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THE USE OF MODERN BIOTECHNOLOGY IN THE PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS 1-IMMUNITY TRAITS AND SERUM PROTEIN THE USE OF MODERN BIOTECHNOLOGY IN THE PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS 1-IMMUNITY TRAITS AND SERUM PROTEIN GELL ELECTROPHORESES

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Two thousands eggs of Bandara strain and 300 Japanese quail birds, 35 days old were used as an attempt to introduce fragment functional (1, 2 and 4 µg/egg) and separated cells (chimera) (100 and 300 cell/egg) from thymus (Thy) and bursa of fabricious (Bur) glands of Japanese quail into the embryonic cells of Bandara eggs at the third day of incubation. The results showed significant differences between treatments concerning white blood cells (WBCs) and monocytes counts. The thy-cell group had the highest WBC's count (33.3 x10 3 /mm 3) compared with control one (26.5 x10 3 /mm 3). While level of 100 cells of thymus group had the lowest average counts of monocytes (1.17X10 3 /mm), Thy-DNA (4 µg) group had the highest one (3.25X10 3 /mm). Concerning phagocytic activity, Bur-DNA group had higher (18.9%; P<0.01) phagocytic activity than that of control group. A marked significant increase in the humoral immune response to NCDV due to the biweekly vaccination was detected for transgenic chicks having Bursa or Thy-DNA. However, Bandara chicks transfused with Bur-DNA surpassed (1024) those of thymus origin (101.8). Chicks having Bur-DNA showed higher maternal antibody titers (p<0.05) against infectious bursal disease virus (IBDV) (570.2) than both thymus (251.2) and control groups (222.8). On the other hand, chimera chicks having bur-cells significantly expressed the maximum antibody titers (15864) followed by those having Thy-cells (14184), There was a marked difference among different samples in the presence and ISSN 110-2047.ALEX.J.VET.SCIENCE.2006,VOL. 24 NO 1 Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F Alex. J.Vet, Vol 24, No.
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THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
227
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
By
Mandour, M. A. M.*; Aly, O. M.**; Zweel, H. S.***; Abdel Aziz, Doaa, F.**
*Dept. of Animal Husbandry and Animal Wealth Development, Fac. of Veterinary
Medicine, Alex. Univ. Egypt.
** Animal Production Research Institute, Agricultural Research Center, Ministry of
Agriculture, Dokki, Egypt.
*** Animal and Fish Production Department, Faculty of Agriculture (Saba Basha), Alex.
Univ. Egypt.
ABSTRACT
Two thousands eggs of
Bandara strain and 300 Japanese
quail birds, 35 days old were used
as an attempt to introduce
fragment functional (1, 2 and 4
µg/egg) and separated cells
(chimera) (100 and 300 cell/egg)
from thymus (Thy) and bursa of
fabricious (Bur) glands of Japanese
quail into the embryonic cells of
Bandara eggs at the third day of
incubation. The results showed
significant differences between
treatments concerning white blood
cells (WBCs) and monocytes
counts. The thy-cell group had the
highest WBC's count (33.3
x103/mm3) compared with control
one (26.5 x103/mm3). While level of
100 cells of thymus group had the
lowest average counts of monocytes
(1.17X103/mm), Thy-DNA (4 µg)
group had the highest one
(3.25X103/mm). Concerning
phagocytic activity, Bur-DNA
group had higher (18.9%; P<0.01)
phagocytic activity than that of
control group.
A marked significant
increase in the humoral immune
response to NCDV due to the
biweekly vaccination was detected
for transgenic chicks having Bursa
or Thy-DNA. However, Bandara
chicks transfused with Bur-DNA
surpassed (1024) those of thymus
origin (101.8). Chicks having Bur-
DNA showed higher maternal
antibody titers (p<0.05) against
infectious bursal disease virus
(IBDV) (570.2) than both thymus
(251.2) and control groups (222.8).
On the other hand, chimera chicks
having bur-cells significantly
expressed the maximum antibody
titers (15864) followed by those
having Thy-cells (14184),
There was a marked
difference among different
samples in the presence and
ISSN 110-2047.ALEX.J.VET.SCIENCE.2006,VOL. 24 NO 1
Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F
Alex. J.Vet, Vol 24, No.1 April 2006
228
absence of some protein bands as
well as the concentration of some
protein. The concentration of α-
globulin due to 4µg of Bur-DNA
decreased compared to the control
group, while 2 g of Bur-DNA
decreased β1-globulin but
increased β2-globulin compared to
the control. In addition, the 2 and
4 µg of bur-DNA resulted in the
greatest concentration of γ1-
globulin when compared to the
control group. The levels of 2 and
4µg/egg showed a sharp increase
in the concentration of γ3-globulin
over the control and the 1µg DNA
level. It could be concluded that
Bur-DNA could improve immune
response to New castle disease
virus, while Bur-cells increased
immune response to infectious
bursal disease virus. The
application of these results may
improve immune response,
decrease losses and improve the
economic return from raising
Bandara chickens.
KEY WORDS: Biotechnology, immune
response, gell
electrophoresis,
chickens, quail.
INTRODUCTION
Modern biotechnology is a new
tool for genetically improving
poultry production and immune
responsiveness. Transgenesis and
chimera approach might be used to
make a poultry species perform
better, or it may introduce entirely
new properties into a different
species. In this approach, a foreign
protein might be produced in a
transgenic or chimeric chicken, or it
may provide new disease resistance
properties, or alter the immune
system to generate new
histocompatability antigens or new
antibodies.
Several techniques have been
applied in chicken embryos to
enhance the immune response and
disease resistance of the hatched
chicks. These attempts included
bursal and thymus tissue
transplantation (Kendle and Jerome,
1970), implantation of bursal
rudiments in quail-chicken embryo
(Le Douarin et al., 1977),
transplantation of quail bursal cells
into 3 days chick embryo (Glick,
1978), and transgenic chickens using
avian retero-virus and reticulo
endotheliosis virus as a gene transfer
vector (Shuman and Shoffner, 1986)
and quail DNA (Mandour, 1996).
Different kinds of WBC's as
lymphocytes, monocytes, basophils,
eosinophils and neutrophils were
counted by Lucas and Jamra (1961)
and Ahmed (1999). Phagocytosis by
macrophages is recognized as the
initial step of the specific immune
response. El-Tahawy (2005) found
that differences between groups
treated with DNA from either quail
or broiler breeds and the control
group in the phagocytic (PA) activity
was significant, although there were
no significant differences between
them in phagocytic index also.
Unfortunately, little is known
about the integration of foreign
Deoxyribonucleic acid (DNA) or
cells from different tissues forming
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
229
antibody cell organs of naturally
resistant quails to some viral diseases
(Weissman and Hitchner, 1978).
Therefore, this work was conducted
as an attempt to introduce fragment
functional DNA (transgenesis) and
separated cells (chimera) with
different doses from thymus and
bursa of fabricious glands of
Japanese quail (Coturnix coturinix
japonica) into the embryonic cells of
Bandara strain at the third day of
incubation. The effect of transgenesis
and chimera on the immunity of the
hatched chicks as well as correlated
hematological parameters were
evaluated.
MATERIALS AND METHODS
This study was carried out
during the period from 2003 to 2005
in El-Sabahia Poultry Research
Station, Animal and Poultry
Production Research Institute,
Agricultural Research Center,
Ministry of Agriculture. The Gel
electrophoreses, and haematological
parameter estimates were done at
Animal Husbandry and Animal
Wealth Development Department,
Faculty of Veterinary Medicine,
Alexandria University.
Two thousands eggs of
Bandara strain and 35 days old
Japanese quail birds (Coturnix
coturinix japonica) were used
through this study. The detailed
management, feeding, and
vaccination programs were described
by Abd El-Aziz (2006).
DNA extraction was done according
to Sambrook et al. (1989) and
modified by Abdel-Hamid et al.
(2002). Determination of DNA
concentration was measured and
calculated according to Charles
(1970).
Separated cells were isolated
from the thymus and bursa of
fabricious glands after the method of
Maeda et al., 1997.
Egg Treatment:
Two thousands eggs in 2
hatches, each of one thousand, were
obtained from Bandara strain. Eggs
were collected daily and stored at 12-
15˚C for 7-10 days. The incubator
was cleaned, fumigated and washed
with a sterilizing solution (Hydrocyle
200 ml/1 L water). Eggs were
fumigated before setting in the forced
draft-type incubator. At the third day
of incubation, the fertile eggs were
randomly assigned into five groups:
a. Thymus and Bursa of
Fabricious gland DNA group:
Each DNA group was divided
into three subgroups of eggs (150
eggs each). Eggs were
disinfected, drilled and injected
with extracted DNA (Salter et al.,
1986) from thymus or bursa
glands in 3 doses (1, 2 and 4
µl/egg for the three subgroups,
Ahmed, 1999), through yolk sac.
b. Thymus and Bursa cells group:
Eggs were disinfected, drilled and
injected with separate cells from
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Alex. J.Vet, Vol 24, No.1 April 2006
230
thymus or bursa glands in doses
100, 300 cells/egg for each group
with 150 eggs/subgroup.
c. Control group: The control
group consisted of four subgroups
each containing 100 eggs:
(Sterilized buffer solution of
DNA & cells, drilled egg groups)
and normal eggs without drilling
or injection.
A total of 464 hatched chicks
were wing banded on the day of
hatch according to the corresponding
DNA or cells group and brooded in
the floor pens. Ambient temperature
was 35˚C during the first week then
it was decreased by 3˚C weekly until
it reaches 24-27˚C by the end of the
second wk. A starter diet (20.6%
crude protein and 2879 kcal/kg of
diet) was fed from hatch to 4 wk of
age, followed by a grower diet
(16.86% crude protein and 2864
kcal/kg of diet) from 8 to 18 wk of
age. Feed and water were offered ad
libtum.
Chicks were vaccinated as
following:
1- They were vaccinated against
Newcastle disease virus (Lingonic
Hitchner B1 + IB), 14, 21(Lasota)
and 35 (Lasota + IB) at 7 days of
age, then every 2 wk (Lasota).
2- They were vaccinated against
Gamboro disease virus (Infectious
bursal disease virus) at 11 and 22
days of age.
Chicks were provided with 24
hours daily of light till the 4th wk and
then reduced to 14 hours during the
growing period. At the 20th wk of age
the lighting period increased
gradually up to 16 hr.
At two wk of age, chicks were
divided into 28 cages in electrically
battery according to the 14
treatments (2 replicates each).
Blood samples were collected from
wing vein before each vaccination,
centrifuged and serum was stored
at -20ºC for later analysis. At 20 wk
of age, blood samples were
collected for hematological pictures
(using citrated blood in the ratio of
0.1/1 ml of 4% potassium citrate
solution to 1 ml of blood (Hawk et
al., 1965).
Studied parameters:
Haematological parameters:
a. White blood cells counts.
b. Differential leucocytic count according to (Schalm, 1986).
c. Phagocytic activity and phagocytic index were estimated according Kawahara
et al. (1991) as following:
Phagocytic activity (PA) = Percentage of phagocytic cells containing yeast
cells.
Phagocytic index (PI) =
yeastsedphagocytoscellsphagocyticofNumber
edphagocytizcellsyeastofNumber
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
331
Immunity Tests:
Blood samples collected
before every vaccination was tested
for Gamboro and Newcastle disease
antibodies. Immune response to IBD
vaccination was done using ELISA
technique (Khafagy et al., 1990).
Titration of hemagglutinating
inhibiting antibodies against NDV
(Heamagglutination Inhibition test)
was carried out according to
Majiyable and Hitchner (1977).
Determination of different protein
fractions by electrophoresis:
The electrophoresis was
carried out according to the
procedure of Laemmli (1970) using
vertical electrophoresis apparatus,
Model DE 600 Hoefer.
50 µl serum from different
treatments and the high molecular
weight markers were thawed at room
temperature and mixed with equal
amounts of sample buffer (pH 7.2). In
a sterile, clean and dry beaker, the
separating gel (10%) and (3%) were
prepared according to Laemmli
(1970). Each pattern profile was
measured with a polar planimeter.
The area of each profile fraction was
calculated as a percentage of the
whole. Comparisons were then made
among profile on the basis of these
percentages for corresponding
numbered fractions. The classification
of bands was done according to Elissa
et al. (1993).
Statistical Analysis:
Data were analyzed by the
General Linear Model (GLM)
procedure (SAS, Institute, Inc, 1990).
The Least Square Mean (LSM) +
standard errors were calculated and
tested for significance using the “t”
test.
Before analyzing the data, all
skewed data were transformed to the
corresponding arc sin values according
to Gomez and Gomez (1984). Results
of phagocytic activity and phagocytic
index were expressed as means +
standard error and different values,
then evaluated by students “t” test.
RESULTS AND DISCUSSION
Results listed in Table 1 show
the effect of introducing foreign
DNA or cells of different sources
and levels into chicken embryos at
the 3rd day of incubation on
differential leucocytic counts of
different blood traits.
1. White blood cells (WBC’s):
The average count of WBC’s of
the treated groups (DNA and cells)
ranged from 22.5 to 36.0 x103/mm3
for high level of bur-DNA (4µg/egg)
and Thy-cells (300cell/egg) groups,
respectively. It was shown that there
was highly significant effect of type
(DNA or cells) of treatments on
WBC's, where Bur-DNA group was
equal to thy-DNA one which had the
lowest count (23.8 x103/mm3) while
thy-cells group had the greater count
of WBC’s (33.3 x103/mm3) and
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Alex. J.Vet, Vol 24, No.1 April 2006
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control group had intermediate value
(26.5 x103/mm3) on the whole
average. It was found that bur-cells
had lower WBC’s count than those
of Thy-cells, but higher than Bur-
DNA and Thy-DNA. These findings
were within the range found by Fritts
et al. (1989) concerning the average
of control group, but higher than
those reported by Hamdy et al.
(2003) who found that averages of
WBC’s ranged from 14.9 to
23.6x103/mm3 and from 18.7 to
25.4x103/mm3 at the 6th wk and 7th
wk, respectively. Moreover, it is
worth noted that Thy chimera chicks
had significantly more WBC’s
(33.3x103) than those of non
transgenic ones (23.8X103), which
could improve immune response and
play more efficient role in defense
mechanism against disease whether
being specific or non specific.
These findings agree with
Kendle and Jerome (1970) after
transplanting Bur and Thy tissue;
LeDouarin et al. (1977) who
implanted bursal rudiments in quail-
chick embryo; Glick (1978) who
transplanted quail bursal cells into 3
days chick embryo; and Mandour
(1996) who produced high immune
transgenic chickens by injecting
embryos with quail bursal (antibody
forming cell organ) DNA that acted
as a dominant organ for gene
resistance to IBD virus infection.
2. Differential Leucocytic Count:
a. Lymphocytes: Averages of
lymphocytes of the different treated
groups under investigation ranged
from 48.0 103/mm for the low level
(100 cell) of Bur-cells to 54.3x103/mm
for Bur-DNA (1µl/egg). Average of
control group was 53.4x103/mm.
Although no significant
differences were detected for any
treatment among the different groups
and control one, there was a 8%
decrease in lymphocytes due to
increasing Thy cell level to 300 and
8.6 and 11.3% due to doubling the
Thy-DNA level to 2 and 4 µg/egg,
respectively. The recorded averages
for transgenic and chimera Bandara
chicks as well as control group were
close to the corresponding values of
transgenic and control Gimmizah
chicks reported by El-Tahawy
(2005).
b. Monocytes counts: Both type and
level of treatments had a significant
(p<0.01) effect on monocyte count.
Low level (100 cells) of Thy cell
group had the lowest average
(1.17x103/mm) and high level of
Thy-DNA group (4 µg/egg) had the
highest average (3.25x103/mm),
meanwhile average of control group
was 1.41x103/mm. Concerning types
of treatments, thy-cells group had
significantly (P<0.01) the lowest
average (1.25x103/mm), while
chicken group of thy-DNA had the
highest one (2.27x103/mm). On the
other hand, transgenic Bandara
chicks group of high level (4 µg/egg)
thy-DNA had significantly (p<0.01)
higher monocytes count
(3.25x103/mm) than those of 1 and
µg/egg DNA of the same gland. In
contrary, low dose (1 µg) of Bur-
DNA significantly increased
monocytes number compared to 2µg.
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
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The significant increase in
monocyte counts due to transgenesis
or chimera over the control one points
to the positive stimulation of antibody
forming cell origin genes to improve
protection against infection. This
finding disagrees with El-Tahawy
(2005) who did not record significant
difference due to inoculation of
pituitary-DNA of the same origin.
However, differences in response
could be expected due to nature of
gland used.
c. Basophils counts: Averages of
basophils counts of all treatments
groups ranged from 7.36 103/mm for
Thy-DNA (2 µg/egg) group to 9.83
103/mm for Thy-cells (300 cell/egg)
group, while that of control group
was 8.26 103/mm. Results showed
that both averages of low and high
levels of Thy-DNA (8.86 103/mm
and 8.33 103/mm, respectively), were
significantly higher than that of 2
µg/egg (7.36 103/mm). On the other
hand, El-Tahawy (2005) did not
detect any significant change in
basophils counts due to transgenisis
of Gimmizah embryos at the 3rd day
of incubation with any level of quail
or broiler breeders DNA, his values
were close to those recorded under
the present investigation.
d. Eosinophils counts: Averages of
eosinophils counts of all transgenic
and chimera groups ranged from
8.33x103/mm for thy-cells
(300cell/egg) group to 10.80x103/mm
for bur-cells (300cell/egg) group,
while eosinophils counts of control
group was 10.50x103/mm. Although,
insignificant effect of type of
treatment was found on this trait
according to analysis of variance,
(Table 5) results showed that chimera
Thy cell group had significantly lower
overall average (8.75x103/mm) while
average of control group and Bur-
DNA were higher (10.5x103/mm)
compared with Bursa and Thy cells as
well as thy-DNA.
No level by source of either
types (DNA or cells) interaction was
detected. The significant reduction
only in eosinophil count due to
chimera of either Thy or Bur and
Thy-DNA compared to the control
group was closer to those detected
by El-Tahawy (2005).
e. Neutrophils counts: Averages of
all treated groups were in range
(23.7-32.5 103/mm) for Thy-DNA
(1µg/egg) and Bur-cells
(100cells/egg) groups, respectively.
Meanwhile, average of neutrophils
counts of the control group was
26.4x103/mm. Statistical analysis
(Table 5) showed significant effect
of level of treatment on this trait. It
was shown that increasing the level
of Thy-DNA from 2 to 4 µl/egg
significantly increase the averages of
neurophil counts (31.0 103/mm and
30.6 103/mm, respectively),
compared to the low level group
(23.7 103/mm). The increase was
amounted to 30.8 and 29.1%,
respectively. Evidently, increasing
Thy-cells to 300 cell significantly
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increased the neutrophilus count by
12.4% compared to 100 cells.
Non significant fluctuation due
to chimera of either sources was
detected. The non significant effect
of DNA or cell inoculation on the
whole average of neutrophil counts
agree with the findings of El-Tahawy
(2005) who recorded closer range of
neutrophils counts (27.66-29.25), but
disagree with El-Naggar (2002) who
recorded significant (p<0.05) effect
of transgenesis in Japanese quails on
neutrophils counts.
Results concerning
lymphocytes, eosinophils, and
basophils are higher than those
reported by Twisselmann (1939)
who found that WBC’count
(103/mm), at 6 wk of age to mature
of chickens were 40.9, 2.7, 4.3 and
16.5 for lymphocytes, eosinophils,
basophils and monocytes,
respectively. In contrary, results
reported herein, were less than the
estimates of Lucas and Jamra (1961)
who found that WBC's of White
Leghorn females were (81.5, 1.5,
2.3, 4.5 103/mm) and (77.8, 3.0, 1.7,
4.9 103/mm) at 6 and 12 wk of age
for lymphocytes, bosinophils,
basophils and monocytes,
respectively. Results of Lillehoj and
Chai (1988) demonstrated that
various lymphoid organs of chicken
glands, such as spleen, thymus, bursa
of fabricius and gut intraepithelium,
contain subpopulations of cells that
can mediate spontaneous
cytotoxicity.
Wide variations were found
between results of Bandara strain
under investigation compared with
those estimated by Sturkie (1986) on
quail except that for monocytes
which was in this range. Fritts et al.
(1989) collected the blood from 7
wk-old broiler chicks with or without
fulminant pulmonary hypertension
syndrome (PHS). Broiler of PHS had
lower percentage of lymphocytes
and eosinophils and higher
percentages of heterophils, while
percentage of monocytes and
basophils were similar between
groups. The ratio between
heterophils and lymphocytes was
substantially higher in broiler with
PHS compared to control (3.99 vs.
0.22). In this study, average of
control group was approximately
equal to the group of treated by low
level of Bur-DNA and higher than
the other injected treatments groups,
findings that disagree with Ahmed
(1999) who found that injection of
foreign DNA into Montazah strain
increased the number of
lymphocytes in all treated groups
compared to those of control.
Moreover, results under
investigation were nearly similar to
those of Hamdy et al. (2003)
concerning lymphocytes cells
percentages at 6 and 7 wk which
ranged from 4.4% to 6.1% and from
4.9% to 6.6%, respectively, but less
than those for monocyte, which
ranged from 18.7% to 27.5% and
from 20.2% to 32.4%, respectively.
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
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Phagocytic activity (PA) and
phagocytic index (PI):
Results listed in Table 2
showed averages of phagocytic
activity (PA) and phagocytic index
(PI). There were highly significant
differences between averages of PA
of the different sources, types and
levels of treatments. In general,
averages of PA ranged between
19.75 for Bur-cells (300cell/egg)
and 26.67 for thy-cells
(300cell/egg). Concerning types of
treatments, Bur-DNA group had the
highest PA (24.40), while control
group had significantly (p<0.01) the
lowest average (20.52). The
increase amounted to 18.9%.
Results indicated that Thy-cells
resulted in significantly higher PA
than Bur-cells and the control group
too. On the other hand, averages of
the two treated groups 1 and 2
µg/egg of Bur-DNA were
approximately similar but higher
(p<0.05) than that of high level
(22.59). In addition, average of high
level (300 cell) of Thy-cells was
higher (p=0.05) than that of low
level (26.67 vs. 21.83),
respectively. The increase
amounted to 22.17%.
Averages of PI are in range
(2.43-4.50) for both high level of Bur
and Thy cells, respectively, while
that of control group was 2.73.
Insignificant effect of level of
treatment was found on this trait
according to analysis of variance,
however, increasing thy-cells to 300
cells/egg increased the PI by 59%
(p<0.05). Moreover, it was showed
that Thy-cells group had significantly
higher average (3.66) than average of
control group which was the lowest
(2.73) compared with the other types
treated. The increase amounted to
34.1%. It is clear that PI of Thy-cells
was significantly higher than that of
Thy-DNA and bur-cells as well.
The detected significant
differences and improvement due to
chimera or transgenic chick
production as well as level effect on
phagocytic activity were also
observed by Kreukniet et al. (1995)
and El-Tahawy (2005) who
recorded higher phagocytic activity
(22.4) than that of control group
(20.33). The interaction of level
with DNA source was also detected
by the last author who found
significant increase of low broiler
breed DNA level (1µg) on
phagocytic activity than the 2µg
level (23.66 vs. 21.16). In addition,
the recorded nonsignificant effect
on phagocytic index was consistent
with the findings of Helal (1999),
and El-Edel (2005) who reported
statistically nonsignificant
differences on phagocytosis.
Accordingly, it could be concluded
that phagocytosis would be possibly
improved in transgenic or chimera
chicks having Bur or Thy-DNA or
cell origin.
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
237
Table (2): Least-squares means + standard errors of phagocytic activity (PA) and
phagocytic index of the different treatments
Source
Type
Level
PA
PI
Bursa
Cells
(cell/egg)
100
21.83+1.23
3.45+0.71
300
19.75+1.50
2.43+0.87
A
Av
ve
er
ra
ag
ge
e
2
20
0.
.7
79
9+
+0
0.
.9
97
7B
B
2
2.
.9
94
4+
+0
0.
.5
56
6B
B
DNA
(µg/egg)
1
25.0+0.95a
3.70+0.55
2
25.11+0.71a
3.52+0.41
4
22.59+0.64b
3.10+0.37
A
Av
ve
er
ra
ag
ge
e
2
24
4.
.4
40
0+
+0
0.
.4
45
5A
A
3
3.
.4
44
4+
+0
0.
.2
26
6A
AB
B
Thymus
Cells
(cell/egg)
100
21.83+1.23b
2.83+0.71
300
26.67+1.57a
4.50+0.54
A
Av
ve
er
ra
ag
ge
e
2
24
4.
.2
25
5+
+0
0.
.8
87
7A
A
3
3.
.6
66
6+
+0
0.
.5
50
0A
A
DNA
(µg/egg)
1
23.36+0.81
3.29+0.46
2
24.92+0.74
2.66+0.51
4
24.17+0.67
2.95+0.64
A
Av
ve
er
ra
ag
ge
e
2
24
4.
.1
16
6+
+0
0.
.4
48
8A
AB
B
2
2.
.9
97
7+
+0
0.
.2
28
8B
B
C
Co
on
nt
tr
ro
ol
l
2
20
0.
.5
52
2+
+0
0.
.3
39
9B
B
2
2.
.7
73
3+
+0
0.
.2
22
2C
C
a-c: Means having different letters in every column and within every factor of treatments
are significantly different (P<0.05).
A-C: Means having different letters between the overall mean (averages) are significantly
different.
Antibody development against
New Castle Disease Virus
(NCDV):
Means of hemagglutinating
inhibiting (HI) antibodies against
NCDV in chimera and transgenic
Bandara chickens injected with quail
DNA or cells of either bursa of
fabricious or thymus at 3d of
embryonic stage are presented in Table
(3).
Residual maternal antibodies
to NCDV were detected in chicken
sera at 7 days of age of the different
groups including the control one,
which might be due to previous
vaccination program of the parental
stock against NCDV. The HI titer
was almost equal, low and ranged
from 3.2 to 4.0. Chimera chicks
having quail Bur or Thy-cells
showed unexplainable low equal
response after 2 wk post vaccination
against NCDV (16.0) which did not
differ significantly (p<0.05) from
the control group (22.6).
On the other hand, a marked
significant increase in the humoral
immune response to NCDV due to
the biweekly vaccination was
detected for transgenic chicks
having Bur or Thy-DNA. However,
Bandara chicks transfused with bur-
DNA surpassed (1024) those of
Thymus origin (101.8). The above
mentioned levels of HI antibodies
for the chimera group were much
less than the range reported by
Goodwin et al. (1992), but those of
Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F
Alex. J.Vet, Vol 24, No.1 April 2006
238
transgenic chicks were within the
levels recorded by Khaliel (1995)
and Mandour (1996).
From the above mentioned
results, the significant increase in
antibody production against NCDV
of transgenic chicks over those of
control group, revealed its
immunostimulant effect. This
finding could be considered as an
advantage, since Bastamy et al.
(1986) reported that chicks given
live NCDV vaccine at 12 days had
lower HI antibody response to
NCDV vaccine at 7 and 21 days of
age.
Table (3): Means of Hemagglutinating Inhibiting (HI) antibodies against New
Castle Disease Virus (NCDV) in chicks inoculated with different
DNA or cell origin
-Means having different letters in the column and within every source or type of
treatments are significantly different (P<0.05).
Antibody development against
Infectious Bursal Disease Virus
(IBDV):
The mean IBD antibody titers
in transgenic and chimera chicks
vaccinated at 7 and 21 days of age
are shown in Table (4). Maternal
antibodies to IBDV were detected
in chicken sera of all the
experimental and control groups of
the Bandara strain at one wk of age,
with low mean titers. They ranged
from 183.0 to 534.5 and 363.3 to
594.0 in the chimera and DNA
injected groups, respectively, while
the control group showed 222.8
level. It was noted accidentally that
chicks having either bur-cells or
DNA showed higher maternal
antibody titers (p<0.05) against
IBDV (570.2) than both thymus
groups especially Thy-cells (183.0)
and control groups (222.8), but
differences was not significant
(p<0.05) between the DNA and cell
of bursa. However, thymus DNA
had higher (p<0.05) value than
thymus cells, however, did not
significantly difference from the
Bur-groups.
Two weeks post vaccination
with IBDV, high immune response
was revealed in all studied groups
due to acquired solid immunity
against the vaccine. Chimera chicks
having Bur-cells significantly
expressed the maximum antibody
titers (15864.0) followed by those
having Thy-cells (14184.0), while
transgenic groups of Thy-DNA
Source
Type
Age
7 days
21 days
Bursa
Cells
4.00.28
16.07.83b
DNA
4.00.23
1024.06.39a
Average
4.00.25
512.02.9A
Thymus
Cells
4.00.28
16.07.83b
DNA
3.20.29
101.86.39a
Average
3.50.45
32.05.05B
Control
3.40.20
22.65.53B
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PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
239
origin produced the least antibody
titer against IBDV (7819.0) which
did not significantly difference from
the Bur-DNA (Table 4). However,
the increase amounted to 36.3%.
Also, the chimera chicks of either
Bursa or thymus followed similar
trend and being higher than the
DNA group. They have 48.9 and
81.4% more antibody titers against
IBDV than chicks having Bur or
Thy-DNA, respectively.
Table (4): Least-squares means + standard errors of infectious bursal disease
antibodies of the different treatments injected with different DNA or
cells source
a-c: Means having different letters in every column and within every factor of treatments are
significantly different (P<0.05).
A-C:Means having different letters between the overall mean (averages) are
significantly different.
Since the aim of the second
vaccination against IBDV is to give
immunity against the disease to
chicks that missed or did not
acquired it in the first one,
therefore, it was noted that there
were no significant variation
between the different groups in
antibody response against IBDV,
whether being of cell or DNA
origin. The above mentioned ranges
were within those reported by
Mandour (1996). It was recorded
that, the vaccinated DNA injected
groups maintained high levels of
virus neutralizing antibodies
produced by vaccination (Ghanem,
1994) in both the two breeds which
were studied, than the
corresponding control levels
(Mandour, 1996). On the other
hand, Abdel-Fattah et al. (1999)
studied the effects of crude thymus
extraction on the immune response
and protection against challenge
with virulent IBDV, ELISA results
showed a marked increase in
antibody titers in thymus treated
Source
Type
Age
7 days
21 days
35 days
Bursa
Cells
534.5+30.4a
15864.0+1158.2a
12350.0+3273.9
DNA
594.0+57.0a
10657.0+4063.3 b
16651.7+2477.8
Average
570.2+54.0A
12739.8+4089.6
16672.3+3560.0
Thymus
Cells
183.0+13.0b
14184.0+10.0a
16922.0+11.0.0
DNA
363.3+203.8a
7819.0+1320.7b
14300.0+4192.2
Average
251.2+21.6B
10365.0+3609.2
15348.8 +3293.9
Control
222.8+221.4B
10779.0+3628.3
16672.0+3560.0
Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F
Alex. J.Vet, Vol 24, No.1 April 2006
240
groups. Chicken administrated
thymus extracts and vaccinated with
IBDV showed 100% protection
against challenge with IBDV.
Accordingly, it could be
concluded that it could be possible
to improve or produce
hyperimmune transgenic chickens
via DNA or cell injection of quail or
bursal origin. However, it is
recommended to produce inbred
lines of transgenic or chimera
chickens, capable to overcome the
interfering effect of the maternal
antibodies against active
immunization with IBDV vaccine.
4.1.11 Gel electrophoretic of
serum proteins:
The electrophoresis patterns of
serum protein prepared from DNA
bursa (1, 2 and 4µg/egg) and control
group are shown in Fig (1). Results
showed that there were marked
differences among the different
samples in the presence and absence
of some protein bands as well as the
concentration of some protein.
Concerning bursal DNA, albumin
concentration in the three levels had
increased over the control group,
especially 2µg/egg. There was a
decrease in the concentration of α-
globulin in the 4µg level of bursal
DNA compared to the control group,
while the second level (2 g)
decreased it than control in the β1-
globulin and increased over the
control in the β2-globulin. A sharp
increase in the concentration of 1
and γ3-globulin was observed due to
2 and 4µg DNA level/egg over the
control and the 1µg.
It was clear from Fig (1) that
the concentration of albumin
reached its least in the 3rd level
(4µg/egg) compared to the control.
Concentration of α-globulin
increased in the 1µg DNA over the
control and the two other levels (2
and 4µg). The second level (2µg)
had lower concentration of β1-
globulin than the control and the
two other levels (1 and 4µg) while
the 1st level (1µg) gave the greatest
concentration of β2-globulin than
the control. The third level of Thy-
DNA (4µg) gave the greatest
concentration of β3-globulin while
the second level gave the lowest
concentration. Injection of 4µg of
Thy-DNA resulted in an increase in
the concentration of γ1-globulin
compared to the control group while
the 1st level increased the
concentration of the γ2-globulin
than the control and the other levels.
Albumin concentration in the
Bur-cell treated group showed that
the 1st level (100 cells) gave the
greatest concentration compared to
the control while the other
concentration gave the lowest
concentration and the control was in
between (Fig 2). The concentration
of α-globulin decreased in the sera
of the 2nd level of Bur-cell (300
cells) as well as the β1-globulin
which decreased in that group. Only
the γ1-globulin increased in this
group compared to the control.
The Thy-cell group (Fig. 2)
showed that the concentration of
albumin increased in the two levels
(100 and 300 cells) compared to the
control group, but this increase was
more drastic in the 2nd level. The
concentration of α-globulin was not
affected by the injection of Thy-
cells while the β1-globulin had
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
241
decreased in the 2nd groups
compared to the control group. The
β2-globulin increased in the 2nd
level (300 cells) more than the
control group and the 1st level (100
cells).
It could be concluded that
injection of DNA from either bursa
or thymus origin at different levels
induced marked increase in serum
protein, while the corresponding
treatments of cell origin did not
express such Thy-cells at 300
cells/egg level which almost
doubled the serum albumin
concentration
Fig (1): Gel electrophoresis of treated and control groups.
Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F
Alex. J.Vet, Vol 24, No.1 April 2006
242
Fig (2): Gel electrophoresis concentration pattern for different treatments
THE USE OF MODERN BIOTECHNOLOGY IN THE
PRODUCTION OF HIGH IMMUNE RESPONSE CHICKENS
1- IMMUNITY TRAITS AND SERUM PROTEIN
GELL ELECTROPHORESES.
Alex. J.Vet, Vol 24, No.1 April 2006
243
Mandour, M. A. M.; Aly, O. M.; Zweel, H. S.; Abdel Aziz, Doaa, F
Alex. J.Vet, Vol 24, No.1 April 2006
244
The marked differences
among different samples in the
presence or absence of some protein
bands as well as the concentration
of some proteins due to DNA
microinjection was pointed out by
Ahmed (1999) and El-Tahawy
(2005). The first author concluded
that treatment with female broiler
DNA resulted in enhancement of
the humoral immune system in the
treated chicks because the serum of
that group contained the highest
level of immunoglobulines. Also,
El-Tahawy (2005) indicated that
introducing foreign DNA (gene or
genes) having different expressions
on protein synthesis in chickens
produced under the same
environmental conditions.
In conclusion, it is possible to
improve immune response, decrease
losses and have better economic
return due to introduce DNA and
Chimera cells into bandara strain
which may be used as a model for
other poultry breeds.
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-       
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