Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
108
MOLECULAR AND SEROLOGICAL DETECTION OF BOVINE
ADENOVIRUS TYPE-3 IN BASRA PROVINCE
Shant Antranik Sinbat; Adnan M. Al-Rodhan; Rasha M. Othman
Department of Microbiology-College of Veterinary Medicine,University of Basrah
Basrah, Iraq
Key words: bovine adenovirus type 3,cattle, basra
ABSTRACT
Investigation of bovine adenovirus type 3 in symptomatic and asymptomatic
cattle were carried out in this study ofdetecting circulating specific bovine adenovirus
type 3 antibodies by Indirect ELISA and standard PCR technique. The study
exhibited these virus have detected by ELISA more than PCR technique. The results
were showed thatthe overall seropositivity ratio of bovine adenovirus antibodies in
animals of Basra was (61.9%) and bovine fecal samples will tested by
PCRwereonly seven fecal positive sample (6.1%) was found.
INTRODUCTION
Adenoviruses (AdVs) are double stranded DNA, non-enveloped viruses with
icosahedral capsid symmetry composed of 252 capsomers, The size of genomes
ranges between 24 and 45 kb (1,2). The virus can cause respiratory and enteric
infections of calves. Bovine adenoviruses (BAVs) cause a variety of clinical signs
including conjunctivitis, pneumonia, diarrhea, and polyarthritis and Bovine
respiratory disease complex (BRDC) is a major problem for cattle and it continues to
cause serious economic losses for cattle industry (3). Adenoviruses were first isolated
from adenoid tissues by two independent groups attempting to identify the causative
agents of acute respiratory infections(4,5).They have been isolated from mammals,
birds, reptiles and other species (6,7). Their replication is mostly limited to single host
species but it can present asymptomatically in species other than the natural host (8).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
109
MATERIAL AND METHODS
The study included 322 symptomatic and asymptomatic sample from different
region of Basra provincewith clinical symptoms of Bovine adenovirus represented by
mild or severe diarrhea, nasal discharge and normal animal.Samples included 115
bovine fecal specimens, 92 bovine blood samples and 115 bovine nasal swab samples
were collected from bovine with clinical signs from different regions (Abi-Elkhasib,
Shutt-alarab, Basra center, Alqarma, Alzubair). Suspected animals were at age group
ranged between2 month -five years of age in the city of Basra.Sample were collected
in cool condition by ice box and used viral transport media (VTM) to keep the sample
in moderate pH and inhibit bacteria, fungal growth until reach the samples to
laboratory to be diagnosed. All samples were tested with standard PCR (AccuPower®
PCR PreMix, Bioneer, Korea)and Indirect ELISA (BIO-X Adenovirus 3 bovine
ELISA Kit 2*48 test, BIO K 063\2, made in Belgium).
Samples collection
Fecal samples and nasal swab samples were collected from 115 bovine of
clinical sign including diarrhea, nasal discharge, fever, weight loss, in appetence.
From diarrheic and non-diarrheic cattle's of different age and sex in Basra province
(Abi-Elkhasib, Shutt-alarab, Basra center, Alqarma, Alzubair).Fecal samples were
varied in amount from 0.2gm to 10gm per sample and collected in sterile disposable
closed plastic containers containing viral transport media.The nasal sampleswere
taken by sterile swabs. Blood sampleswere collected from the same animals in sterile
10 ml test tube. were transported under cold conditions to the laboratory where the
necessary tests were performed or stored frozenuntil use.
ELISA Test
Indirect ELISA (Adenovirus 3 bovine ELISA Kit, BIO X, BIO K 063\2, Belgium)
was performed for detection antibodies against Bovine adenovirus type 3 in serum
samples of cows, according to the manufacturer's instructions. The signal read for
each samples were divided corresponding positive control serum odd and multiply
this result by 100 to express it as a percentage. Delta OD Sample
Val = ________________*100
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
110
Delta OD positive
PCR Analysis
DNA extraction was performed by using Stool DNA Extraction Kit (Stool DNA
extraction kit, LOT no.1302k, REF.K-3036, Bioneer, Made in Korea),for purification
of viral DNA from stool, according to the manufacturer's instructions. The viral
nucleic acid extraction kit III was designed for efficient purification of viral DNA
from cell free samples, which uses the specific sequence of two pairs of primers,
designed E2Afwd and E2Aseq1, which located in E2A region of BAV-3. The
primers E2Afwd (5’-GAG ATG GAT GTG AAC AGC GA-3’) and E2Aseq1 (5’-
ACA TTC TGA TGC TGG TAC TG-3’) amplified an approximately 644 bp product
from the BAV-3 DNA.The reaction was heated in athermocycle for 3 min at 93°C and
then submitted to 30 cycles of amplification. The conditions of amplification
were 45 s at 95°C, 50 s at 55°C, and 1 min at 72°C. The final extension step was done
at 72°C for 10 min.DNAconcentration was complete by Nano drop instrument, and
the horizontal agarose gels was used for analysis of DNA virus amplification product
size. The concentration of agarose used was 1.5%; gels were prepared as percentage
weight / volume solutions.
RESULT
According to different regions of Basraprovince
Results show that out of (92)serum samples.Wereseropositivity ratio of bovine
adenovirus antibodies in animals of Basra were (61.9%). Two regions out of five
under survey recorded high seropositivity ratio including, Basra center (83.3%) and
Shut-Alarab (71.4%). The other threeregions had a lower seropositivity ratio ,Qurma
(50%), Abi-Elkhasib (56%) and Zubair (37.5%),compared to other districts (Table 1).
The difference among these regions was considered to be statistically not significant (
P> 0.05 ).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table(1): Distribution of Bovine adenovirus infection according to ELISA
results in bovine of different region of Basra province.
Animals clinical picture
Fifty eight (58) animals show clinical signs and 34 animal had been
asymptomatic were investigated by ELISA and resultswererevealed that74.1% were
seropositive for antibodies encountered in clinically diseased animals in Basra
regions, followed by Shut-Alarab (87.5%) showed high rate of seropositivity.
However 34 asymptomaticanimals were show 14 ( 41.2 %) seropositive samples. The
high rate of seropositivity was observed in the animals of Basra center (70 %). The
difference among animals in different Basra regions was considered to be not
statistically significant ( P > 0.05 ).
ELISA results
Regions Negative %
n.
Positive %
n.
Examined
n.(%).
Abi-Elkhasib 25(27.2) 14 56 11 44
Shut-Alarab 21(22.8) 15 71.4 6 28.6
Basra center 18(19.6) 15 83.3 3 16.7
Qurma 20(21.7) 10 50 10 50
Zubair 8(8.7) 3 37.5 5 62.5
Total 92 57 61.9 35 38.1
P>0.05 ( X²:5.14;p-value:0.273:DF:4)
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table(2): Bovine adenovirus results in symptomatic animalsofdifferent region of
Basra province based on ELISA.
Table(3): Bovine adenovirus results in asymptomatic animals ofdifferent region
of Basra provincebased on ELISA
ELISA results
Regions Negative %
n.
Positive %
n.
Examined
n.(%).
Abi-Elkhasib 17(29.3) 11 64.7 6 35.3
Shut-Alarab 16(27.6) 14 87.5 2 12.5
Basra center 8(13.8) 8 100 0 0
Qurma 14(24.1) 8 57.1 6 42.9
Zubair 3(5.2) 2 66.7 1 33.3
Total 58(100) 43 74.1 15 25.9
P>0.05 ( X²:4.301;p-value:0.3667:DF:4)
ELISA results
Regions Negative %
n.
Positive %
n.
Examined
n.(%).
Abi-Elkhasib 8(23.5) 3 37.5 5 62.5
Shut-Alarab 5(14.7) 1 20 4 80
Basra center 10(29.4) 7 70 3 30
Qurma 6(17.7) 2 33.3 4 66.7
Zubair 5(14.7) 1 20 4 80
Total 34(100) 14 41.2 20 58.8
P>0.05 ( X²:2.881;p-value:0.5779:DF:4)
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Determination of serum’s positivity degree
Symptomatic bovine positive samples 14 (32.5%) were considered moderately
positive where their serum’s positivity degree values ranged between >56% -≤ 79 %,
while the lowest positivity degree were detected in 2 cases (4.7%). Their positivity
degree ranged between 10% -≤ 33 %, On the other hand the highly serum’s positivity
degree were detected in 16 positive samples (37.2%) where their positivity degree
was >102 % (+++++).Most of asymptomatic bovine positive serum samples 7 (50%)
showed lowest positivity degree ranged between 10 -≤ 33 % and the moderate
positivity degree (>56 - ≤79 %) was observed in 2 cases (14.3%), while the highly
serum’s positivity degree were detected in 4 positive samples (28.6%).The difference
between symptomatic and asymptomatic bovine serum’s positivity degree was
considered to be statistically highly significant(X²:27.92;p-value:0.0009:DF:9).
PCR amplification results
The result of PCR amplification that performed on the extracted DNA was
confirmed by electrophoresis as the strands of the DNA which are resulted from
successful binding between primers and the extracted DNA. These successful binding
appear as a single band under U.V illuminator using ethidium bromide as a specific
DNA stain. Bands with expected size (644bp) were observed.
Figure.(
1) Positive and negative results of PCR amplification ; Lane (1) laddermarker ; Lane
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
114
(5,6,7) positive BAV-3 specific gene (644bp ); Lane (2,3,4,8) negative Broadspectrum
AtAdV on 1.5% agarose .
DISCUSSION
Bovine respiratory disease complex (BRDC) is a major problem for cattle and it
continues to cause serious economic losses for cattle industry. The causes of BRDC
are multiple and complex, but the three factors of stress, viral infection and bacterial
infection are almost always involved in cases of severe disease (9).This is the first
study about the molecular and serological detection of BAV-3 in cattle Basra of
Clinical samples
The PCR analysis was performed on (230) fecal and nasal swab samples (115
for each). Depending on the results of the PCR analysis only 7 (6.1%) fecal samples
were PCR positive for BAV-3 specific gene, whereas 0 (0%) of nasal swab samples
were negative for BAV-3 specific.
Table(4): Distribution of BAV-3 specific gene results according to different
region of Basra based on PCR.
PCR results
Basra regions Fecal samples
Tested
samples
n.(%).
Negative
n.(%).
Positive
n.(%)
Abi-Elkhasib 38(33.1) 1(2.6) 37(97.4)
Shut-Alarab 27(23.5) 1(3.7) 26(96.3)
Basra center 22(19.1) 5(22.7) 17(77.3)
Qurma 20(17.4) 0 20(100)
Zubair 8(6.9) 0 8(100)
Total 115 7(6.1) 108 (93.9)
X²:13.533;p-value:0.001:DF:4
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Iraq,will be a good beginning for further studies on BAV-3. On the basis of this work,
a broad pathogen epidemiological investigation of BAV- 3 might be carried out and
serological methods would be established for detecting antibodies against BAV-3 in
Iraq. Vaccine for BAV-3 might be developed using the new identified BAV-3. All of
these efforts would greatly improve the investigations on BRDC in Iraq, On the other
hand, recombinant BAV-3 is being developed as a live vector for animal vaccination
and for human gene therapy (10). Therefore the BAV-3 might be developed as an
appropriate expression vector.
Results of the present study revealed that the seropositivity of BAV-3 was
61.9%, these results were lower than those reported by (11) and (12) whom detected
antibodies against BAV-3 in tested (91.6 and100 % respectively) against BAV-3 in
sera of young and adult cattle of Iran . Beside that serologic evidence in United
States indicated high prevalence of BAV-3 in cattle. (13)determined antibodies in
(81.38 %) of unvaccinated cattle using serum neutralization (SN) test.
The ubiquitous exposure to bovine adenoviruses makes determination of the specific
type involved in an outbreak of respiratory or enteric disease, The critical assessment
of BAV, broadly and by genotype, as indicators of BAV infection and environmental
contamination from cattle manure requires improved knowledge of their prevalence,
shedding, and genetic diversity. Many previously published PCR assays targeting
BAV suffer from poor sensitivity, limited breadth of detection (14,15). In the present
study the examination of bovine nasal and fecal samples using specific BadV-3,
broad-spectrum PCR primer revealed the shedding of most prototype BAV(except
BadV-3, -9,) by cattle, significantly expanding the genetic diversity of BAV detected
to date by PCR (16). The current revealed that7(6.1%)out of 115 head of symptomatic
cattle showed BAV 3 based PCR positive results but serologic data showing that
43(74.1%) BAV 3 based ELISA positive results and 14(41.2%) animals of
asymptomatic cattle.
The current detection of BAV 3 in 7(6.1%) fecal samples only ,agreed with
(17). who mentioned that frequencies determined by using PCR have a wide range,
from 0% to 30% for samples collected from single animals whereas, it is disagreed
with (18), who reported frequencies ranged from 50% to 75% for samples pooled
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
116
from multiple animals. The present finding of BAV3 in fecal samples and its
negativity in PCR analysis of nasal swabs was disagreed with (19) who found positive
PCR result where he used similar BAV3 having the same nucleotide sequence in the
amplification DND previously extracted from nasal swabs collected from a group of
feedlot cattle with acute respiratory disease.
التشخیص الجزیئی والمصلی لفایروس الغدی البقری- 3 فی محافظھ البصرة
شانت انترانیک سنباط؛ عدنان موسى الروضان؛ رشا منذر عثمان
فرع الاحیاء المجھریھ، کلیھ الطب البیطری، جامعھ البصرة، البصرة، العراق
الخلاصھ
تم الکشف والتحقیق عن الفایروس الغدی البقری - 3لعینات الابقار السریریھ المصابھ والغیر مصابھ فی ھذه
الدراسھ والتی تم فحصھا بتقنیھ الممتز المناعی المرتبط بالإنزیم وتقنیھ تفاعل سلسلة البلمره، اظھرت ھذه
الدراسھ بتشخیص الفایروس بتقنیھ الممتز المناعی المرتبط بالانزیم اکثر من تقنیھ تفاعل سلسلة البلمرھحیث
لوحظ ان معدل الایجابیھ للاجسام المضاده لفایروس البقری الغدی- 3 فی الابقار فی محافظھ البصرة
کان(% 61.9 ) وعینات البراز البقریھ تم فحصھا بواسطھ تقنیھ تفاعل سلسلة البلمره حیثما کانت فقط سبع عینات
براز بقریھ ایجابیھ.
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