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Development of Dot – Enzyme Linked Immunosorbent Assay for Detection of
Antibodies to Infectious Bursal Disease, Hydropericardium Syndrome and
Chicken Anaemia Viruses
K.V.Subramanyam*1, V.Purushothaman2, B.MuraliManohar3, G.Ravikumar4 and S.Manoharan5
Department of Veterinary Microbiology, Madras Veterinary College, Vepery, Tamil Nadu Veterinary and Animal
Sciences University, Chennai 600007, Tamil Nadu, India
*
Corresponding Author: K.V.Subramanyam
1
E-mail: vkothapalli2001@yahoo.com, 2E-mail: drvpurushothaman2001@yahoo.com,
3
E-mail: drbmmin@yahoo.com, 4E-mail: capsidvet@rediffmail.com, 5E-mail: ulagaimano@yahoo.com
ABSTRACT
A rapid and sensitive dot-ELISA test was developed for the simultaneous detection of antibodies against infectious
bursal disease (IBD), hydropericardium syndrome (HPS) and chicken anaemia (CAV) viruses in a single serum
sample. The developed dot-ELISA was compared with the virus neutralization test (VNT). For IBD and HPS,
viruses isolated and purified from field outbreak materials were used while VP1 ORF3 recombinant protein
expressed in DH5α E.coli was used for CAV as coating antigens. Sera samples from 302 breeder chicken were
screened for antibodies to the three viruses. A total of 195, 205 and 104 serum samples were found positive for
IBDV, HPSV and CAV respectively. By comparing with VNT sensitivity, specificity and accuracy of the test were
calculated. The dot-ELISA test was found to be highly sensitive, specific with a K value of 0.99 and hence could be
very useful for serological diagnosis of IBDV, HPS and CAV in a single serum sample.
Keywords: Chicken anaemia virus - Diagnosis - Dot-Enzyme linked immune assay - Hydropericardium syndrome
virus - Infectious bursal disease virus - Virus neutralization test
1. INTRODUCTION
The important immunosuppressive viral infections of poultry viz., infectious bursal disease virus (IBDV) and
chicken anaemia virus (CAV) are paving path for so many other infections. Infection with IBDV causes impaired
immune response to many viral infections like Newcastle disease virus [1-2], making the chickens highly
susceptible to other viral infections like inclusion body hepatitis [3], Marek’s disease [4-5], infectious bronchitis [6],
infectious laryngotracheitis [7], chicken anaemia virus [8] and reoviruses [9]. Along with viral infections, IBDV
infection also increases susceptibility to secondary bacterial infections like S.typhimurium and E.coli [10], S.aureus
[11], and coccidial infections [12-13]. Infectious bursal disease (IBD) causes high mortality in 3 to 6 week old
chicken, where as less acute or subclinical disease is common in 0 to 3 week old chicken [14] and IBDV is
immunosuppressive at an early age. CAV causes disease in 2 to 4 weeks old chicken and affected flocks show
retarded growth and mortality is generally between 10-20% [15-18]. The appropriate breeder flock
hyperimmunization will transfer adequate levels of maternal antibodies to hatchlings until chicks develop age –
resistance. The fine-tuning of breeder flock immunization can be achieved by screening with serological tests.
Serological diagnosis based on ELISA has been a rapid and reliable method of screening large number of samples.
However, these methods can only be performed under laboratory conditions causing delay in serological screening
of large flocks. There is a need for a simple, rapid yet accurate method of diagnosis that can be performed at farm
level. Hence, an immunocomb based dot –ELISA was developed with specially designed trough was developed to
assess the antibody status for IBD, HPS and CAV in single test sera.
2. MATERIALS AND METHODS
VIRUSES AND ANTIGENS
Virus isolation and Purification:
The field isolate of IBDV from an outbreak in the Namakkal poultry belt of Tamil Nadu, India was used and the
virus was isolated as per the method described by Thangavelu [19]. Briefly, the bursae collected from the disease
outbreak were made into 50 per cent (w/v) suspension in phosphate buffered saline (PBS, pH 7.2) by triturating in
sterile sand and the homogenate was centrifuged at low speed for 20 minutes. The virus was extracted from the
supernatants with chloroform [20]. After treating the clear aqueous phase with antibiotics, the virus presence was
confirmed by agar gel immunodiffusion (AGID) and counter immune electrophoresis (CIE) and stored in small
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aliquots at -700C until further use. The virus obtained from 50% bursal suspension was further propagated in 12 day
old specific pathogen free (SPF) embryonated chicken eggs by chorioallantoic membrane route (CAM) and in
chicken embryo fibroblast (CEF) cultures obtained from SPF eggs. The virus material has produced characteristic
lesions in embryos by 3 to 5 days and the harvest was further adapted to CEF cultures which has produced
characteristic cytopathic effects (CPE). The cell culture virus was further purified as per the method described by
Dobos et al [21] with few modifications. The purified viral proteins were analyzed by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS –PAGE) as described by Laemmli [22].
The 50% and 25% hydropericardium syndrome virus suspensions were extracted with chloroform from the livers of
affected chicken in an outbreak near Coimbatore poultry belt of Tamil Nadu, India. The clear extract supernatants
were used for further work after treating with penicillin (5000 I.U/ml) and streptomycin (5000 μg/ml) and the virus
was confirmed by both AGID and CIE as per method described by Roy et al [23]. Further, the HPSV was adapted to
chicken embryo liver cell (CEL) culture from SPF eggs [24] and the CEL adapted HPSV was purified as described
by Ahmad and Burgess [25] and purified viral proteins were analyzed by SDS – PAGE [22].
The CAV VP1 recombinant clones were obtained from department of animal biotechnology, Madras Veterinary
College, TANUVAS, Chennai, India.
PCR for CAV:
The VP1 ORF3 was constructed in pPROEXHTb plasmid and expressed in DH5α E.coli. The clones were further
confirmed for the presence of CAV VP1 insert by extracting the plasmid DNA by alkali lysis method [26]. The
plasmid DNA was amplified for the VP1 gene of CAV in a 50 µl reaction by polymerase chain reaction (PCR). The
forward primer is 5’- TGG CAA GAC GAG CTC GCA GAC C-3’ and reverse primer is 5’- CCC AGT ACA TGG
TGC TGT TCG-3’. The cycle conditions employed were 940C for 2 minutes, 940C for 1 minute, 600C for 1 minute
and 720C for 2 minutes for 34 cycles and a final extension of 720C for 10 minutes followed by holding at 4 0C. The
PCR product was electrophoresed on 1% agarose with DNA marker.
Restriction digestion of CAV:
The PCR product was subjected to restriction enzymes digestion using Sal I and Xho I enzymes after purification by
EZ – 10 spin column PCR products purification kit (Bio Basic Inc., Canada). After confirmation of VP1 clones, the
recombinant protein was induced in bulk with 1% isopropylthio-β-D-galactoside (IPTG) and the expressed
recombinant protein was purified by using Ni CL agarose columns (Genei, Bangalore, India) and western blot was
performed with CAV specific antiserum from CR Laboratories, USA.
ANTIBODIES AND ANTISERA
A group of 6 chickens each for IBDV and HPSV were immunized by mixing 500 mg/100 µl quantity of both viruses
separately with 1 ml of Freund’s complete adjuvant (Sigma, USA) subcutaneously by keeping 4 chickens each for
both viruses as control. After one week first booster was given and second booster was given 14 days after 1 st
booster dose with Freund’s incomplete adjuvant (Sigma, USA). The presence of antibodies was observed by test
bleeding the birds after two weeks of last injection by AGID and CIE. Then, a large amount of blood was collected
from chicken and the separated serum was stored in small aliquots at -200C until further use. Antiserum against
CAV VP1 was raised in rabbits, approximately 1 mg per ml of purified recombinant VP1 was mixed thoroughly
with an equal volume of Freund’s complete adjuvant and the total quantity was given into two rabbits
subcutaneously at different places. One week of first dose, a second dose was given with Freund’s incomplete
adjuvant and rabbits were test bled one week after giving second booster and the serum was separated and stored
until further use.
TEST SERUM SAMPLES
A total of 302 serum samples were collected from the broiler breeder flocks located around Coimbatore of Tamil
Nadu state, India and these serum samples were inactivated at 56 0C for 30 minutes and stored at -800C until further
screening by VNT and dot-ELISA.
DESIGN OF IMMUNOCOMB AND DOT-ELISA
The antibodies to IBDV, HPSV and CAV at a time in a single serum sample by dot - ELISA were detected as per
the method described by Manoharan et al [27]. The purified antigens of IBDV, HPSV and CAV at 1 µl volume (150
ng / µl) were spotted onto a nitrocellulose membrane of immunocomb having 3 rows on 12 flanges. The purified
antigens of three viruses were spotted on the first, second, and third rows respectively and the strips were incubated
at 370C for 1 hour in a humid chamber. One hour after incubation the combs were washed three times with wash
buffer (PBST) and incubated for 60 minutes in blocking or serum dilution buffer. In the present study, a 7.5% of
skim milk powder (Himedia, India) in PBST was used instead of routine 5%. The combs were again washed three
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times with PBST and then incubated at 370C for 45 minutes with serum samples from the field diluted 1:100 in
serum dilution buffer. After incubating with serum samples washed three times in PBST and incubated with antichicken Ig Y HRP conjugate (Sigma, USA) diluted 1:1000 in conjugate diluent. Finally, the combs were exposed to
the substrate (5 mg of diaminobenzidine (Sigma, USA) in 10 ml of distilled water and 10 µl of hydrogen peroxide)
for 10 minutes. The enzymatic reaction was stopped by washing the combs with tap water. The positive reaction was
indicated by the appearance of a brown dot within 10 min. A special trough was designed and fabricated with acrylic
material that allowed the immersion of dot-ELISA comb completely.
VALIDATION OF THE TEST
Virus neutralization test:
The 302 serum samples collected from broiler breeders were screened for the presence of antibodies to IBDV and
HPSV by VNT. The VNT for detection of IBDV antibodies was carried out as per the procedure described [14].
Briefly, 50 µl of virus containing 100 TCID50 was placed in each well of a tissue-culture microtitre plate (Nunclon).
The test serum samples were serially diluted by two fold with the virus placed in wells. After incubating for 30
minutes at 370C, 0.2 ml of chicken embryo fibroblast cell suspension, with a cell density allowing confluent
monolayer to be obtained after 24 hours of incubation, was dispensed into each well. Plates were sealed and
incubated at 370C for 4-5 days, after which the monolayers were observed microscopically for typical CPE. The
end-point is expressed as the reciprocal of the highest serum dilution that did not show CPE.
The VNT for HPSV was carried out with chicken embryo liver cell culture [28]. Initially, doubling dilutions of
serum were made in tissue culture medium and then 50 µl of each dilution was added to microtitre plate already
containing 100 TCID50 units of virus. Following reaction time of 1 hour at 37 0C 100 µl of freshly prepared CEL
culture was added to all wells. Plates were sealed and incubated at 37 0C for 4-5 days, after which the monolayers
were observed microscopically for typical CPE. The end-point was expressed as the reciprocal of the highest serum
dilution that did not show CPE.
STATISTICAL ANALYSIS
The sensitivity, specificity and accuracy of dot ELISA, flow through were compared with the virus neutralization
test as described by Venkatesh [29].
Sensitivity:
a / (a+c)
Specificity:
d / (b+d)
Accuracy:
a+d / (a+b+c+d)
The results obtained from the tests were analyzed for the percentage of agreement with virus neutralization test with
the use of Kappa statistics. The kappa statistics is a decimal measure of agreement between two tests, especially in
the absence of a standard and is defined as kapp or K. K = (a+d – P) / 1- P, where P= (a+b) (a+c) + (c+d) (b+d) and
P is the probability, a: is the number of samples positive by both i.e., test to be compared and gold standard test, b: is
the number of samples positive by standard test whereas negative by test to be compared, c: is the number of
samples negative by standard test and positive by test to be compared and d: is the number of samples negative by
both.
Guide lines for interpretation of K values are as follows:
k value < 0.1 indicates poor agreement
k value between 0.1 and 0.2 indicates slight agreement
k value between 0.21 and 0.4 indicates fair agreement
k value between 0.41 and 0.6 indicates moderate agreement
k value between 0.61 and 0.8 indicates substantial agreement
k value > 0.81 indicates perfect agreement
3. RESULTS
The IBDV showed five structural polypeptides with molecular weights of 90,000 Da (VP1) a RNA dependent RNA
polymerase of the virus playing key role in encapsidation of the viral particles, 40,000 Da (VP2) had been identified
as host protective antigen and induced the neutralizing antibodies, 32,000 Da (VP3) a group specific antigen and
recognised by non-neutralizing antibodies, 28,000 Da (VP4) a minor, non-structural polypeptide and involved in the
auto-processing of the polyprotein as a virus-encoded protease producing VP2a, VP3 and VP4 itself and a precursor
polypeptide of 49,000 Da (VPx) were detected in the SDS-PAGE [21 and 30]. The purified viral proteins of IBDV
were shown in Fig.1.
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In the present study, the polypeptides of purified virus HPSV were resolved by SDS-PAGE. In 12.5% acrylamide
gel, the purified virus yielded 12 polypeptides. This finding was in agreement with some of the workers Rajesh
Kumar and Rajesh Chandra [31]. On the contrary, some workers got 8 polypeptides on resolution in 10% gel [32].
This difference in number of polypeptides might be attributed to difference in concentration of resolving gel and
various other physical conditions. The purified viral proteins of HPSV were shown in Fig.2.
The VP1 insert PCR amplified product of CAV was of approximately 1.3 kbp Fig.3. The restriction enzyme
digestion of VP1 cloned plasmid of CAV yielded approximately 6 kbp and 1.3 kbp size fragments Fig.4. The peak
expression of VP1 recombinant protein was noticed after 5 hours of expression Fig.5 and the purified recombinant
protein was of approximately 51 kilodaltons (kDa) Fig.6. The Western blot analysis of CAV VP1 recombinant
protein was performed using CAV specific antiserum. The recombinant protein blotted nitrocellulose membrane
after reacting with CAV antiserum showed a single band with a size of 51kDa as shown in Fig.7.
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A total of 302 serum samples were screened for the presence of IBDV and HPSV antibodies by VNT. The serum
samples were diluted serially from 1:10, 20, 40, 80, 160, 320 and 640 (log 2 3.3 to log2 9.3). The neutralization titres
less than 4.9 on log2 scale were considered as negative and above 5.0 as positive for IBDV [33]. The results are
presented in Table 1. Out of 302 serum samples screened 182 serum samples were positive and 120 serum samples
were negative. In case of HPSV, the neutralization titres less than 5.9 on log 2 scale were negative and above 6.0
were positive [34]. A total of 165 serum samples were positive for HPSV antibodies and 137 were found to be
negative as presented in Table 2. As the MDCC MSB cell line was not available, VNT for CAV antibodies was not
carried out and compared with that of dot-ELISA.
TABLE 1: Screening of serum samples by VNT for IBDV antibodies
1
Numerical
dilution
1/10
2
1/20
4.3
61
3
1/40
5.3
41
4
1/80
6.3
66
5
6
1/160
1/320
7.3
8.3
54
15
7
1/640
9.3
6
S.No.
Log2 dilution
No. of samples
3.3
59
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TABLE 2: Screening of serum samples by VNT for HPSV antibodies
1
Numerical
dilution
1/10
2
1/20
4.3
39
1/40
5.3
56
4
1/80
6.3
84
5
1/160
7.3
64
1/320
8.3
12
1/640
9.3
5
S.No.
3
6
7
Log2 dilution
No. of samples
3.3
42
The same 302 serum samples were screened for the presence of antibodies to three viral antigens viz., IBDV, HPSV
and CAV by dot-ELISA and the results were presented in Table 3. A total of 195, 205 and 104 serum samples gave
positive reaction in dot-ELISA for IBDV, HPSV and CAV antibodies respectively Fig.8. The comparison of dot –
ELISA with VNT (for IBDV and HPSV) was provided in Tables 4 & 5, along with statistical analysis. The trough
specially designed to carry out dot – ELISA comb was provided as Fig. 9.
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TABLE 3: Screening of serum samples by Dot ELISA
Positive
195
205
104
IBDV
HPSV
CAV
Negative
107
97
198
TABLE 4: Comparison of VNT and Dot ELISA for IBDV antibodies
VNT
Dot-ELISA
Positive
164a
18c
182
Positive
Negative
Total
Specificity
Sensitivity
Accuracy
K value
:
:
:
:
Negative
31b
89d
120
Total
19555
107
302
90.1%
74.1%
83.7%
0.99
TABLE 5 :Comparison of VNT and Dot ELISA for HPSV antibodies
VNT
Dot-ELISA
Positive
Negative
Total
Specificity
Sensitivity
Accuracy
K value
a
b
c
d
:
:
:
:
Positive
160a
5c
165
:
:
:
:
Negative
45b
92d
137
Total
205
97
302
96.9%
67.1%
83.4%
0.99
no. of samples positive by both tests
no. of samples positive by standard tests whereas negative by test to be compared.
no. of samples negative standard test and positive by test to be compared.
no. of samples negative by both.
DISCUSSION
The objective of the present study was to develop immunodiagnostics for the detection of antibodies to IBDV,
HPSV and CAV in poultry flocks. The early chick mortality is an important problem to be looked in and the breeder
flock vaccination plays crucial role in curtailing the early chick mortality. Age limiting diseases like IBD and CAV
can be controlled by transfer of enough antibodies to the chicks and this is achieved by hyper immunization of
breeder flocks. The vaccination schedule of breeder flocks can be fine tuned based on the antibody status. The
conventional serological tests are time consuming and cumbersome and a pen-side test is highly suitable for this
purpose. Hence, the dot-ELISA was developed for screening antibodies to three viruses’ viz. IBDV, HPSV and CAV
at a time. The combination of these three viruses is so important in that the IBDV and CAV cause
immunosuppression and the adenoviruses, especially the HPSV, take upper hand during the phases of
immunosuppression further worsening situation. The monitoring of breed flock antibody status reveals optimum
protection to infections, to know the improvement of vaccination programme and to have an idea regarding the
infections status of flock. For these reasons, seromonitoring has to be done on a permanent basis [35].
The virus neutralization test is the reliable serological test which besides differentiating IBDV isolates into antigenic
serotypes and subtypes [36], the time of vaccination of breeder flocks can also be determined [37]. The IBDV
neutralizing antibodies were detected as early as 3 or 4 days post infection [38] and even low levels of circulating
antibodies (titres of 16 to 64) can be detected by VNT. In spite of the sensitivity of neutralization test, it is time
consuming, demanding sophisticated facilities, requires expertise both for testing and interpretation. In the present
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study, out of 302 serum samples screened for antibodies to IBDV by VNT, 182 serum samples were positive and
120 serum samples were negative (Table.1). With respect to the HPSV, in the present study, 302 serum samples
were screened and 165 serum samples were found to be positive where as 137 serum samples were negative for
HPSV by VNT (Table.2). The neutralization assay gives information on both serotype and neutralization titre and
most of the neutralization assays with regard to adenoviruses were done for serotyping of isolates [39] and
classification [40] but not for obtaining neutralization titres.
It could be surmised that the chicken with negative titre must have not transferred enough immunity to their
hatchlings which will be under constant threat to IBDV and HPSV infection till they cross the susceptible age. The
chicken with ‘just’ positive titres also must be considered as they were in border line. The breeder flock
seromonitoring is the only solution in such situations and the tests that are equally sensitive to that of VNT were the
need of the hour which saves time, reagent cost and easy reading of results.
The previous study for detection of antibodies to three viruses’ viz., Newcastle disease (ND), IBD and infectious
bronchitis (IB) by dot-ELISA, is a semi - quantitative method developed by Manoharan et al [27], but the results
were not compared with any gold standard test. The positive samples could be classified as strong, moderate and
weak positives by comparing the colour reaction given by the known strong and weak positive controls. In the
present study, dot-ELISA test for detecting the antibodies to three viruses gave results that were in good agreement
with VNT (for IBDV and HPSV only). Since the MSB1 cell line was not available, the dot-ELISA results of CAV
were not compared and just detected CAV antibodies using recombinant antigen. The kappa (K) value for both
IBDV and HPSV when compared with VNT was 0.99 (Tables 4 & 5), which indicated that they were in perfect
agreement.
The purified antigens used in the present study should have reduced the non-specific reactions. In comparison to
plate ELISA, the developed dot-ELISA can be performed without sophisticated equipment like ELISA reader and
the dot-ELISA can be performed at farm level with minimum reagents without any specialized instruments. The
farm personnel can perform the test as per the protocol and read the results without much effort. Moreover,
antibodies to three viruses can be screened at a time and which reduces the cost of the dot-ELISA. The dot-ELISA is
a semi quantitative assay in which the antibodies level can be quantified up to some extent based on the colour
intensity of the dot. Even a score card can be constructed with different intensities of dot, coding for the
corresponding antibody level with VNT. By knowing the approximate antibody concentration, the breeder flock
vaccination can be rescheduled so as to get uniform, high level of maternal antibodies in all hatchlings.
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