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~ 1206 ~
Journal of Entomology and Zoology Studies 2017; 5(6): 1206-1211
E-ISSN: 2320-7078
P-ISSN: 2349-6800
JEZS 2017; 5(6): 1206-1211
© 2017 JEZS
Received: 12-09-2017
Accepted: 13-10-2017
Ahmed Saadi Khalaf
Msc. Veterinary Medicine
Collage, Baghdad University,
Iraq
Atheer Abd Alrazaq Aldoori
Phd. Veterinary Medicine
Collage, Baghdad University,
Iraq
Correspondence
Ahmed Saadi Khalaf
Msc. Veterinary Medicine
Collage, Baghdad University,
Iraq
Genomic diversity and prevalence of Rotavirus in
cow and buffalo calves in middle area of Iraq
Ahmed Saadi Khalaf and Atheer Abd Alrazaq Aldoori
Abstract
A total of 335 suspected fecal sample were collected from calf of cattle and buffalo with age in between
(3 days to 4 months) from middle area of Iraq between November 2016 to May 2017.
Immuno-chromatographic rapid tests (FASTest® Strips) were used for the detection of Bovine, rotavirus,
which gave us 159 positive results, while when we took the 159 positive samples and tested by ELISA
(cosabio) kit gave us 111 positive results. Extraction of RNA and PCR methods from these 111 positive
samples for detection Rota virus using vp7, vp4 primers were done and gave us 19 positive samples, The
nucleotide sequence of the VP7 gene of the strains which we got were X57852.1, X52650.1, KP013395.1
& AB457636.1 from the buffalo and cattle calf feces most closely identity to that American G10 P(11),
Maxico, UK G10 P(5) and Iranian G10 bovine rotavirus genotype
Keywords: Rotavirus, genotype, calves, genotype
Introduction
Rotaviruses are the most common cause of neonatal diarrhea in calves [8], The virus is present
in most cattle herds and typically causes diarrhea in calves up to 3 years. However, periodic
asymptomatic re-infection and shedding occurs in older cows and calves [33], There are
eight species of this virus, referred to as A, B, C, D, E, F, G and H. Young animals like calves
are primarily infected by species A, B. Two surface proteins VP4 and Vp7 of rotaviruses, are
important in serotype determination of rotavirus [15], and in inducing neutralizing antibodies
and protective immunity, The two structural outer capsid proteins, VP7 (Glycoprotein) and
VP4 (Protease sensitive protein) [22], Define as the G and P serotype/genotype of rotavirus,
there are At least 27 G genotypes and 35 P genotypes have been identified.
The VP6 gene has been classified into 2 subgroups (SGI and SGII); SGII is the most prevalent
and SGI is more commonly found in animals [34]. The virus is transmitted by the fecal-oral
route also via surfaces and objects, contaminated food or water. Viral diarrhea is highly
contagious. It infects and damages the cells that line the small intestine and
causes gastroenteritis and causing dehydration, electrolyte disturbances, fever, shock and
death, The diarrhea is caused by multiple activities of the virus. Malabsorption occurs because
of the destruction of gut cells called enterocytes. This may result in decreased intestinal
absorption of sodium, glucose, and water, and decreased levels of intestinal lactase, alkaline
phosphatase, and may lead to isotonic diarrhea [17, 21].
The nucleotide sequences of the 11 RNA segments from different rotavirus strains are known
and the complete nucleotide sequence [10].
Sequencing includes any method or technology that is used to determine the order of the four
bases—adenine, guanine, cytosine, and thymine—in a strand of DNA, The advent of rapid
DNA sequencing methods has greatly accelerated biological and medical research and
discovery [14].
Sequencing helped us to determined the rotavirus ID in the NCBI and for detect the genotype
identity in our governorates with other world rotavirus genotype by use sangar method that by
send dsDNA and forward primers of VP7and VP4 for sequencing [16, 27].
Materials and Methods
Sample collection
A total of 355 fecal samples were collected between November 2016 to May 2017 from
animal stations located in different governorates regions in middle of Iraq: (Baghdad, Dyala,
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Journal of Entomology and Zoology Studies
Salah aldden, Babil, Wasit) cattle and buffalo calf with acute
diarrhea associated with fever and/or dehydration) were
enrolled in the study. Stool specimens were tested for the
presence of rotavirus antigen, fecal sample were centrifuged
at 6000 rpm for 5 minutes to remove particulates, and stored
the supernatant at -20oC until use.
Immuno-chromatographic: (rapid tests): used (ABON-
FASTest® Strips) The method described according to the
protocol provided by the kit.
ELISA: The results were obtained from Chromatographic
Immunoassay kits were tested by ELISA method to reveal the
presence of rotavirus antigen, The method described
according to the protocol provided by the kit and calculated
the result according to the following equation:-
Cut-off value = the average value of ODnegative+ 0.10
The samples were considered as positive when ODsample is
more or equal cut-off value while it was considered as
negative when ODsample is less than cut-off value
Extraction of Rotavirus dsRNA from fecal Suspension
The method described according to the protocol provided by
the kit manufactured by Qiagen.
Estimation RNA concentration and purity
The viral nucleic acid concentration and purity of the sample
extracted from feces were estimated by using Nano drop
(Acta/USA). 1µl of the extracted RNA was put in the
machine. The purity was (~2.0) that means RNA is high
purity according to Acta device protocol
Reverse Transcription (RT) for VP7 and VP4 Genes
The pure RNA which we revealed subjected to RT -PCR. to
get cDNA. At first dsRNA was incubated at 56 °C for 5
minutes, A mixture of 8 μl of the extracted dsRNA with 2.5 μl
of dimethyl sulfoxide,1 μl of VP7 antisense (VP7R) and 1 μl
of VP7 sense primers (VP7F),same procedure used with VP4
primers, The method described according to the protocol
provided by QIAGEN kit by utilize Omniscript and
Sensiscript Reverse Transcriptases which included in the
QIAGEN and provide highly efficient and specific reverse
transcription, and also by utilize Hot Star Taq DNA
Polymerase included in the QIAGEN OneStep RT-PCR kit
which provides hot-start PCR for highly specific
amplification. During reverse transcription, reactions were
heated to 95 °C for 15 min to activate Hot Star Taq DNA
Polymerase and to simultaneously inactivate the reverse
transcriptases. The protocol provided by the kit [26].
Table 1: program of cDNA Qiagen.
Cycles
Time
Temperature
Step
No.
X1
30 minutes
50 oC
Reversetranscriptase (RT)activation
1
X1
15 minutes
95 oC
Polymerase activation &RT inactivation (HotStarTaq)
2
X35
30 second
94 oC
Denaturation
2
30 second
48 oC
Annyling
3
60 second
72 oC
Extenation
4
X1
7 minutes
72 oC
Final-Extention
5
-
-
4 oC
Hold
6
[24].
Amplification of VP7 and VP4 Gene
For amplification of VP7 gene, forward (VP7F) and reverse
(VP7R) primers, were used in the first round PCR to generate
the 896 bp partial length VP7 gene product. For each sample,
5 μl of cDNA was added to a 0.5 ml PCR tube containing 2.5
μl of 10XPCR buffer [200 mM Tris-HCl, (pH8.3), 500 mM
KCl], 0.75 μl MgCl2 2H2O (50 mM) 0.5 μl dNTP mixture
[0.2 mM dATP, dACTP, dGTP, dTTP]; 1 μl of reverse primer
(VP7R), 1 μl of forward primer (VP7F) is shown below and
14 μl sterile triple distilled water. Above mixture was mixed
properly and given a small spin. Then subsequently 0.25 μl of
Taq polymerase was added to each reaction tube. The tubes
were placed in a thermal cycler and the PCR reaction was
cycled 35 times maintaining the proper conditions. Same
procedure was used for VP4 gene amplification by use VP4
forward (VP4F) and reverse (VP4R) primers which is shown
below [22].
The primers which used in the tables below
Table 2: (the forward and reverse vp7 primer were we used, [14]
896 bp
ATGTATGGTATTGAATATACCAC
VP7 Forward
9CON 1F
1
896 bp
AACTTGCCACCATTTTTTCC
VP7 Reverse
VP7 R
2
Table 3: the forward and reverse vp4 primer were used [29].
876 bp
TATGCTCCAGTNAATTGG
VP4 Forward
CON3 F
1
876 bp
ATTGCATTTCTTTCCATAATG
VP4 Reverse
CON 2 R
2
Nucleotide sequencing
Amplified products of the VP7 and VP4 genes we obtained
from conventional PCR, with the forward primers (VP7F),
(VP4F) sent to (macrogen) Korean company for sequencing,
direct sequencing of each amplicon was carried out using the
Sanger dideoxynucleotide chain termination.
Results
Immuno-chromatographic rapid tests
The purpose of qualitative detection of Rotavirus in faeces
specimen by chromatographic immunoassay (Monoclonal
antibodies) to capture the presence of virus antigen. In our
study samples were collected from 335 suspected animals
gave us 159 positive infection rotavirus as in the table below.
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Journal of Entomology and Zoology Studies
Table 4: Results were obtained using Chromatographic Immunoassay kits for rotavirus in 335 fecal samples according to age of tested calves.
AGE
Region
Total
14 wk.s
12 wk.s
8 wk.s
7 wk.s
6 wk.s
5 wk.s
4 wk.s
3 wk.s
2 wk.s
1wk.
Dyala
4
0
1
0
0
0
1
0
0
0
2
Positive
22
1
1
3
0
1
0
4
2
12
7
Negative
26
1
2
3
0
1
1
4
2
12
9
Total
Salahalden
8
0
0
0
0
0
0
0
0
2
6
Positive
27
0
0
0
1
1
1
4
1
6
6
Negative
35
0
0
0
1
1
1
4
1
6
12
Total
Babel
7
0
0
1
0
0
0
0
1
1
4
Positive
57
0
3
5
11
1
12
4
2
10
10
Negative
64
0
3
6
11
1
12
4
3
11
14
Total
Wasit
85
0
4
10
8
8
5
10
11
10
19
Positive
34
10
10
2
2
3
2
0
0
2
3
Negative
119
10
14
12
10
11
7
10
11
12
22
Total
Baghdad
55
0
5
2
5
4
8
9
10
13
Positive
36
9
11
3
5
0
2
2
3
2
Negative
91
9
16
5
10
4
10
11
13
15
Total
wk. = week
((Enzyme-linked Immunosorbent Assay (ELISA)
In the current study, all the 159 samples from
chromatographic immunoassay were confirmed by ELISA
tested, our study gave us 111 (70%) positive samples. The
highest positive results were recorded in Wasit 71 positive 84
%, and the lowest 3 only were in Babil 42 %)from these 159
positive sample.
Conventional Polymerase Chain Reaction
In this study, PCRtechnique were used for detection the two
outer layer protein’sVP7and VP4. All bovine stool, which had
been tested using Chromatographic and ELISA, were
confirmed by PCR and gave us 19 (45%) positive result
Fig 1: The gel electrophoresis of PCR products of VP7 (896bp) in and VP4 (876bp) on agarose gel with 100bp DNA ladder (line 1) at 70
voltage for (60) min. lane 1, 2: VP7 (896bp) and lane 9, 10: VP4 (876bp).
896 bp
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Journal of Entomology and Zoology Studies
Fig 2: The gel electrophoresis of PCR products of VP7 (896bp) in and VP4 (876bp) on agarose gel with 100bp DNA ladder (line 1) at 70
voltage for (60) min. lane 2, 3: VP7 (896bp) and lane 8, 9: VP4 (876bp).
Sequencing and Genotyping of VP4 and VP7
In this study multiplex polymerase chain reaction technique
and sequencing were used for genotyping of both VP7 and
VP4. However we took the highest 42 titer from Elisa test to
use it in molecular detection PCR technique reveal 19 positive
result which were send for sequencing, the result of
sequencing gave us 12 positive result were done in south
Korean macrogen sequencing company, these are ID:
X52650.1 (G10P [11]), KP013395.1 (G10), AB457636.1,
(G10P [5]) and X57852.1 (G10P [11]) according to the Ncbi.
The nucleotide sequence of the VP7 gene of the strain were
most closely related to that of a Iranian G10, American G10
P(11) and British G10 P(5) bovine rotavirus, from the cattle
calf feces. Identity 98% to 97% G10P (11) were most
frequently detected were 7 G10 P(11) in bovin samples
genotype specially in Wasit provenance, That by six samples
(four in Wasit, one in Dyala and Baghdad were X52650.1
which we found was identity 98% to Rotavirus A RVA/Cow-
tc/USA/B223/1983/G10P [11] Sequence ID: LC133552.1 and
we found two samples from calves in Wasit and Baghdad
have the Sequence ID: KP013395.1 identity, partial cds to
Rotavirus A isolate Markazi-G10 and identity 97% to Iranian
Rotavirus A strain where three samples two from Baghdad
and one in Babel with ID sequence AB457636.1 identity 99%
to Rotavirus A strain RVA/Vaccine/USA/BRV-KC-
1xUK/2009/G10P [5] segment 9 capsid glycoprotein VP7
(VP7) gene, Sequence ID: KC215545.1. four in Wasit, one in
Dyiala and one from Baghdad were X52650.1 which identity
98% to Rotavirus A RVA/Cow-tc/USA/B223/1983/G10P [11].
Where in Wasit and Baghdad have the Sequence were ID:
KP013395.1 identity, partial cds to Rotavirus A isolate
Markazi-G10 and identity 97% to Iranian Rotavirus A strain
and only one sample from Salah alden was ID: X57852.1
(strain B223) identity 98% to Rotavirus A RVA/Cow-
tc/USA/B223/1983/G10P [11] VP7 gene for structural protein
VP7, complete cds Sequence ID: X52650.1.
Discussion
Diarrhea is a leading cause of economic losses to the beef
industry and major cause of calf mortality and morbidity
during the first few weeks of life in most countries [27]. Most
infected age of positive calves was between8-15 days of age [6].
Defining the map of calve diarrheal-causing entero pathogens
allows for application of effective and targeted preventative
measures. In middle area of Iraq, our study reported the
prevalence incidence of the group A rotavirus in cattle and
buffalo calves, Immuno-chromatographic rapid tests (FAS
Test® Strips) which we used as a screening test gave as 159
positive from 335 fecal samples, the result revealed 71% (85
of 119 samples) were high positive in Wasit, but the lowest
positive was in Dyala 15% (4 of 26 samples). The first week
of age of infection gave us higher titer (44 positive case)
which were the highest result from 159, the lowest infected
age were 14 week and didn’t give positive result, Most
infected age of positive calves was between 3-15 days aged
because the calf was born with no possesses natural
antibodies to fight disease, high levels of serum estrogen and
maternal-fetal cortisol produced at the end of pregnancy and
calving have immunosuppressive effects on the cellular
components that participate in the innate Immune Response
(IR) [12]. In general previous evaluation studies suggested that
rapid field test could be considered as a helpful tool for fast
and effective diagnosis of common entero pathogens
associated with neonatal calve diarrhea [11].
Thereafter we took 159 from the positive samples tested using
ELISA as confirmatory tests to reveal the presence of
rotavirus antigen. ElISA is more specific, more accurate, and
rapid assay that have been used for detection of Rotavirus
Antigen. Enzymes produce the signal and related to the
detection reagents in fixed proportions to permit accurate
quantification [1].
Chromatographic Immunoassay and ELISA are the simple
and good standard methods for detection of rotavirus. These
methods, however, require low cost equipment and simple
experience, which is available in many laboratories. Some
researchers for detecting rotavirus infection have used ELISA
and PCR. These methods are expensive and requires long
time if they have been used together [13].
The highest 42 positive titter of ELISA from fecal bovine
samples were taken due to the molecular detection roots
which were very difficult and high cost. These 42 ELISA
higher positive titter agree with [11] who's got high selective
result by ELISA. Then we utilize VP7 and VP4 primers for
RT PCR and amplification which gave us 19 positive samples
876 bp
896 bp
876 bp
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Journal of Entomology and Zoology Studies
(17%) because of the Iraqi hot climate and molecular lab
difficulties, were these positive samples for molecular
detection sent to sequencing and genotype our result gave us
12 positive.
In the present study we got ID sequence are KP013395.1,
AB457636.1, X52650.1 and X57852.1 according to the
NCBI, G10 were observed as a highly prevalent strain
circulating in both buffalo and bovines calves, Specially
among buffalo that agree with calves rotavirus Indian study
[22] and 3 of P typing isolate were p [5] and that agree with
Italian study [25].
Three isolates in this study were G10P [5] that agree with
Indian project result [29]. in Our study the highest G and P
genotype were G10P(11) in 7 isolates this agree with the other
related regional researches [22].
Also the identity of sequence ID: LC133552.1, agree with [5]
in Brazil which was 98% to our Sequence ID: X52650.1 in
recent study.
Our study agree with the regional similar researches like
Iranian study (Determination and distribution of the G and P
genotypes of group A bovine rotavirus) were investigated on
386 fecal samples collected from calves with diarrhoea using
a semi-nested RT-PCR typing assay, they got by ELISA. 75
positive samples were selected randomly and subjected to
typing assay. G10 (50.6%) and P[11] (64%)) [4]. that may
explain why G10 was the most distribution in our area.
While that different from turkey study which found genotype
G1, G2, and G9, VP7 specificities and genotype P[4], P[6] and
P[8] VP4 specificities and The most common strain was G2P[4]
[3]. Also there is study in Saudi Arabia showed the distribution
of serotype G1-G4, G9, G12, P [4], P [6], and P [8]. [19] these tow
related studies showed genotype difference from our
genotype.
Various hypotheses previously supported that BRVs may
cross the host species barrier and circulate among neonates or
adults and can cause zoonotic transmission and can infect
human beings [35] like G10 P(11) was highly distributed and
can transmit from animal to human and opposite by Indian
project [16].
Acknowledgement
I would like to express my appreciation and gratitude to My
dear supervisor, assistant professor Dr. Atheer aldoori, Dr
Amin sabar, Dr. Faisal Ghazi Alhamadani and the team of
laboratory of the health centre of the ministry of health
specially Mrs Iman. M. Aufi.
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