Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
363
EVALUATION OF HUMORAL AND CELLULAR IMMUNE
RESPONSES TO EIMERIA TENELLA OOCYST PROTEIN AS
VACCINE TO BROILER
Suhair R. Al-Idreesi*, Mahmoud Kweider**, Mahmad M.Katranji***,
*Department of Microbiology , Faculty of Veterinary Medicine, Basrah University, Iraq.
**Department of Animal Biology, Faculty of Sciences, Damascus University, Syria.
***Faculty of Veterinary Medicine, Al-Baath University, Syria.
Keywords : Vaccine, Eimeria tenella, Western blot.
ABSTRACT
To determine the type of immune response to oocyst vaccine in broiler against coccidiosis,
broilers were vaccinated with two doses of prepared oocyst protein from the local strain of
Emeria tenella parasite. The vaccine was applied on 3rdand 16th day of age subcutaneously at
a dose (25 μg per chicken), vaccinated birds were challenged at 30 day of age. Blood samples
were collected at (7th, 28thand 39th) day of age. The immunogenicity of vaccine was studied
by using SDS-PAGE and Western blot. Fourteen polypeptides had been estimated more
immunogenic after probing with immunized chicken serum at 39th days of age, their
molecular weight are (167.8, 114.5, 83.4, 78.2, 73.5, 53.3, 44.1, 38.8, 36.4, 28.2, 20.5, 18,14.9
and 13.9) KD. In addition, the levels of ˠ -IFN and IL-4 were estimated in the serum of
immunized chickens by using ELISA kits. The results demonstrated two types of immunity,
cellular and humoral responses against E.tenella oocyst vaccine.
INTRODUCTION
Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. Worldwide
economic losses due to the parasites have been estimated to exceed 1.2 billion U.S dollars per
annum [1]. Eimeria tenella is the most virulent species, causing severe hemorrhagic enteritis
by infection of the epithelium and the sub mucosa of the ceca and, eventually, death of
infected chickens [2].
Many studies have been done to understand the type of immunity to Eimeria for controlling
this disease. Immune responses to Eimeria are complex and involve many facets of
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
364
nonspecific and specific immunity, the latter encompassing both cellular and humoral
immune mechanisms [3,4]. The lymphocytes, macrophages, and other effector cells act in
harmony to secrete cytokines and proinflammatory molecules, mediating the appropriate
immune responses to the invading parasite. In contrast to the mammalian cytokines, only a
little chicken homologs have been described, the main ones being interferon (IFN)-γ,
transforming growth factor (TGF), tumor necrosis factor, interleukin (IL)-1, IL-2, IL-6, IL-8,
and IL-15 (LILLEHOJ et al., 2004). Recently, a number of cytokines including IL-17, IL-18,
IL-16, IL-12, IL-10, and the Th2 type IL-3, IL-4, IL-13, granulocyte macrophage colony
stimulating factor, and IL-5 have been described in chicken [5].
Alterations in lymphocyte subpopulation and cytokine production during Eimeria infections
in animals have been investigated to clarify the nature of protective immunity [6,7,8].
Various cytokines are produced by macrophages following coccidial infection [9].
T-helper (Th) Th-1 secret ˠ -IFN and also tend to secret IL-2, while Th-2 cells secrete IL-4,
and also tend to secrete IL-5, IL-6 and IL-10.The cell type whose secretions dominates may
help to determine the outcome of certain parasitic infections [10]. IFN production in chickens
has been used as a measure of T-cell responses to coccidial antigens[11,12,13].
Wallach [14] demonstrated the ability of antibodies (raised by live immunization or against
purified stage –specific Eimeria antigens) to inhibit development of parasite in vitro and in
vivo. There are three principal classes of antibodies are known in birds, IgM, IgA and IgY,
the presence of other antibody classes such as IgD or IgE in birds has not been documented
[15].
The aim of the current study is to investigate the type of immune response when use oocyst
of Eimeria tenella antigen as vaccine from local strain against coccidiosis, because strain
differences in Eimeria – induced IFN-ˠ production were observed [13]. And also to study the
type of oocyst antigencity by western blot technique.
MATERIALS AND METHODS
Parasite propagation:
Local isolate of Eimeria tenella was obtained from (Dr. Katranji M.M.,Parasit
Lab./College of Veterinary medicine /Hama/Syria )and propagated throughout 3 weeks old
chickens (Broiler,Ross.308).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
365
Oocysts were collected from the ceca of infected chickens at the 7th day post infection .After
sporulation with potassium dichromate at 28 C◦ for 6-7 days , oocysts were purified by
standard salt flotation technique and sterilized by sodium hypochlorite treatment as described
previously [16]. Sporulated oocysts were stored in phosphate buffer saline (PBS PH=7.6) at 4
c◦ until further use.
-Preparation of oocyst protein (vaccine):
About 2 ml of suspension containing about (4x 107) of purified sterilized oocysts (Pic. 1).
Had previously been vigorously mixed with glass beads for 10 min. on vortex, then glass
beads washed with minimal amount of PBS. The suspension of oocysts, sporocysts,
sporozoites and walls was frozen at -196 c◦ in liquid nitrogen and defreeze in water bath at
45 c◦ for 3 times .Lysate buffer (0.5%Nonidet P40, Tris-HCL 10Mm , Aprotinen 0.1 U/ml
,1% Triton X -100) were added to the suspension (200 μl /1.5 ml)and incubated for 24h. at 4
c◦ with vortex . Centrifugation was done for the suspension at 2000 rpm for 10 min and the
supernatant were taken as a source of protein( vaccine) .Concentrations of protein were
determined by the method of Bradford assay[17].
- Chickens field experiment:
Eighty chicks of Broiler (Ross 308) at age of one day- old, coccidiosis free, were obtained
from (Hama,Syria)hatcheries. The source of drinking water was from the main water supply
and the feeding was on non medicated broiler diet (according to animal nutritional
requirement of local feed tables) [18] as mash ad libitum. Throughout the study, birds were
maintained in three separated floor pens and on litter composed of wood shaving to a depth5
cm. Temperature in the floor pens was maintained at 20-30 C◦ .Extreme mangement was
taken to avoid accidental exposure of chicks to coccidia during immunization period. In
addition feces were examined periodically by the flotation technique for the absence of
coccidial oocysts. The birds were grouped (20-30 chicken per group) at first day of hatching
as in table (1):
Table (1) Groups of experimental design
Groups Type of groups
G1 Vaccinated with oocyst protein, challenged group(20 Birds)
G2 Unvaccinated, challenged group(30 Birds)
G3 Unvaccinated, Unchallenged group(30 Birds)
-Immunization:
A total number of 80 broiler chicks (Ross, 308) one-day old were divided into 3
groups( G1,G2 and G3) (Table 1). G1 was immunized subcutaneously(S/C )in the neck with
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
366
two doses: first dose at 3rd day of age with 25μg antigen (oocyst protein) , and booster dose
was given at 16th day of age with the same dose of protein. After two weeks of last
immunization an oral inoculation with 104 of virulent Eimeria tenella sporulated oocysts was
done for all groups except G3 which was kept as unimmunized unchallenged control. Chicks
in group G2 challenged only but were not immunized.
Blood collection:
Blood was collected from the wing vein from all chickens post first dose of 7th days
old chicken and after second dose of oocyst protein antigen at 28th days old chicken and also
collected at the end of experiment at 39th day of age chicken post challenge. Sera were stored
at – 20 c◦ until use.
Antigens characterization:
E.tenella oocyst antigens were identified by Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE).
The extraction of E.tenella oocyst protein (50 μg) per lane were lysed by boiling in
reducing loading buffer {LB;25 %glycerol , 5% β-Mercaptoethanol , 10% sodium dodecyl
sulfate (SDS), 0.01% Bromophenol blue in Tris /Hcl (PH=6.8)16mM}.Then separation was
done by using (SDS-PAGE) on a 5-15 gradient gel. Polypeptide in the gel were transferred
electrophoretically to nitrocellulose paper (BA85; 0.4 μM; Scheleiche&Schull,Inc.)in a
transblot transfer cell(Bio-Rad Laboratories).Electrophoresis was done with transfer buffer at
4 ˚c for 1.15 h at constant 250 A .After transferring, the nitrocellulose paper containing the
polypeptid was washed two times for 5 min each time ,with distilled water .Excess binding
sites on the nitrocellulose paper were blocked by washing the paper with Tris buffer salin –
tween(TBS-T)PH=7.5{10mM Tris-Hcl ,154mM Nacl and 0.1 % Tween -20} plus to 3%
povine serum albumin ,for 24 h at 4 ◦c.
The nitrocellulose paper was further washed 3 times for 5 min. with (TBS-T)
buffer on rotator shaker. The membrane was then cut into strips which were separated into
individual container and exposed to a 1:200 dilution of the experimental chicken sera in
(TBS-T) buffer for 1h .Following washing, the nitrocellulose membrane was incubated for 1h
in a 1:1000 dilution of{ Rabbit anti – chicken IgG (H&L) conjugated to hores radish
peroxidase (Invitrogen Company/California/USA ) },washed (3x5 min) in TBS-T buffer ,and
finally incubated in OPD(Ortho Phenil diamin)substrate solution for 15-20min with rotated
shaker until band had been appeared. The reaction was stopped by washing nitrocellulose
stripes for 2 times with D.W
Production of interleukin -4(IL-4) and interferon -ˠ (IFN-ˠ) induced by oocyst protein in
chicken:
Serum chicken sample measured at 7th, 28th and 39th days of age with ELISA kits
(Life Science Inc. USCN) for IL-4 and IFN-ˠ according to the manufacturer's instructions.
Optical densities of kit standards and test samples were read at (450 nm) using an ELISA
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
367
plate reader (HumaReader HS, Human,Germany ). The results were described as pictograms
of IL-4 and IFN-ˠ per 100 μl of samples.
RESULTS AND DISCUSSIONS
Picture(1): Oocyst of Eimeria tenella (100x)
In the current study we prepared oocyst protein from the local strain of E. tenella
oocysts as a vaccine to immunize broiler. It was generally accepted that asexual stages
produce the strongest stimulus for development of immunity [18]. For a better understanding
of strain variation is needed for any vaccine to give promising results against local field
strains of Eimeria [20]. So, the present study has tested the type of immune response against
the asexual stage of oocyst protein of local strain of E. tenella as a vaccine.
There was no contamination by extraneous coccidian throughout this study, that
demonstrated the success of the field experiment.
Antigens analysis
Polypeptides of E.tenella were obtained for analysis by the separation of oocyst
antigens by SDS-PAGE. There are 21 polypeptides were prominent stained strongly with
coomassie blue. Their molecular weight were 174.5,165.3,129,113.8,89.7
83.9,65.4,57.7,42.5,41.1 ,33.2 ,27.5, 25.9, 22.9, 16.8, 13.9 12.3, 11.6, 10.9 and 9.6 KD. Pic.
(2).
Oocyst wall
Sporocyst wall
Sporozoites(2)
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Picture (2): SDS-PAGE(5-15%) stained with Coomassie blue after electrophoretic
separation of oocyst proteins( OP).
Murray and Glausk [21] detected thirteen polypeptides with MW (235,175, 105, 94, 88, 82,
80, 68, 60, 50, 45, 28 and 26 )KD. from oocyst of E.tenella by using SDS-PAGA. In
comparison. Talebi [22] found eleven polypeptides with molecular weight (116, 84, 66, 58,
48, 45, 36, 24, 22, 20 and 14) KD during his study on E.tenella oocyst by use SDS-PAGE.
The apparent differences in molecular size are presumably due to minor technical
differences associated with measurement at these sizes with gel [23]. Stotish et al. [24]
reported that polypeptide was somewhat heterogeneous in molecular size , possibly because
of the random cleavage of smaller peptide fragment during solubilization.
In western blot, the reaction of parasite-specific IgG( H&L) antibodies with oocyst antigens is
differ when each of 3 immune sera are used as a probe in the immunobloting procedure .
These differences were most obvious between molecular weight 12.3 and 167.8KD Pic. (3).
Immunized serum of 39 day age of chicken consistently identified and reacted more intensely
with more numbers of antigens in oocyst preparations than immunized serum at 28-7 day of
150
100
80
60
40
30
10
11.6
10.9
9.6
12.3
13.9
16.8
22.9
25.9
27.5
33.2
41.1
42.5
55.8
57.7
65.4
83.9
89.7
113.8
129
165.3
OP Standard
174.5
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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age of the chicken. These antigens with a molecular weight (167.8, 114.5, 83.4, 78.2, 73.5,
53.3, 44.1, 38.8, 36.4, 28.2, 20.5, 18,14.9 and 13.9) KD were identified with immune serum
post of challenge dose, by E. tenella parasite .
The immunized serum at 7 days of age was reacted slightly when used as a probe. Six
antigens have been reported with a molecular weight (116.2, 110,99,59.8,58.2,40 and 14.1 )
KD .In comparison eight reacted immunized serum were identified at 28 day age.Their
molecular weight were (167.8,73.5,53.3, 41.3, 38.8, 34.1, 15.8 and 12.3) KD. Immune serum
of 28 and 39 days age reacted with five common antigens (167.8, 73.5, 53.3, 38.8 and 15)KD.
Picture(3): Immunoblot of proteins transferred from a 5-15 %SDS-PAGE. Lanes are
immunoblots of Oocyst extract preparations probed with immune serum (7th days)
:after first dose of vaccine ; (28th days): after second dose of vaccine and (39th days) after
challenge with E.tenella oocysts.
In this study, there were differences in the identification of oocyst antigens and staining
intensities among sera that were collected from chickens at (7, 28, 39) days of age. We found
that the number of proteins and intensity of reaction was increased after second dose of oocyst
antigen and after challenge with E.tenella parasite which might be due to the type of using
IgG conjugate.
10
30
40
60
80
150
100
167.8
114.5
83.4
78.2
73.5
53.3
44.1
38.8
36.4
28.2
20.5
18
14.9
13.9
167.8
73.5
53.3
41.3
12.3
15.8
34.1
38.8
99
110
116.2
59.8
58.2
40
14
7th days 28thdays 39th days Standard
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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IgM produced in largest amount during primary response, with more IgG being produced
during subsequent exposures to the stimulating antigen. Responses are generally stronger and
more rapid at second exposure [25]. Tress et al. [26] found during his study that IgM is the
class of antibody which appears first following E. tenella infection before IgA and IgG types
of antibody are produced, and serum IgM and IgG were peaking at 13th and 17th days post
infections with E. tenella parasite. Another study demonstrated that after E. tenella infection,
significantly higher levels of IgM were produced in the cecum at 7 and 14 days post infection
[27]. In contrast Davis [28] reported that the maximal antibody production varies (from 2 to 4
week pi) as humoral antibody responses to avian coccidian which was depended on
immunogenicity of the Eimeria species, being earlier in infection with E. maxima than with
E.tenella .
Murray and Glausk [21] observed seven polypeptids that reacted strongly with anti sporozoite
sera when used oocyst protein as an antigen in western blot analyses ,these were
235,105,94,71,64,45 and 26 )KD molecular weight. This finding indicates that those proteins
are strong antigens. Another study found four immunogenic bands (45,22,20 and 14 )KD in
oocyst proteins of E.tenella [22]. There proteins were slightly differed from those observed by
Hasbullah et al. [29] who revealed three common immunogenic bands (23,21 and 14KD)
between oocyst and seconed generation merozoites of this species. These variations in size of
immunogenic protein bands might be due to antigencity of coccidial strains which vary
geographically [30]. In addittion, certain strain of Eimeria exhibits immunological variation
[31] Experiment of Passive immunizations showed a good correlation between the intensity of
IgG and IgM antibodies that binding to gametocyte antigens by both western blot and ELISA
with the ability of those sera to provide passive protection in vivo[17].
Evalution of IFN-ˠ and IL-4 production:
After immunization with oocyst protein, IFN-ˠ and IL-4 levels in the serum samples were
examined using an ELISA assay. The average expression level of IFN-ˠ was estimated to be
87.8 pg/100 μl at day 39, which was more than fifteen fold increase as compared with control
negative group (the data shown in fig.1).
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure(1):Level of IFN-ˠ in serum of broiler after first vaccine dose(firs.dos),second dose(sec. dos) with
oocyst protein and after challenge(aft.chall) with 104 oocyst of Eimeria tenella .
Li et al. [32] found that IFN-ˠ levels in the serum samples were significantly higher when
compared with those of the PBS group using rhomboid- like protein of E. tenella as a subunit
vaccine in protective immunity against homologous challenge,and also a marked increase in
IL-2 level in the serum samples as compared with the control group. Laurent et al.[33]
showed that IFN-ˠ expression in the cecum and jejunum of White Leghorn chickens increased
more than 200-fold at 7 days post primary infection with E. tenella and E. maxima .Hong et
al. [34] supported these observations by finding a significant up regulation of IFN-ˠ
expression in the duodenum of E.acervulina infected chickens and ceca of E. tenella infected
chickens, probably due to the recruitment and stimulation of TCR2+ and CD4+ IELS
(intraepithelial lymphocytes), respectively. Lillehoj et al. [15] found during their study on
cytokine, it does inhibit E. tenella development in vitro. In the peripheral blood, a transient
but sharp increase in the proportion of CD8-expressing T cell was found in White Leghorn
chicken at 8 days after a primary infection with E. tenella [35]. This increase was found to be
concurrent with a marked increase in interferon IFN-ˠ and in nitric oxide (NO) production
upon in vitro stimulation of PBL of T-cell mitogens and E. tenella sporozoite antigen[35].
The effect of IFN-ˠ against E. tenella by using immunohistochemistry showed that
macrophages were surrounding E. tenella sporozoites within 48h after intra cecal inoculation
[36]. There is experimental evidence from in vitro data that IFN-ˠ may stimulate macrophage
and neutrophils to produce reactive oxygen intermediated which kill Eimeria sporozoites [37].
Production of NO2ˉ + NO3ˉ and IFN-ˠ in serum increased during host response to
infection of Eimeria [38,37].
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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In this study we, observed high level of IFN-ˠ production after
challenge as compared with control negative group, which increase after first and second dose
of vaccine from control negative group. Because host response to the infection after the
challenge and the intestinal lesions in coccidiosis are caused, in part. During infection,
cytokines such as IFN-ˠ can stimulate inflammatory cells like macrophage to synthesize
highly reactive free radical, NO. These NO are not only toxic the invading parasite but also
can damage the host tissue [39].Therefore, IFN-ˠ is highly post challenge.
The average expression level of IL-4 was estimated to be 158 pg/100 μl at day 39, which
appeared higher than control negative group which was 41.7 pg/100 μl (the data shown in
fig.2).
The results showed that there were an increase level of IFN-ˠ and decrease in the level of
IL-4 in serum samples after second dose of vaccination.
Figure (2):Level of IL-4 in serum of broiler after first vaccine dose(firs.dos),second dose(sec.dos) with
oocyst protein and after challenge(aft.chall) with 104 oocyst of Eimeria tenella .
Chicken Th2 cells are necessary for inducing the humoral response to combat parasite
invasion [40,41]. In this study the level of IL-4 in serum of immunized chicken was higher
after the first vaccine dose and then drastically decreased after the second dose of vaccination,
and return to high level after challenge. Hong et al. [34] found during their study on analysis
of chicken cytokine and chemokin gene expression following E.acervulina and E. tenella
infections, IL-4 and IL-13 mRNAS were decreased 25 to 2 x 105- fold after primary and
secondary infection.
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The differences between IL-4 and IFN-ˠ might be due to the mutual antagonism of them
action, IFN-ˠ acts on B cells, T cell, NK cells and macrophage. In addition, IFN-ˠ stimulates B
cell to produce of IgG2a and lowers production of IgG3, IgG1b in mice. It enhances T cell
production of MHC class I molecules but not the production of MHC class II molecules. It
induces Th1 cells to produce both IL-2 and IL-2R.It acts on Th2 cells to inhibit the production
of IL-4 and as a result blocks IgE production in vitro. IFN-ˠ enhances the functions of NK
cells and activates macrophages and greatly increases their ability destroys ingested
microorganisms. It promotes antibody –mediated phagocytosis as well antibody –dependent
cell- mediated cytotoxicity (ADCC) reactions. IFN-ˠ increases MHC class I expression on
tumor cell lines and induces the appearance of MHC class II molecules on endothelial cells,
and fibroblasts, as well as on macrophages [42].
A previous study on this oocyst vaccine has reported (67-69%) protection, number of oocyst
per gram of feces and cecal lesion score from chickens in the immunized groups with oocyst
protein decreased significantly from control groups . The body weight gain was not affected,
also when estimated anti-coccidia index (ACI) which has been revealed the oocyst protein
slightly effective [43].
The immune response to our experimental vaccine demonstrated humoral and cellular
protection. Li et al.[32] suggested during their studied, specific IgG antibody responses
against E. tenella was generated in the chickens immunized with recombinant rhomboid like
protein expressed in E.coli and this protein is capable of eliciting humoral response and
activating cell- mediated immunity in birds. Akhtar et al. [44] showed the humoral and
challenge responses when use supernatant from sonicated sporulated oocyst that induced a
strong protection as immune chicks contain high level of antibodies to resist heavy dose of
challenge .Similar finding was also observed by [9,14]. .In contrast, another study found the
antibody responses have a minor role in protective immunity to Eimeria spp. because
agammaglobulinemic chickens produced by hormonal and chemical bursectomy are though
resistant to reinfection with Eimeria spp.[45]. Therefore, Th1 responses seem to be dominant
during coccidiosis, as best manifested by proving the involvement of IFN-γ [5]. Subramanian
et al. [39] found during his study on the (EtMIC1)a recombinant E. tenella sporozoite antigen
in birds could induce high antibody and strong cell mediated immunity responses. Previous
studies agree with this observation when used different antigens [46,47] . In contrast, Wakelin
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and Rose [48] found both humoral and cell-mediated mechanisms are involved in anticoccidial
immunity but it has been suggested that the latter plays a major role in protection.
Therefore, different antigens preferentially stimulate different immune responses [49].
Finally it was found that in order to a chief protective immunity by using parasite extracts, it
requires the inclusion of the correct antigens and exclusion of the irrelevant ones [17]. This
indicates while some parasite-specific antigens induce protective immunity, others actually
induce an exacerbation of the infection. Therefore, in the design of any parasitic vaccine, it is
crucial that the combination of various antigens maximizes their inhibitory effect of the
parasite growth and development [50].
CONCLUSION
oocyst protein vaccine has many immunogenic antigens which reacted more intensely from
other protein. We recommend to use these antigens as a vaccine of single band to determine
which band has a most immunogenic pattern. In addition during this study, we determined
two types of immune responses to our use oocyst protein vaccine to locale Syria isolate of
E.tenella parasite which was humoral and cellular immune responses.
تقییم الاستجابة المناعیة الخلطیة والخلویة للقاح البروتینی للکیسات البیضیة لطفیلی الایمیریة فی
دجاج اللحم Eimeria tenella تنلا
*سھیر ریاض الادریسی، **محمود قویدر،*** محمد محسن قطرنجی
* فرع الاحیاء المجھریة ، کلیة الطب البیطری ,جامعة البصرة- العراق.
** قسم علم الحیاة الحیوانیة , کلیة العلوم , جامعة دمشق , سوریة.
*** کلیة الطب البیطری, جامعة البعث-سوریة.
الخلاصة
ترمی ھذه الدراسة تحدید نوع الاستجابة المناعیة للقاح الکیسات فی دجاج اللحم ضد طفیلی الایمیریة تنلا .اذ تم تلقیح
دجاج اللحم بجرعتین من اللقاح المحضر من السلالھ المحلیة للایمیریة تنلا لطور الکیسات البیضیة کبروتین. تم اعطاءه
بواقع جرعتین ( 25 میکروغرام لکل دجاجة)، فی الیوم الثالث والسادس عشر من عمر الدجاج تحت الجلد،واعطیت جرعة
التحدی بعمر 30 یوم،سحب الدم بعمر ( 7و 28 و 39 ) یوم من عمر الدجاج ، درست القابلیة المستضدیة للقاح بأستعمال
الرحلان الکھربائی العمودی والتبصیم المناعی وحدّد اربعة عشر نوع من متعددة الببتیدات وکانت اکثر استجابة مناعیة بعد
-53.3 -78.2 -83.4-114.5- فحصھا ضد امصال الدجاج الممنع بعمر 39 یوم حیث تراوحت اوزانھا الجزیئیة( 167.8
( INFγ) 13.9 ) کیلو دالتن وتم تحدید مستوى الانترفیرون – غاما -14.9 -18 -20.5 -28.2 -36.4 -38.8 -44.1
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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فی امصال الدجاج المحصن بأستعمال کیت الالیزا. اوضحت النتائج نوعین من الاستجابة (IL-4) والانترلوکین - 4
المناعیة الخلطیة والخلویة ضد لقاح البروتینی للکیسات البیضیة لطفیلی الایمیریة تنلا.
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