinated 

Control

Trial 1 

Stage 6 – Field trials (Industry) 

• ND D 58 vaccine field trial at Udumalpet 

• Farm A 

Sl. No. Age T1-Trial T2-Control 

1 Day-old H 120 Spray/Ocular H 120 Spray/ocular 

2 Day 5 ND D 58 Ocular F Strain or B1 ocular 

3 Day 12 Gumboro Ocular drop Gumboro ocular drop 

4 Day 21 ND D 58 Ocular Lasota ocular

Trial 1 


• ND D 58 vaccine field trial at Udumalpet 

• Farm B 

Sl. No. Age T1-Trial T2-Control 

1 Day-old H 120 Spray/Ocular H 120 Spray/ocular 

2 Day 5 ND D 58 Ocular F Strain or B1 ocular 

3 Day 12 Gumboro Ocular drop Gumboro ocular drop 

4 Day 21 Lasota Ocular Lasota ocular

Trial 1 


Performance index Farm A 

Particulars Trial Control Difference 

No. Of Chicks Placed 2750 2750 

Mean Age of lifting (Days) 44.92 45.27 -0.35 

Average Daily weight gain (gms). 43.28 43.43 -0.15 

Average weight(Kg) 1.944 1.966 -0.022 

Mortality% 5.75 5.27 +0.48 

FCR 2.207 2.217 -0.01 

Medication Cost/Kg 1.16 0.53 +0.63 

Production Cost/Kg 34.47 33.85 +0.62 

European Economic Factor(EEF) 185 186 -1

Trial 1 


Performance index Farm B 

Particulars Trial Control Difference 

No. Of Chicks Placed 3548 3547 

Mean Age of lifting (Days) 45.35 46.13 -0.78 

Average Daily weight gain (gms). 41.59 40.54 +1.05 

Average weight(Kg) 1.886 1.87 +0.016 

Mortality% 12.63 12.49 +0.14 

FCR 2.217 2.288 -0.071 

Medication Cost/Kg 1.38 0.675 +0.705 

Production Cost/Kg 35.57 35.77 -0.2 

European Economic Factor(EEF) 164 155 +9

Trial 1 


Seroconversion

Trial-II 


Age Group1 Group2 Group3 Route 

Farm A 

Day 1 IB H120 IB H120 IB H120 

Hatchery 

Spray 

Day 5 ND D58 ND D58 Live B1 Eye drop 

Day 13 IBD IBD IBD Eye drop 

Day 21 ND D58 LaSota LaSota Eye drop 

Farm B 

Day 5 ND D58 ND D58 Live B1 Eye drop 

Day 13 IBD IBD IBD Eye drop 

Day 21 ND D58 LaSota LaSota Eye drop

Trial-II 

Farm A 


Chicks 

housed 3600 3720 13560 

Mean age 43.01 43.01 43.01 

Day gain 48.67 45.01 47 

Avg wt 2.10 1.910 2.029 

Mort% 5.46 4.56 6.17 

FCR 2.000 2.060 2.190 

Med cost 0.51 0.52 0.54 

Podn cost 30.87 31.94 33.5 

EEF 230 204 202

Trial-II 

Farm B 


Chicks 

housed 2100 2460 2405 

Mean age 43.09 43.09 43.09 

Day gain 50 48 49 

Avg wt 2.210 2.078 2.042 

Mort% 6.47 6.2 7.81 

FCR 2.000 2.020 2.122 

Med cost 0.5 0.5 0.51 

Podn cost 30.56 32.61 31.18 

EEF 239 205 223

Stage 7 – Transfer of Technology 

• Sustained effort of the University and increasing the quantity of 

data on D58 strain 

• Interest evinced by Globion of Suguna Group 

• Controlled field trials with 50000 doses 

• The sero-conversion of TANUVAS vaccine was in 

acceptable range. 

• There is no respiratory complication in birds vaccinated 

with TANUVAS vacccine 

• TANUVAS vaccine does not affect the performance 

parameters of broilers 

• Production cost may be curtailed on large scale 

production 

• Transfer on non-exclusive basis has been carried out

Immunity to turkeys 

• Incidence of ND in turkeys 

• Present use of F and LaSota vaccines 

• Experimental trial design 

• Three groups of day old 36 Beltsvile small white turkeys 

• Groups – 1: 

• 12 birds receive F vaccine on day 10 as eye/nasal drops and LaSota in water 

as booster on day 42; 

• 12 birds received F vaccine alone on day 10 as eye/nasal drops 

• 12 birds remain as unvaccinated controls 

• Group – 2: Same design as Group 1 except that instead of F vaccine as 

nasal/eye drop LaSota or thermostable D58 strains were used 

• Humoral immune response – studied by HI and ELISA from serum 

sample collected at weekly intervals

Log 10 titre 


RDVF Vs D58 Vs LaSOTA - ELISA 

7.000 

6.000 

5.000 

4.000 

3.000 

2.000 

1.000 

0.000 

1 2 3 4 5 6 7 8 9 10 11 12 

Age in w eeks 

RDVF+LaSOTA D58+LaSOTA LaSota+LaSota

Log 10 titre 


RDVF Vs D58 Vs LaSOTA - HI 

2.500 

2.000 

1.500 

1.000 

0.500 

0.000 

-0.500 

1 2 3 4 5 6 7 8 9 10 11 12 

Age in w eeks 

RDVF+LaSOTA D58+LaSOTA LaSota+LaSota

Log 10 Titre 


6.600 

6.400 

6.200 

6.000 

5.800 

5.600 

5.400 

5.200 

5.000 

4.800 

4.600 

4.400 

4.200 

4.000 

3.800 

3.600 

3.400 

3.200 

3.000 

2.800 

2.600 

2.400 

2.200 

2.000 

1.800 

1.600 

1.400 

1.200 

1.000 

0.800 

0.600 

0.400 

0.200 

0.000 

-0.200 

-0.400 

1 2 3 4 5 6 7 8 9 10 11 12 

Age in w eeks 

D58+La D58 No Vacc D58+La D58 No. Vacc

Development of Vaccine 

• Vaccine production 

• Lactose pellet technology 

• Lactose 33%, Starch 33%, Virus 34% and 

Amaranthus (food grade) dye 

• Air dried and stored in containers 

• Approximately 5mm 

• EID50 of one grain - 10 6.5 

• Dosage 

• 2 grains per bird

Standardization of Vaccine 

• Followed the standards specified for live ND vaccine – 

OIE 2000 

• Standardized - safety, purity and potency 

• Safety – 

• 20 grains given to unprimed birds 

• No harmful effects, symptoms or death 

• Purity – Bacterial, fungal and viral (IB) 

• Free from aerobic, anaerobic, fungal and negative for IB 

• Potency – Challenge based potency 

• Single grain given to birds 

• Birds developed serum antibody titre of > 16 at 21 days

Post vaccination trials (Lab) 

• Three types of trials conducted 

• Unprimed and mat. Ab negative chicks 

• Primed with D58 and Oral vaccine given as booster 

• Primed at hatchery + oral vaccine given instead of K 

• Sero-conversion studies 

• HI and ELISA 

• Serum collection – 0,7,14,21 PV and 7,14 PC 

• Four fold increase in serum antibody titre 

• Challenged with virulent NDV (obtained from IVPM) – 10 6 

EID 50 

• 100 % protection in vaccinated and varying mortality in control 

birds with exhibition of symptoms

Log 

titre 

Log2 

Titre 

LOG 

titre 


Immune response in unprimed mat.Ab negative chicks 

3 

2.5 

2 

1.5 

1 

0.5 

0 

2.3995 

1.9115 

1 

0 7 21 

Days post vaccination 

3 

2.5 

2 

2 

Oral pellet vaccine instead of K 

2.4136 

2.7180 

2.3780 

Mean HI titre 

1.5 

1 

3.5 

3 

2.5 

2 

D58 as primiving vaccine and oral pellet vaccine as 

booster 

3.0066 

2.3372 

2.8028 

0.5 

0 

0 7 21 35 

Days post vaccination - 0, 7, 21, 35 

1.5 

1 

1 

0.5 

0 

0 21 28 42 

Days

Vaccine consumption pattern 

• Proventriculus 

• Lymphoid cell aggregation – 2 nd day 

• Mononuclear infiltration – 4 th day 

• Diffused lymphoid aggregation – 5 th day 

• Spleen 

• Increase in germinal centres – 2 nd day 

• Trachea 

• Mild hypertrophy – 6 th day 

• Lungs 

• Mild congestion with lymphoid aggregation – 2 nd day 

• Ileocaecal junction 

• Mild lymphoid depletion – 6 th day

Post vaccination trials (field) 

• Controlled field trials 

• Desi birds 

• Erode Dt, Namakkal Dt, Kancheepuram Dt 

• >3000 doses 

• Unvaccinated birds varying serum antibody titres were 

observed 

• Four fold increase in titre 

• So far 

• No reports of untoward post-vaccinal reaction 

• Still supplying

Other work carried out 

• Phylogenetic analysis of D58 using all the six genes 

• Cloned and expressed NP, P, HN and F genes of D58 isolate of NDV 

• Antibody profile against individual proteins of NDV under different 

conditions 

• Single serum dilution ELISA to predict antibody titre in the serum of 

birds vaccinated against NDV 

• Utility of the D58 vaccine in Japanese quails 

• Isolation of NDV from water birds

Summary 

• Developed a theromostabilized live Newcastle disease vaccine 

• The vaccine was found to be safe, pure and potent in lab and 

controlled field trials 

• The vaccine seed was also tested under field conditions 

• priming vaccine in broilers gave satisfying immunity 

without affecting production parameters 

• produce satisfactory immunity in turkeys 

• Molecular characterization studies also proved the low 

pathogenicity of seed correlating with ICPI value

Log10 titres 

11 

13 

15 

17 

19 

21 

23 

25 

27 

29 

31 

33 

35 

37 

39 

41 

43 

45 

47 

49 

51 

53 

55 

57 

59 

61 

63 

65 

67 

69 

14.000 

12.000 

10.000 

8.000 

6.000 

4.000 

2.000 

0.000 

-2.000 

1 

3 

5 

7 

9 

Sample # 

WVP HN NP P F HI

Acknowledgements 

• To the Tamil Nadu Veterinary & Animal Sciences University and the 

Indian Council of Agricultural Research 

• To the coworkers 

• Ramani Pushpa, Ananth, Varalakshmi, Logeswari, Ponnusamy, Vivitha and 

Sadhwi Sharma 

• Prabhakar, Indra, Latha

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