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The Journal of the Egyptian Public Health Association (JEPHAss.)
Vol.81 No. 1& 2, 2006
COMPARATIVE STUDY BETWEEN ELISA,
IMMUNO-DIFFUSION AND CELL BOUND IMMUNO ASSAY TECHNIQUES FOR
DETECTION OF
ANTI-BOVINE VIRAL DIARRHEA ANTIBODIES
IN CALVES OF SOME FARMS IN ALEXANDRIA
AND BEHIRA GOVERNORATES
Bahgat Z Youssef*
*Microbiology Department, Animal Health Research Institute, Dokki, Alexandria Branch.
ABSTRACT
Rapid, highly sensitive and specific technique is essential need for early diagnosis of
Bovine Viral Diarrhea (BVD) infection in calves to prevent, control and eradicate the
persistently infected animals; so, ELISA, Immuno-diffusion (ID) and Cell bound immuno
assay (CBIA) techniques were compared for detection of anti-Bovine Viral Diarrhea (antiBVD) antibodies in 240 calves blood samples of some farms in Alexandria and Behira
Governorates (120 each). Out of total 240 tested blood samples; 143 (59.6 %), 47 (19.6 %), and
155 (64.6 %) were positive for anti-BVD antibodies using ELISA, ID and CBIA respectively.
The highest detection rate was found in diseased calves at Behira Governorate (83.5 %) using
CBIA technique, while the lowest detection rate was found in contact calves at Alexandria
Governorate (3.2 %) using ID technique.
The percentage of agreement between CBIA and each of ELISA and ID was 95% and 55%
respectively for detection of anti-BVD antibodies in Calve sera.
The percentage of sensitivity between CBIA and each of ELISA and ID was 100 %,
while the percentage of specificity between CBIA and each of ELISA and ID was 87.6 % and
only 44 % respectively for detection of anti-BVD antibodies in calves sera.
This study showed that, the CBIA as well as ELISA techniques were highly sensitive
and specific tests for detection of anti-BVD antibodies in calves sera when compared to ID
technique. Moreover, CBIA has many advantages over ELISA technique; Firstly, CBIA is
rapid, simple, cheap and not need to ELISA reader as in ELISA technique. Secondarily; the
microtitre plate containing BVD infected tissue culture monolayer can be preserved for long
time in comparison to prepared ELISA plates.
Key words: bovine viral diarrhea, cell bound immunoassay, immuno-diffusion.
INTRODUCTION
Since 1946, when Olafson et al (1946)
(1),
recognized Bovine Viral Diarrhea virus (BVD-V)
in the United States and up till now, a variety of serological techniques had been used to detect
BVD viral antigen or its antibodies as ; Serum neutralization test (SNT)
technique (IF)
(3),
Complement fixation test (CF)
Immuno-peroxidase test (IP)
histochemistry (IHC)
(8,9)
(6),
(4),
(2),
Immuno-fluorescent
Immuno-diffusion technique (ID)
Enzyme linked immuno-sorbent assay (ELISA)
(7),
Immuno-
and Reverse transcriptase- polymerase chain reaction (RT-PCR)
However, the neutralization test which used as a standard technique
(12),
(5),
(10,11).
has many
disadvantages; as it is time consuming, bacterial contamination and toxic effect of sera on tissue
Correspondence to:
Bahgat Z Youssef
Microbiology Department,
Animal Health Research Institute
Dokki, Alexandria Branch
E-mail: Bahgat27@hot mail.com
The Journal of the Egyptian Public Health Association (JEPHAss.)
Vol.81 No. 1& 2, 2006
.
culture and the presence of non-cytopathogenic BVD-V in bovine sera may interfere with the
growth of indicator virus used in neutralization test and result in false positive neutralizing
antibody assays (4).
ELISA technique had been developed and used to detect most infectious viruses and their
antibodies in sera and other specimens with high sensitivity and specificity as well as in
detecting anti-BVD antibodies (13).
Many workers applied ID to detect anti-BVD antibodies in sera samples of diseased cattle,
they reported that, the results by this technique were more specific when the titer of antibodies
in
sera
high
is
(5,14,15).
Cell bound immuno-assay (CBIA) is a modified form of cell ELISA. It has been used for
detection of anti-Canine distemper virus in dog sera,(16) anti-Bovine herpes virus in bovine sera
(17),
anti- infectious bural disease virus in chicken sera (18).
The aim of this work is to compare between ELISA, Immuno-diffusion and Cell bound
immuno assay techniques for detection of anti-BVD antibodies in calves on some farms in
Alexandria and Behira Governorates.
MATERIAL AND METHODS
Sampling
The collection of the samples of this study was carried out during the period of Autumn
2005, it included 240 calves collected from some farms of Alexandria and Behira Governorate
(120 each). Out of 120 calves blood samples collected at Alexandria Governorate; 89 and 31
were collected from diseased and apparently healthy contact calves respectively. While out of
120 calves blood samples collected at Behira Governorate; 91 and 29 were collected from
diseased and apparently healthy contact calves respectively. The diseased calves suffered from
watery diarrhea in all and pyrexia, mild mucoid nasal discharge or mild buccal ulceration in
some of them.
Sample preparation
All 240 blood samples were withdrawn from jugular vein by vein puncture, each sample
placed in centrifuge tube to get serum. All serum samples were divided into aliquots and
frozen in deep freeze until used for detection of anti-BVD antibodies using ELISA, ID and CBIA
techniques.
Serological tests
1- ELISA technique
ELISA technique which used in this study to detect anti-BVD antibodies was carried out
according to Hyera et al. (1987)(19), Pacheco and Lager (2000)(13) after optimization of antigen,
serum and peroxidase conjugate dilutions according to Bahgat (1999)(20).
30
The Journal of the Egyptian Public Health Association (JEPHAss.)
Vol.81 No. 1& 2, 2006
.
2-Immuno-diffusion technique
Agar gel diffusion technique which used in this study to detect anti-BVD antibodies was
carried out according to Darbyshire et al (1961)(21) and Hosny et al (1996)(5).
3- Cell bound immuno-assay (CBIA)
CBIA which used in this study to detect anti-BVD antibodies was carried out according to
Zaghawa (1993)(16) as follows:I-Preparation of infected monolayer cell culture in microtitre plates
Madin Darby bovine kidney (MDBK) cell cultures (kindly provided by Egyptian Organization
of Biological Product and Vaccine-Abbasia Cairo), were grown in microtitre tissue culture plates at a
seeding concentration of 600 cells per well. Cells were inoculated simultaneously with BVD virus
except for one vertical raw of the plate which remained without virus inoculation and was used as
negative control. The plates were then incubated in a CO2 incubator at 37OC/72 hours, then the plates
were washed 3 times with phosphate buffer saline-Tween 80 (PBS-T80) and fixed by heat at 80OC/2
hours and the plates were kept in refrigerator till used.
2-Test proper
1. One hundred μ of each sera sample was dispensed into corresponded well of the
microtitre plates containing the infected monolayer cell culture. The plates were
incubated at room temperature for 2 hours and then washed 3 times with PBS-T80 .
2. One hundred μ of 10-3 anti-bovine immuno-globulin peroxidase conjugate (prepared in
rabbit, Sigma product no. A7417) was added
to each well, incubated at room
temperature for 2 hours and then washed 3 times with PBS-T80 .
3. One hundred μ of freshly prepared substrate solution ( O phenylene diamine
dihydrochloride Sigma product no. P8806) was added to each well, incubated at room
temperature for 30 minutes and then washed 3 times with PBS-T80 .
4. The plates were then examined under an inverted microscopy where positive wells
showed pink to reddish cells, while negative cells remained unstained.
Statistical analysis
The percentage of agreement, sensitivity and specificity were calculated according to
Knapp & Miller (1991)
(22).
RESULTS
Table 1, 2 & 3 show the comparative study between ELISA, ID and CBIA techniques for
detection of anti-BVD antibodies in some farms at Alexandria and Behira Governorates. The
highest detection rate was found in diseased calves at Behira Governorate (83.5 %) using CBIA
technique, while the lowest detection rate was found in apparently healthy contact calves at
Alexandria Governorate (3.2 %) using ID technique. The highest detection rate in Alexandria
Governorate was found in diseased calves (66.3 %) using CIBA technique .
31
The Journal of the Egyptian Public Health Association (JEPHAss.)
Table (1):
Animal case
Diseased calves
Contact calves
Total
180
60
240
ELISA
No. of
positive
samples
122
21
143
Percent
(%)
67.8
35.0
59.6
ID
No. of
positive
samples
45
2
47
CBIA
Percent
(%)
25.0
3.3
19.6
No. of
positive
samples
135
20
155
Percent
(%)
75.0
33.3
64.6
Comparative Study Between ELISA, ID and CBIA Techniques for Detection of AntiBVD Antibodies in calves Sera Collected from Some Farms at Alexandria
Governorate
Animal case
No. of
tested
samples
Diseased calves
Contact calves
Total
89
31
120
Table (3):
.
Comparative Study Between ELISA, ID and CBIA Techniques for Detection of AntiBVD Antibodies in calves Sera Collected from Some Farms at Alexandria and Behira
Governorates
No. of
tested
samples
Table (2):
Vol.81 No. 1& 2, 2006
ELISA
No. of
positive
samples
57
10
67
Percent(
%)
64.1
32.3
55.8
ID
No. of
positive
samples
21
1
22
CBIA
Percent(
%)
23.6
3.2
18.3
No. of
positive
samples
59
11
70
Percent(
%)
66.3
35.5
58.3
Comparative Study Between ELISA, ID and CBIA Techniques for Detection of AntiBVD Antibodies in calves Sera Collected from Some Farms at Behira Governorate
Animal case
No. of
tested
samples
Diseased calves
ELISA
ID
CBIA
No. of
positive
samples
Percent(
%)
No. of
positive
samples
Percent(
%)
No. of
positive
samples
Percent(
%)
91
65
71.4
24
26.4
76
83.5
Contact calves
29
11
37.9
1
3.5
9
31.0
Total
120
76
63.3
25
20.8
85
70.8
Table 4 & 5 show the percentage of agreement between CBIA and each of ELISA and ID
techniques for detection of anti-BVD antibodies. The percentage of agreement between CBIA
and ELISA was 95% while it was only 55% between CBIA and ID techniques for detection of
anti-BVD antibodies.
\
32
The Journal of the Egyptian Public Health Association (JEPHAss.)
Table (4):
Vol.81 No. 1& 2, 2006
The Percentage of Agreement Between CBIA and ELISA Techniques for Detection of
Anti-BVD Antibodies in calves Sera Collected from Some Farms at Alexandria and
Behira Governorates
No. of examined
sera
CBIA
technique
ELISA
technique
143
12
85
240 (total)
Positive
Positive
Negative
Positive
Negative
Negative
Results of
Agreement
Disagreement
143
00
85
228
Percentage of agreement =
Total No. of agreement
 100
Total No. of examined samples
Percentage of agreement =
228
 100 = 95%
240
Table (5):
.
00
12
00
12
The Percentage of Agreement Between CBIA and ID Techniques for Detection of
Anti-BVD Antibodies in calves Sera Collected from Some Farms at Alexandria and
Behira Governorates
No. of examined
sera
CBIA
technique
ID
technique
47
108
85
240 (total)
Positive
Positive
Negative
Positive
Negative
Negative
Percentage of agreement =
Results of
Agreement
47
00
85
132
Disagreement
00
108
00
108
Total No. of agreement
 100
Total No. of examined samples
Percentage of agreement =
132
 100 = 55%
240
Table 6 & 7 show the results of sensitivity and specificity of CBIA and each of ELISA and
ID techniques. The sensitivity between CBIA and each of ELISA and ID techniques was 100%.
While the specificity between CBIA and ELISA was 87.6 %, and it was only 44 % with ID
technique.
Table (6): The Percentage of Sensitivity and Specificity of CBIA in Comparison to ELISA
Technique for Detection of Anti-BVD Antibodies in calves Sera Collected from Some
Farms at Alexandria and Behira Governorates
CBIA technique results
ELISA technique results
Positive
Negative
Total
Positive
155
143 ( A )
12 ( B )
155
Negative
00
00 ( C )
85 ( D )
85
143 (A+C)
97 (B+D)
240
Total
Percentage of sensitivity
Percentage of specificity
A
143
 100 =
 100 = 100 %
AC
143
85
D
 100 = 87.6 %
100 =
=
97
BD
=
33
The Journal of the Egyptian Public Health Association (JEPHAss.)
Vol.81 No. 1& 2, 2006
.
Table (7): The Percentage of Sensitivity and Specificity of CBIA in Comparison to ID Technique
for Detection of Anti-BVD Antibodies in calves Sera Collected from Some Farms at
Alexandria and Behira Governorates
CBIA technique results
ID technique results
Positive
Negative
Total
Positive
155
47 ( A )
108 ( B )
155
Negative
00
00 ( C )
85 ( D )
85
47 (A+C)
193(B+D)
240
Total
A
47
 100 =
 100 = 100 %
AC
47
Percentage of sensitivity
=
Percentage of specificity
85
D
 100
100
= BD
= 193
= 44 %
DISCUSSION
BVD Virus which belongs to family flaviviridae, genus pestivirus is associated with a
variety of disease manifestations, causing a world wide economic impact and a considerable
threat to the live stock industry
(23,24).
Therefore, a rapid, cheap and accurate tool for diagnosis
of BVD virus and their antibodies is very important to prevent, control and eradicate the
persistently infected animals (25,26).
The results of this study showed that, out of 240 tested blood samples; 143 (59.6 %), 47
(19.6%) and 155 (64.6%) were positive for anti-BVD antibodies using ELISA, ID and CBIA
techniques respectively. The highest detection rate was found in diseased calves at Behira
Governorate (83.5 %) using CBIA technique and the lowest detection rate was found in contact
calves at Alexandria Governorate (3.2 %) using ID technique.
The detection rate of anti-BVD antibodies using ELISA technique was 64.1 % and 71.4 % in
diseased calves at Alexandria and Behira Governorates respectively. These results were in
agreement with results of Pacheco & Lager (2000)(13) & Nifisah (2003)(27), they found 64.9 % - 78 %
were positive for anti-BVD antibodies in diseased animals using the same technique. While the
detection rate by ID technique was 23.6 % and 26.4% in diseased calves at Alexandria and Behira
Governorates respectively. These results were very low than those obtained by ELISA or by CBIA
(66.3 % and 83.5%) at Alexandria and Behira Governorates respectively.
ID technique is an insensitive technique as reported by many other worker
(5,15,28),
they
reported that ID technique requires high titer of antibodies for detecting the precipitin line.
Bolin et al. (1985)(14), reported that ID technique results were more specific when antibodies titer
in the sera was very high (1:256 or higher).
The percentage of agreement between CBIA and ELISA was very high (95%) while it was
very low when compared with ID technique, which yielded only 55%.
The percentage of sensitivity for CBIA in comparison to each of ELISA and ID techniques
was 100 %, while the percentage of specificity for CBIA in comparison to each of ELISA and ID
34
The Journal of the Egyptian Public Health Association (JEPHAss.)
Vol.81 No. 1& 2, 2006
.
techniques was 87.6 % and only 44 % respectively. CBIA proved to be sensitive and specific
technique as well as ELISA for detection of anti-BVD antibodies, but ID technique was less
specific test for detecting anti-BVD antibodies. Regarding the sensitivity and specificity of CBIA
technique for detecting anti-BVD antibodies in the sera is in agreement with results of Zaghawa,
(1993)(16) & (1997)(17), when they compared CBIA with serum neutralization test in detecting
anti-canine distemper virus and anti-infectious bovine rhinotracheitis virus. Also is in
agreement with the result of other workers who compared CBIA with serum neutralization test
in detecting anti-infectious bursal disease (18) and anti-bovine viral diarrhea (29).
This study proved that; the ID technique was less specific in detecting anti-BVD
antibodies from calves sera, because it need too high titer of antibodies to give the precipitin
lines, while both ELISA and CBIA techniques were sensitive and techniques for detecting antiBVD antibodies from calves sera, the later technique has many advantages over ELISA
technique. Firstly; CBIA is rapid, simple cheap and does not need ELISA reader, there fore it
can be used in field study. Secondarily; the microtitre plates containing BVD-V infected tissue
culture monolayer can be preserved for long time without change (3 months in this study) in
comparison to microtitre plates coated with BVD viral antigen which should be used within
one week after coating.
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