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Research Arcle
Volume: 3, Issue: 3
December 2019
Pages: 57-63
Invesgaon of bovine coronavirus and bovine rotavirus by rapid
diagnosis kit and RT-PCR in diarrheic calf feces*
Journal of Istanbul Veterınary Scıences
Gülşah Uyunmaz Saklı 1, Oya Bulut2 , Mustafa Hasöksüz3, Hasan Hüseyin Hadımlı4
1. Republic of Turkey Ministry of Agriculture And Forestry Veterinary Control Central Research Instute,
Department of Virology, 06020 Etlik-Ankara, Turkey. 2. Department of Virology, Faculty of Veterınary
Medicine, Selcuk University, Konya, Turkey. 3. Department of Virology, Faculty of Veterinary Medicine,
Istanbul University, Avcılar, İstanbul, Turkey. 4. Department of Microbiology, Faculty of Veterinary Medicine,
Selcuk University, Konya, Turkey.
Uyunmaz Saklı, G.: ORCID: 0000-0002-7807-9869; Bulut, O., ORCID: 0000-0002-2407-7390;. Hasöksüz M.:
ORCID: : 0000-0003-3185-6453; Hadımlı H.H. ORCID: 0000-0002-7665-687X.
ABSTRACT
This study has invesgated bovine coronavirus (BCoV) and bovine rotavirus (BRV), which
are among the most important causes of diarrhea in calves leading to nancial losses in
Turkey and all over the world BCoV and BRV were detected by Reverse Transcriptase
Polymerase Chain Reacon (RT-PCR), which is one of the most reliable method of
diagnosis, The results obtained by RT-PCR were compared to the sensivity of the
commercial Rota-Corona Rapid Test Kits used by clinical veterinarians in elds. In this
study, 96 fecal samples were examined from diarrheic calves in cale farms in the cies
of Konya and Afyon for BRV and BCoV rstly by BoviD-5 Ag rapid test kit, and then we
applied the RT-PCR test. A comparison of the rapid test kit with the RT-PCR in terms of
sensivity and specicity revealed the 83% sensivity and 100% specicity of the BRV
and 7.6% sensivity and 100% specicity of BCoV. In conclusion the praccal and rapid
diagnosis of the disease using of Rapid Diagnosis kit used by the clinician veterinarians
may be useful, but the results must be interpreted with cauon since the sensivity of
the test decreases due to the reducon in the number of viruses in the later stages of
the infecon.
Keywords: BCoV, BRV, calf diarrhea, rapid test kit, RT-PCR
DOI: ---
To cite this arcle: Uyunmaz Saklı G., Bulut, O., Hasöksüz, M., Hadimli, H.H. (2019). Invesgaon of bovine coronavirus
and bovine rotavirus by rapid diagnosis kit and RT-PCR in diarrheic calf feces. Journal of Istanbul Veterinary Sciences. 3
(3), 57-63, Abbreviated Title: J Ist Vet Sci
*This study was summarized from PhD thesis of rst author. This study was presented at the 2nd Internaonal Congress of Veterinary
Microbiology (16-19 October 2018).
Neonatal calf diarrhea caused by viral, bacterial and
protozoon agents is one of the infecons characterized
by enteris leading to weight loss and deaths in calves
under one month of age (Murphy et al.,1999).
Neonatal calf diarrhea is among the most important
reasons for nancial losses in the meat and milk
industry all over the world (Boileau et al.,2010).
Although its causes show variaons depending on the
regional and stable condions, the role of rotavirus
and coronavirus in the cases of calf diarrhea have been
found to reach up to 50% and 80% respecvely.
Rotaviruses generally cause infecons characterized by
diarrhea in dairy calves (Al Mawly et al., 2015) and
beef calves (Cho et al., 2013) up to 9-21 days old.
Bovine group A rotavirus, bovine coronavirus,
enterotoxigenic K99+ Escherichia coli (K99),
Cryptosporidium parvum and Salmonella spp. are
reported to be the most common enteric infecon
*Corresponding Author: Gülşah Uyunmaz Saklı
E-mail: gulsah_uyunmaz@hotmail.com
hps://dergipark.org.tr/tr/pub/hp-www-jivs-net
Arcle History
Received: 05.08.2019
Accepted: 22.11.2019
Available online:
02.12.2019
Creative Commons Attribution 4.0 International License
Introducon
agents (Bartels et al., 2010; Izzo et al., 2012). Among
the factors that aect the course of rotavirus and
coronavirus infecons are whether the newborns
received colostrum, me of weaning, climate
condions, their immune condions, and other
present enteropathogenic agents. The main mode of
transmission of rotaviruses is the fecal-oral route.
Through the feces of the infected animals, a high level
of viral parcles (approximately 1011 parcle/g) is
shed around. This shedding reaches the highest level
on the third and fourth days, and the virus can survive
in the feces for several months (Murphy et al., 1999).
Virus isolaon (Mebus et al., 1969; Hasoksuz et
al.,2002; Gulyaz et al., 2005),
immunochromatographic rapid diagnosc assays (Al-
Yousif et al., 2001; Uhde et al., 2008; Klein et al., 2009;
Bartels et al., 2010; Altug et al., 2013), IEM
(Immunelektron microscopy) (Saif et al., 1980), ELISA
(Alkan, 1998; Murphy et al., 1999; Gulyaz et al., 2010)
and RT-PCR (Cho et al., 2001; Hasoksuz et al., 2002;
Aich et al., 2007; Decaro et al., 2008; Asano et al.,
2010; Bok et al., 2015) are among the most preferred
methods to diagnose rotavirus and coronavirus
infecons.
Materials and Methods
Specimens: The samples were collected from calves (1
to 30 days old) with acute diarrhea cases in stables in
Konya and Afyon. Total of 96 fecal samples were
collected for this study. The age distribuon of the
collected samples is presented in Figure 1. The fecal
samples were collected from the rectums of the
animals with sterile coon swabs. One swab taken out
of the rectum was put in the soluon in the rapid test
kit, and another was homogenized by pung it into
Phosphate Buer Saline (PBS), and stored at -20ºC
unl it was used for RT-PCR. The samples for the rapid
test kits were examined under stable condions, and
their results were reported to the owners in 15
minutes. Necessary notes were taken and the posive
samples were taken into account for the next
examinaon.
Rapid Diagnosc Test: In this study, we used the
Bionote BoviD-5 Ag (Cat. No: RG13-02) rapid diagnosis
kit. We followed the test procedure of the producer.
In line with the procedure, rstly the swab
contaminated with the feces was placed in the
soluon included in the kit during the sampling and
was homogenized. Then one drop of the soluon was
added onto the arrays and according to the change of
color, coronavirus or rotavirus was interpreted as
posive or negave.
RT- PCR Materials: Extracon of the Viral RNA: We
used High Pure Viral RNA isolaon kit of Roche
(Roche, Cat. No: 11858874001). The fecal samples
were suspended at a rate of 1/10 in PBS including
25000 U/ml Penicillin and 20 mg/ml Streptomycin,
centrifuged at +4oC, 3000 rpm for 15 minutes, and
then supernatant was transferred into a sterile tube.
Following the centrifugaon 200 μl of the supernatant
was taken and transferred into a 1.5 ml RNase-free
sterile tube. Each sample was mixed with working
soluon containing 4 μl Poly A and 400 μl Binding
Buer. The working soluon and the sample mixture
were treated with the Removal Buer, Wash Buer,
Eluon Buer included in the kit by using the silica gel
spin column. At the end of the extracon process, 50
µl of viral nucleic acid was isolated, and stored at
-80oC.
cDNA Synthesis: We used Reverse Transcripon
System (Promega A3500) to obtain cDNAs. In order to
synthesize cDNA from the isolated viral RNA, we used
the Promega Reverse Transcripon System synthesis
kit (Promega A3500), and followed the recommended
protocols. 5 µl of the isolated viral RNA was
transferred into the PCR tubes and was incubated at
70ºC for 10 minutes. Following the incubaon, it was
kept in ice for 2 minutes. Master Mix (15 µl for each
sample) was prepared in a dierent PCR tube and 15
µl of Master Mix was added onto each of the 5 µl RNA
samples.
Viral RNA and Master Mix mixture totaling to 20 µl
was put into the Thermal Cycler and amplied at 22ºC
for 10 minutes, at 42 oC for 15 minutes, at 95 oC for 5
minutes at 4oC for 5 minutes, and then the cDNA was
synthesized. The resulng products were stored at -
20oC.
Polymerase Chain Reacon (PCR): We used Promega
Go Taq Flexi DNA Polymerase (Promega M8305) To
detect the presence of bovine rotavirus in the fecal
samples of the calves with the one-step RT-PCR
method. The primers reported by Hasoksuz et al.,
(2008) and Chang et al., (1997) were used. These
primers are specic to the VP7 gene region of the
group A rotaviruses. We used the primers reported by
Cho et al., (2013) for the detecon of bovine
coronavirus. These primers are specic to the N
protein gene of the virus. We used S-Beg5-GGC TTT
AAA AGA GAG AAT TTC-3, End-9, 5-GGT CAC ATC ATA
CAA TTC TAA TCT AAG-3 primers of 1062 bp for bovine
rotavirus and NOF-5-GCA ATC CAG TAG TAG AGC GT-
3, NOR-5-CTT AGT GGC ATC CTT GCC AA-3 primers of
730 bp for bovine coronavirus.
Uyunmaz Saklı G. et al., 2019/ Journal of Istanbul Veterinary Sciences. Volume 3, Issue 3, pp: 57-63
BRV: We used the one-step RT-PCR method for the
viral RNAs obtained. 0.8 μl DMSO, 0.6 μl End-9 of the
rotavirus primers and 0.6 μl S-Beg were added onto
each of the 5 μl RNA products, and they were mixed
with a straw to homogenize. The mixture was
incubated at 94oC for 5 minutes, and then kept in ice.
Following the incubaon, 43 μl Master Mix composed
of Primer F (20 pmol), Primer R (20 pmol), and
soluons of the Promega M8305 kit and the Promega
A3500 cDNA kit was treated with 7 μl RNA and DMSO
mixture. It was amplied at 42oC for 60 minutes, at
94oC for 3 minutes, (at 95oC for 1 minute, at 55oC for 2
minutes, at 72oC for 1 minute at 35 cycles), and at
72oC for 10 minutes.
BCoV: cDNAs of the samples were treated with
the Master Mix mixture of the Promega M8305 kit
including Primer F (50 pmol) and Primer R (50 pmol)
and, for the PCR reacon, Go Taq Flexi DNA
Polymerase, 5X buer green exi color, MgCl2 (25
mM) and dNTP. It was amplied at 94oC for 3 minutes
(at 94oC for 1 minute, at 52ºC for 2 minutes and at
72oC for 1 minute at 35 cycles), at 72oC for 7 minutes.
To display the amplicaon products, 1.5%
agarose gel containing ethidium bromide was
prepared. The PCR products were run at 100 V for 30-
45 minutes and the amplied DNA bands were
controlled under UV light.
Stascal analysis: We used the chi-square test (χ2)
for the stascal analysis of the diagnosc tests. We
recorded p<0.05 as stascally signicant.
Results
We examined 96 diarrheic fecal samples in total for
BCoV and BRV by rapid diagnosc test and RT-PCR
method. The collecve results of the study are
presented in Table 1. According to the results, the
rapid diagnosc test revealed 15 samples as BRV
posive (15.62%) and 1 sample as BCoV posive
(1.04%). The RT-PCR method detected 18 cases of BRV
presence (18.75%), 13 cases of BCoV presence
(13.54%), and 4 cases of both BRV and BCoV presence
(4.16%). The electrophoresis images are presented in
Figure 2 and Figure 3. According to these results, four
samples were found to be both BRV and BCoV posive
by RT-PCR method. However, only one sample was
found to be BRV and BCoV posive with the rapid
diagnosc test.
Figure 1: Distribuon of the samples of calve feces by
age (days)
This sample showed a quite strong DNA band
appearance aer the agarose gel electrophoresis
using RT-PCR. (Figure 3). For the 0-5, 6-15, 16-20 and
21-30 days old calves found to be BRV posive by the
rapid test kit, the rates of posivity were 67%, 24%,
0% and 13% respecvely. Only 4% of the 6-15 days old
calves were found to be BCoV posive. For the 0-5, 6-
15, 16-20 and 21-30 days old calves found to be BRV
posive by the RT-PCR method, the rates of posivity
were 67%, 31%, 6% and 13% respecvely. In terms of
BCoV, while RT-PCR found no posive samples in the 0
-5 days old group, the other groups found to be 34%,
13% and 2% posive respecvely (Figure 4).
Figure 2: Electrophoresis image of the BRV (1062 bp)
posive samples, DNA Ladder (100 bp Fermentas), PC
(Posive Control), NC (Negave Control), DNA bands
of the samples; 27, 28, 12, 13, 14, 15, 16 and 17
Figure 3: Electrophoresis image of the BCoV (730 bp)
posive samples; DNA Ladder (100 bp Fermentas), PC
(Posive Control), NC (Negave Control), DNA band of
the samples 16, 17, 19, 22, 23 and 25
Table 1:
-
Pathogen Rapid Test Kit RT-PCR
- -
Uyunmaz Saklı G. et al., 2019/ Journal of Istanbul Veterinary Sciences. Volume 3, Issue 3, pp: 57-63
Discussion
Rapid and accurate diagnosis of BRV and BCoV
infecon is important for the control and eradicaon
of the disease in newborn animals in cale farms in
many developed and developing countries. Therefore,
it is important to diagnose BRV and BCoV rapidly in
the eld, and to detect it through rapid and eecve
test techniques in veterinary diagnosc laboratories.
Among these methods, isolang the RNA of the virus
and converng it into DNA (cDNA) and mulplying the
cDNAs by using specic primers (RT-PCR) has the
highest sensivity and originality. However, as these
techniques can only be applied under laboratory
condions and require me, clinical veterinarians
need rapid test kits to diagnose the infecon under
eld condions. These rapid test kits are important in
terms of determining the treatment process and
avoiding wrong anbioc use, but the use of rapid test
kits is unfortunately behind the desired levels.
The most important reason behind this is the
righteous suspicion about the sensivity and
specicity of these rapid test kits. In recent years, the
rapid immunochromatographic tests, which are more
advantageous under led condions, it has become
possible to diagnose dierent enteropathogens in the
feces of calves in approximately such short me
periods as 10 to 15 minutes, and to plan prophylaxis
and treatment (Klein et al., 2009).
Figure 4. Percentage distribuon of the BRV and BCoV
posive calves by age (days)
Many invesgators have reported that the
immunochromatographic rapid test kits are a simple
and easy-to-apply method for the diagnosis of
enteropathogens in feces, and that they may be
preferred by clinical veterinarians and invesgators
more oen as they do not require specialist and fully-
equipped laboratory, are cheap and rapid in
comparison to other techniques, and can be applied
under any laboratory or oce condions which can be
found in each private clinic and even under eld
condions (Thorns et al.,1992; De la Fuente et al.,
2009; Klein et al., 2009). Klein et al. (2009) examined
the fecal samples collected from 1 day to 42 days old
180 calves (98 of them had the symptoms of diarrhea)
both with immunochromatographic rapid test kit and
RT-PCR method. Compared to RT-PCR, the
invesgators (Klein et al., 2009) found the sensivity
of the rapid test kit for BRV as 71.9% and the
specicity for the same as 95.3%, and its sensivity as
60% for BCoV and its specicity as 96.4% for the same.
In their study comparing the commercial rapid test
kits with the mulplex PCR method, Cho et al. (2012)
found the sensivity of the rapid test kits as 60% for
BCoV, and 42.3% for BRV, and they, therefore, stated
that the rapid test kits had to be interpreted carefully
in terms of originality and sensivity. In a study
Table 2. Sensivity, Specicity, PPV and NPV rates of the Rapid Test Kit versus RT-PCR in terms of BRV/BCoV
Reference Test Sensivity (%) Specicity (%) PPV (%) NPV (%)
BRV Rapid Test
BRV RT-PCR
83 100 100 96
BCoV Rapid Test
BCoV RT-PCR
7.6 100 100 88
PPV: Posive Predicve Value, NPV: Negave Predicve Value
Table 3. Stascal comparison of the posive results
by the tests applied
Rapid Test Kit (n) RT-PCR (n)
BRV Posive 15/96a 18/96a
BCoV Posive 1/96b 13/96a
BRV-BCoV Posive 0/96b 4/96b
a, b: Dierent leers within the same column are sta-
scally dierent. (p<0.05) a is stascally higher than
b.
Uyunmaz Saklı G. et al., 2019/ Journal of Istanbul Veterinary Sciences. Volume 3, Issue 3, pp: 57-63
In a study comparing real-me RT-PCR, ELISA and
immunochromatographic tests, Izzo et al. (2012)
found the sensivity of the rapid test kit as 32.7% for
BRV and as 28.2% for BCoV in comparison to RT-PCR
technique. The invesgators reported that the
sensivity and specicity levels of the
immunochromatographic rapid test kits were very low
in comparison to real-me PCR, and that it was
possible to interpret the course of the disease at the
clinic since the viral RNA amount is known due to real-
me PCR method. In their study on the rapid
eological diagnosis of neonatal calf diarrhea by
immunochromatographic test kits, Altug et al. (2013)
reported 14 cases of BRV (27.5%) and 1 case of BCoV
(1.96%) among the samples from 51 diarrheic calves.
In this study, among the samples examined by rapid
test kit, we found 15.6% (15/96) to be BRV-posive
and 1.04% (1/96) to be BCoV-posive. The same
samples were tested using RT-PCR method and the
posivity rates for BRV and BCoV were found 18.75%
(18/96) and 13.5% (13/96), respecvely. A
combinaon of BRV and BCoV infecons was detected
in 4% of the diarrheic feces (4/96).The results
obtained in this study were found to be compable to
those of Altug et al. (2013). Besides, in comparison to
RT-PCR technique, the sensivity of the
immunochromatographic rapid test kits for BRV was
83% and the specicity of the same was 100%, its
sensivity for BCoV was 7.6% and specicity for the
same was 100%. The results for bovine rotaviruses
were found to be compable with those of (Klein et
al., 2009), who have previously contrasted the
immunochromatographic rapid test kits to RT-PCR in
terms of sensivity and specicity, while they were
determined to be higher than those of Cho et al.
(2012) and Izzo et al. (2012). In terms of bovine
coronavirus, our results were signicantly lower than
those of many other invesgators (Klein et al. 2009;
Cho et al., 2012; Izzo et al., 2012) who have studied
the same subject maer. The possible reason for this
might be the fact that the sampling is carried out in
the late course of the disease when the level of virus
shedding and the amount of viral parcles are low.
The rapid immunochromatographic diagnosc
method is based on the aachment by the agent
within the sample dropped on the test stripe to the
conjugated specic anbodies. Therefore, it is
essenal to carry out the sampling during the peak
me of virus shedding. It is necessary to collect the
samples within 72 hours aer the onset of the
disease, because virus shedding decreases in me.
However, it is possible to detect even very low levels
of viruses by the RT-PCR method. The diagnosc
ability of the rapid test kit can be inferior to that of RT
-PCR in samples containing small amount of virus. “In
this study, we idened both BRV and BCoV by RT-
PCR in four samples. Only one sample was found
posive in terms of BoCV using the rapid test kit. The
electrophoresis images from the RT-PCR diagnosis of
this posive sample presented/showed a stronger
DNA stripe image in comparison to the other posive
samples. This indicates that the rapid test kit
determines posive results if there is high amount of
coronavirus in the fecal samples. Therefore, it is
necessary to support the results with a lot of samples.
Examining the age ranges of the calves and the
infecon-posive results by RT-PCR for these age
ranges, we see that the highest level of BRV-posivity
was found as 67% in calves of 0-5 days of age. This
rate was idened as 31% in the 6-15 days age group,
6% in the 16-20 days age group and 13% in the 21-30
days age group. In our invesgaons by RT-PCR for
BCoV, we idened no posivity in the 0-5 days age
group, but 34% in the 6-15 days age group, 13% in the
16-20 days age group, and 2% in the 21-30 days age
group (Figure 4). While the rates idened for BRV by
this study are close to those reported by Al Mawly et
al. (2015) (20% in calves of 1-5 days of age, and 19% in
calves of 9-21 days of age), but in terms of BCoV, the
results of Al Mawly et al. (2015) (5.4% in calves of 1-5
days of age, 6.1% in calves of 9-21 days of age) are
lower than those of this study. Alkan (1998) has
pointed out that this situaon can be associated with
the colostrum that calves receive from their mothers.
Alkan (1998) has reported that one of the most
important factors aecng the average infecon age
is maternal immunity. In this study, we know that the
calves from which we collected the samples had
generally received colostrum from their mothers.
Ellens et al., (1978) and Wood et al., (1975) have
reported that there were no rotavirus specic
anbodies in the second week aer birth, but
anbodies specic to coronavirus reached signicantly
high levels in the third week. Contemplang on the
fact that the coronavirus anbodies are secreted for a
long me in milk, Wellemans and Van Opdenbosch
(1981) have explained it with the fact that mothers
were considerably infected with coronavirus during
the diarrheic periods, their immune systems were
smulated as they shed the virus through their feces
on the day of giving birth, inducing the mammary
gland to secrete Ig anbody. Therefore, the total rate
of BRV-posivity in the rst two weeks (0-15 days) in
this study is 34% while it decreases to 6% and 13% in
Uyunmaz Saklı G. et al., 2019/ Journal of Istanbul Veterinary Sciences. Volume 3, Issue 3, pp: 57-63
the third and fourth weeks, respecvely. With regards
to BCoV, the posivity rate in the rst 3 weeks is 26%
while it decreases to as low as 2% in the fourth week.
The distribuon of posivity by the age groups
idened in this study was found to support the ideas
of Ellens et al. (1978), Wood et al. (1975) and
Wellemans and Van Opdenbosch (1981). This fact
shows that in this study colostrum received from the
mothers of calves smulated the maternal immunity,
and aected the posivity rate by age specied in the
study. Although this study idenes a low level of
sensivity for immunochromatographic rapid test kits,
one might think that the most important advantage of
these kits for clinical veterinarians with regard to BRV
diagnosis is to avoid wrong treatment with anbiocs.
Nevertheless, as the amount of virus decreases in the
late course of the disease, one must not ignore the
fact that it is not a very eecve method. This might
lead to an inaccurate interpretaon of the disease.
The presence of subclinical carrier animals is another
important issue to bear in mind while evaluang the
disease. This is a point a good veterinarian would not
like to ignore while assessing the cases of diarrhea in
calves posing a problem especially in big farms. As a
permanent soluon, molecular methods such as RT-
PCR play an essenal role in idencaon of these
animals. İt is possible to idenfy even one virus
parcle in the feces by RT-PCR (Klein et al. 2009).
Thus, veterinarians will be able to interpret the
disease accurately, and to take such protecve
measures as vaccinaon. It is of paramount
importance to idenfy the eld strains of infecons
such as bovine rotavirus and bovine coronavirus
present in Turkey. Apart from the group A rotavirus
idened in this study, idenfying the G and P type
rotavirus strains present in Turkey is of signicant
importance for the eecveness of vaccinaons. The
results of this study indicates that the rapid test kits
used by veterinarians under eld condions to
diagnose the diseases quickly can be benecial, but a
careful interpretaon is advisable since the sensivity
of the rapid test kits has been found to be low
(especially for BCoV) in comparison to RT-PCR. In
order to be able to interpret diseases more eecvely
and to seek more permanent soluons, it is advisable
to support the results through molecular techniques
such as RT-PCR.
Acknowledgement
The present work was supported by the Research
Fund of Selcuk University. Project No. 13202016
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