Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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ASSESSMENT OF IMMUNITY INDUCED BY NEWCASTLE DISEASE
VIRUS VACCINES AND DETERMINE THE BEST VACCINATION
PROGRAM IN BROILER CHICKEN
Douaa Y. Talib*, Hazim T. Thwiny**
*Department of Animal Production, College of Agriculture, University of Sumer, Thi Qar, Iraq
**Department of Microbiology, College of Veterinary Medicine, University of Basrah, Basrah,
Iraq.
Corresponding author : hazimthwiny@gmail.com
Key words: chicks, vaccination, hem agglutination, Newcastle diseases.
ABSTRACT
The objectives of current research were evaluate the efficacy of commercially available
Newcastle disease vaccine and determine the best vaccination program. A total number of 150
one-day-old unvaccinated chicks were divided equally into 5 groups. One vaccination program
were used for each group which differ from each other while group 5 was unvaccinated control
group. Serum were collected from all groups and five chickens from each group were sacrificed.
Afterward immunization HI geometric mean titer (GMT) rates were observed that both
seroconverted birds in Group 1 to Group 4 have risen statistically significantly, with statistical
significant changes. (p < 0.05). However, the birds in group 4 which had the best HI titers
(147). The levels of ChIFN-γ was measured by ELISA, there were also higher in the vaccinated
groups (group 1, 2, 3 and 4) than in the non-vaccinated group. Group 4 also had the best ChIFN-
γ level. The higher values of lymphoid organs (spleen, Bursa of Fabricius and thymus) indices
were in vaccinated groups are compared to non-vaccinated groups, while between vaccinated
groups there was no significant different (P < 0.05). Commercial ND vaccines are effective and
vaccination scheme of group 4 (live ND vaccine at 7th day of age by eye drop as primary
vaccine followed by live ND vaccine at 21st day of age by drinking water as booster dose) has
more protective effects in broiler chicken.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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INTRODUCTION
Newcastle disease (ND) is an extremely infectious and generally lethal viral poultry
disease infecting mostly chickens, turkeys, and other birds (1). It originated by (NDV); an avian
paramyxovirus type1 NDV subdivided into 3 path types according to the virulence in fowls
which are: lentogenice, mesogenice and velogenice (2, 3). The clinical signs include respiratory
signs such as coughing, panting, sneezing and rales. Additional signs such as falling wings,
tedious legs, enlargement of tissues around neck and eyes, twisted neck, rotating and interruption
of egg creation (4). Prevention and controlling of the infectious disease in poultry industry by
vaccines applied . Consequently, an appropriate strict vaccination program necessity be followed
toward prevention the occurrence of clinical sings at farm house level (5). Though, ND
immunization programs include both of inactivated and attenuated vaccines to improve
protection from infectious diseases. Attenuated vaccines organized with lentogenic strains of
NDV and chemically deactivated strains, mixed with adjuvant are commonly used (1,6). In fowl,
live vaccines applied by eye dropping or orally to stimulate defensive local immunity managed
by (IgA) antibodies (7). While killed vaccine applied by injection has been provide great levels
antibodies creating humeral immunity which will defend the chicks from contagion with NDV.
Disadvantage of inactivated vaccines are not stimulate mucosal immunity in the respiratory and
digestive tracts because oily feature, then the immunity is established slowly. Other disadvantage
of it is expensive and hard to applied more than live vaccines (8). Despite of presence of many
commercial types of live and inactivated vaccines worldwide, ND is stay a great risk to the fowls
trade in developed countries containing Iraq (9). In Iraq, several live vaccines having lentogenic
strains of NDV such as LaSota are carried by several importers but efficiency of these vaccines
in related with climatic state, dissemination and transport are not every time inspected
appropriately and carefully either by the trader or by the handler. Occasionally, the owner are
doubtful about the efficacy of those imported NDV vaccines. Many of related questioned are
faced the veterinarians in this country such as the immunogenicity, virus titer, stability and like
other qualities of those vaccines. Aim of this study is to estimate the efficiency of obtainable ND
live and killed vaccine and select an immunization program that will develop thigh immune
response.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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MATERIALS AND METHODS
Chicks: A whole number of 150 1 day of age unvaccinated mixed-sex birds procured from a
local commercial field. Totally birds were cultivated in their cages and nourish and water were
provided ad-libitum during the experiment. Beforehand immunization the fowls subdivided in to
5 groups, each one involves 30 birds. The chickens of fifth group were reserved as control
without vaccine.
Commercial ND vaccines: Two NDV vaccines are castoff in research. These are Izovac NDV
LaSota (live vaccine) and Nobilis ND BROILER (inactivated vaccine) manufactured
commercially are bought. By using lentogenic strain La So ta for both immunizing and
formulating Ag.
Experimental design: The experimental design as show in Table1. The experimentation are
persisted 35 days. The vaccination schedule (created on the local recommended regimens) was as
follows:
The G 1: thirty chicks were vaccinated at 7th days old with killed ND vaccine subcutaneously
then at 21st days old with attenuated NDV vaccine via drinking water.
The G 2: thirty chicks were inoculated at 7th days old with killed ND vaccine subcutaneously
then at 21st days old with attenuated Newcastle disease virus via eye drop.
The G 3: thirty chicks vaccinated at 7th days old with live attenuated Newcastle disease virus
vaccine via drinking water then at 21st days old with live attenuated Newcastle disease virus
vaccine also via drinking water.
The G 4: thirty chicks were vaccinated at 7th days old with live attenuated Newcastle disease
virus via eye drop then at 21st days old with attenuated Newcastle disease virus vaccine via
drinking water.
The G 5: thirty chicks were kept as an unvaccinated control.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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Table (1). Experimental design with vaccination schedule of different groups
Vaccination
day
G 1 G 2 G 3 G 4 G5
(control group)
7th day
ND killed
vaccine
(S/C)
ND killed
vaccine
(S/C)
ND live
vaccine
(D/W)
ND live
vaccine
(I/O)
unvaccinated
21th day ND live
vaccine
(D/W)
ND live
vaccine
(I/O)
ND live
vaccine
(D/W)
ND live
vaccine
(D/W)
unvaccinated
S/C = Subcutaneously, I/O= Intra Ocular, D/W= Drinking Water
Sample collection and sampling schedule: Two ml of blood was obtained aseptically from
wing vein or jugular vein of each chick and let it clot and serum parted using bench centrifuge at
1500 rpm for 10 min. The sera were parted and kept at -20 o C till the serological tests were
carried out. Before applied vaccine collected serum as of 25% from chicks castoff randomly on
seventh day following hatch to evaluate their maternal immune status. Sera samples are collected
before and after 14 days of each immunization and on 7, 21 and 35 days of age from
unvaccinated control. Six chick ens were sampled randomly from each group.
Microplate hemagglutination inhibition (H I) test: Microplate H I implemented to determine
the antibodies level of the obtained sera as of the bird in the five collections. IgG level of the
NDV in the serum was evaluated by H I and cross HI tests in U-bottom micro titer plates using
constant 8 H A units of LaSota strain as antigen with two-fold serum dilutions ( β method). HI
titers equal to or greater than 1/ 16 were considered positive as recommended by the World
Organization for Animal Health (2).
Chicken interferon gamma (ChIFN-g) ELISA assay: Serum samples were tested to determine
the level of ChIFN-g using commercial ELISA kit (Chicken IFN-γ ELISA Kit, Catalog No : EEL-
Ch0026). This ELISA kit applies to the in vitro quantitative determination of Chicken IFN-
γ concentrations in serum of chicken. The INF-γ examine was done based on the ELISA
manufacturer’s protocol.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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Lymphoid organ indexes: On day 35 of age, chicks were separately weighted as of each group
to evaluate their body mass. Five birds as of each collection were sacrificed. Then a thorough
optical assessment, the thymus, bursa of Fabricious and spleen were directly removed,
dehydrated then discretely weight up. Later significant lymphoid organ weight variation
estimated, their directories were planned (10).
Statistical analysis: A documents collected as of these research in numerous groups were
statistically investigated via analysis methods of alteration (ANOVA). Pstatistically significant.
RESULTS
Hemagglutination inhibition (HI) test.
The last dilution of antigen which gave hemagglutination was 1:128 therefor the dilution of
antigen have 8 HAU was 1:16. This antigen titer (8 HAU) was used in hemagglutination inhibition test.
Ab response of the chicks to vaccination patterns were given in Table 2 and figure 1. The mean prevaccination
H I antibody titer was found to be 4.8 at age 7 days then decline to 2.8 and 0 at age 21 and
35 days correspondingly. HI geometric mean titer (GMT) values after immunization firm and it detected
that in 21 day of age (14 days post vaccination) wholly the chicks in the Group 1 to Group 4 sero
converted with statistically substantial improved (p < 0.05).
H I antibody titers in serum that was protecting. However, the birds in group 4 which had a
higher HI titers (179.2). The other groups as the following: 57.6 in group 1, 64 in group 2, 44.8 group 3.
Later the supporter vaccination, the GMT standards documented as 230.4 in group1, 256 in group 2,
115.2 in group 3 and 358.4 in group 4 on day 35 of age (14 days post booster vaccination dose).
Statistically analyzed of HI GMT and the variance between both was substantial (P < 0.05).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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Table (2): GMT of Ab titers against NDV in experimental groups measured by HI test
Group NDV antibody titers
At 7 days(maternal immunAitty 2)1 days At 35 days
Group 1
4.8±1.78
57.6±14.31b 230.40±57.24bc
Group 2 64±0.00b 256±0.00b
Group 3 44.80±17.52bc 115.20± 28.62 c
Group 4 179.20 ±70.10 a 358.40±140.21a
Group 5 2.8±1.09 c 0±0.00c
P≤0.05, small letter describe the significant different between groups.
Figure (1): HI antibody titers in experimental groups.
0
50
100
150
200
250
300
350
400
day 21 day 35
group 1
group 2
group 3
group 4
group 5
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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Titer of chicken interferon gamma (ChIFN-γ) production in response to vaccination
The mean ChIFN-γ levels, expressed as pg./mL were presented in Table 3. ChIFN-γ measured at
7, 21 and 35 day-old birds from group 1, 2, 3, 4 and 5 by ELISA revealed that the levels ChIFN-γ were
60 pg./mL pre vaccination (at day 7 of age). On day 14 post vaccination (at day 21 of age), the levels
ChIFN-γ were 200 pg./mL in G 1, 180 pg./mL in G 2, 190 pg. /mL in G 3, 250 pg. /mL in group 4, 90
pg./mL in G 5 while on day 14 post booster vaccination dose (at day 35 of age), the levels ChIFN-γ
were 250 pg. /mL in G 1, 300 pg./mL in G 2, 230 pg./mL in G 3, 400 pg./mL in G 4, 75 pg./mL in G 5.
The levels ChIFN-γ were higher in the vaccinated groups (group 1, 2, 3 and 4) than in the nonvaccinated
group (group 5), especially in group 4 (Figure 2).
Table (3): The concentration of chicken interferon gamma (pg./mL) against NDV in
experimental groups measured by ELISA test
Group concentration of interferon gamma (pg./mL)
At 7 days At 21 days At 35 days
Group 1
60.00 ±14.14
200±28.28a 250±21.21b
Group 2 180±21.21a 300±42.42b
Group 3 190±14.14a 230±28.28b
Group 4 250±42.42a 400±28.28a
Group 5 90±28.28b 75±21.21c
P≤0.05, small letter describe the significant different between groups.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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Figure (2): ELISA chicken interferon gamma concentration (pg./mL) in experimental groups.
Lymphoid organs indices: As in Table 4. The highest standards shown the immunized groups in
comparing with non- immunized groups at day 35 of age while between vaccinated groups there was no
significant different in lymphoid organs indices.
Table (4) Lymphoid organs indices of experimental groups
Group Indices of Lymphoid Organs at day 35 of age
Spleen Bursa of Fabricious Thymus
Group 1 0.093±0.005a 0.095±0.02 0.171±0.01
Group 2 0.061±0.01ab 0.086±0.01 0.182±0.01
Group 3 0.077±0.02ab 0.084±0.02 0.197±0.02
Group 4 0.093±0.01a 0.084±0.01 0.194±0.02
Group 5 0.045±0.01b 0.076±0.02 0.142±0.02
P≤0.05, small letter describe the significant different between groups.
0
50
100
150
200
250
300
350
400
450
7 days 21 days 35 days
group 1
group 2
group 3
group 4
group 5
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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DISCUSSION
In the present study, the primary immunization was approved on the time as soon as the
parental antibody titer was (GMT 4.8). The results shown that the parental antibody titer in 7th
day of old was 4.8, the drop in antibody titer was documented at 21th day of age and reached to
undetectable at 35th day of age by estimated using hemagglutination inhibition test diagnostic
method for Newcastle Disease.
The incessant risk of ND occurrences in commercial fowls herds requires early protection
with conservative ND vaccines managed at an early stages of age. Our study values different
vaccination patterns for a commercial presented attenuated vaccines counter to NDV used
beneath confined situations. Our results revealed that the Ab titer was diverse considerably
between studied groups and best increase antibodies levels was in group 4 which used live
vaccine by eye drop at 7th day of age and by drinking water at 21st of age. Our results show that
vaccine given by eye drop method is better than drinking water method and live vaccine is better
than killed vaccine. This result is also reported in a research that for total vaccines intraocular
management improve highest security than drinking water route (11). To hand was diverse
vaccines offered for regulatory of N D. Live vaccines are simple applied and relatively cheap and
provide good immunity (12). Many investigators have record that attenuated ND vaccines give
superior security and healthiness more than inactivated vaccines (13, 14). (15) also other types of
N D vaccines managed by eye drop or orally that improved additional mucosal resistance
presented by IgA antibodies. The immunity detected in immunized chicks specified that the
vaccines were effective. The twofold proliferation in humeral responses of the birds following
‘primer dose’ and ‘booster dose’ was detectable with the efficiency of the trade in vaccines. This
is in tandem with the work(1,16, 17).
The result of the existing study exposed that antibody tires of diverse immunized groups
were significant different. This difference based on the kind of methods of immunization, as a
result the cause of high antibody response in group 4 (eye drop rout) of 7 day old provide
enough resistance to keep chickens this because attenuated virus vaccine replicates rapidly in
mucous membrane of the conjunctiva and nostrils and induce the IgA in the tears (1, 18) such as
a cause of local immunity all these details originate from using the attenuated vaccine by eye
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq.
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drop. The booster dose (orally vaccinated) using for longer-lasting immunity (19). Both humeral
and cell-mediated immune responses play important roles in protecting chickens against NDV
infection (20, 21).
Therefore in this study we evaluated the cellular immunity as well as antibody titer.
Cytokines such as IFN-g are formed by stimulated specific T cells. ChIFN-g capture ELISA
more specific and sensitive than the presented bioassay (22). ChIFN-g ELISA as an alternate to
the propagation test was inspected after mitogen stimulation of chicks spleenocytes. All
vaccinated groups showed high titer of ChIFN-g compared with unvaccinated group. Also group
4 showed higher ChIFN-g titer than other groups. This result also reported by other study that
showed maximum of the chicks immunized by the attenuated NDV vaccine formed ChIFN-g
later induce stimulus, and this from the time when 2 - 4 weeks after immunization. While other
study showed no local correspondence between ChIFN-g produced and humeral reaction (HI
titers) could be established after NDV vaccination. The ChIFN-g ELISA has cool prospective for
determining the character of cellular immunity for security in contradiction of fowls contagious
diseases in the future and will facilitated the research of the role of ChIFN-g in numerous avian
immune machineries. The lymphoid organ weight and their indices are beneficial pointers of
immunological status (10) and show on the animals' capacity to transmit infection and the
providing of lymphoid cells through an immune response (23).The results of our study revealed
the immunized fowls have highest spleen directories than non- immunized birds, while other
lymphoid organ (Bursa of Fabricious and thymus) showed no significant difference between
studied groups. lowest lymphoid organs indices in non-immunized groups referred on little
security (24). The clarify in height Ab reaction has been related with a bigger bursa size in White
Leghorn chicken strains (25). The available commercial ND vaccine are effective and the best
vaccination program for broiler chicken are primary vaccination with live ND vaccine at 7th of
age via eye drop followed by booster dose at 21st day of age with live ND vaccine via drinking
water. Investigators revealed that infection, shedding, and transmission of virulent NDV in
vaccinated birds may occur without clinical signs (26, 27). Agreed this probability we have
confidence in that, if defensive immunization pattern are to be employed, they would go
composed by a checking program confirming that adequate herd immunity levels are succeeded.
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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