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Investigation of bovine coronavirus and bovine rotavirus by rapid diagnosis kit and RT-PCR in diarrheic calf feces

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  • İstanbul University-Cerrahpaşa Faculty of Veterinary Medicine

Abstract and Figures

This study has investigated bovine coronavirus (BCoV) and bovine rotavirus (BRV), which are among the most important causes of diarrhea in calves leading to financial losses in Turkey and all over the world BCoV and BRV were detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), which is one of the most reliable method of diagnosis, The results obtained by RT-PCR were compared to the sensitivity of the commercial Rota-Corona Rapid Test Kits used by clinical veterinarians in fields. In this study, 96 fecal samples were examined from diarrheic calves in cattle farms in the cities of Konya and Afyon for BRV and BCoV firstly 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 sensitivity and specificity revealed the 83% sensitivity and 100% specificity of the BRV and 7.6% sensitivity and 100% specificity of BCoV. In conclusion the practical 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 caution since the sensitivity of the test decreases due to the reduction in the number of viruses in the later stages of the infection. .
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
Research Arcle
Volume: 3, Issue: 3
December 2019
Pages: 57-63
Invesgaon 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 Instute,
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 invesgated 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 Reacon (RT-PCR), which is one of the most reliable method of
diagnosis, The results obtained by RT-PCR were compared to the sensivity 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 cale farms in the cies
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
sensivity and specicity revealed the 83% sensivity and 100% specicity of the BRV
and 7.6% sensivity and 100% specicity of BCoV. In conclusion the praccal 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 cauon since the sensivity of
the test decreases due to the reducon in the number of viruses in the later stages of
the infecon.
Keywords: BCoV, BRV, calf diarrhea, rapid test kit, RT-PCR
DOI: ---
To cite this arcle: Uyunmaz Saklı G., Bulut, O., Hasöksüz, M., Hadimli, H.H. (2019). Invesgaon 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 Internaonal Congress of Veterinary
Microbiology (16-19 October 2018).
Neonatal calf diarrhea caused by viral, bacterial and
protozoon agents is one of the infecons characterized
by enteris 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 variaons depending on the
regional and stable condions, the role of rotavirus
and coronavirus in the cases of calf diarrhea have been
found to reach up to 50% and 80% respecvely.
Rotaviruses generally cause infecons 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 infecon
*Corresponding Author: Gülşah Uyunmaz Saklı
E-mail: gulsah_uyunmaz@hotmail.com
hps://dergipark.org.tr/tr/pub/hp-www-jivs-net
Arcle History
Received: 05.08.2019
Accepted: 22.11.2019
Available online:
02.12.2019
 Creative Commons Attribution 4.0 International License
Introducon

agents (Bartels et al., 2010; Izzo et al., 2012). Among
the factors that aect the course of rotavirus and
coronavirus infecons are whether the newborns
received colostrum, me of weaning, climate
condions, their immune condions, 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 parcles (approximately 1011 parcle/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 isolaon (Mebus et al., 1969; Hasoksuz et
al.,2002; Gulyaz et al., 2005),
immunochromatographic rapid diagnosc 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
infecons.
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 distribuon of the
collected samples is presented in Figure 1. The fecal
samples were collected from the rectums of the
animals with sterile coon swabs. One swab taken out
of the rectum was put in the soluon in the rapid test
kit, and another was homogenized by pung it into
Phosphate Buer Saline (PBS), and stored at -20ºC
unl it was used for RT-PCR. The samples for the rapid
test kits were examined under stable condions, and
their results were reported to the owners in 15
minutes. Necessary notes were taken and the posive
samples were taken into account for the next
examinaon.
Rapid Diagnosc 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
soluon included in the kit during the sampling and
was homogenized. Then one drop of the soluon was
added onto the arrays and according to the change of
color, coronavirus or rotavirus was interpreted as
posive or negave.
RT- PCR Materials: Extracon of the Viral RNA: We
used High Pure Viral RNA isolaon 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 centrifugaon 200 μl of the supernatant
was taken and transferred into a 1.5 ml RNase-free
sterile tube. Each sample was mixed with working
soluon containing 4 μl Poly A and 400 μl Binding
Buer. The working soluon and the sample mixture
were treated with the Removal Buer, Wash Buer,
Eluon Buer included in the kit by using the silica gel
spin column. At the end of the extracon process, 50
µl of viral nucleic acid was isolated, and stored at
-80oC.
cDNA Synthesis: We used Reverse Transcripon
System (Promega A3500) to obtain cDNAs. In order to
synthesize cDNA from the isolated viral RNA, we used
the Promega Reverse Transcripon 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 incubaon, it was
kept in ice for 2 minutes. Master Mix (15 µl for each
sample) was prepared in a dierent 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 amplied 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 resulng products were stored at -
20oC.
Polymerase Chain Reacon (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 specic to the VP7 gene region of the
group A rotaviruses. We used the primers reported by
Cho et al., (2013) for the detecon of bovine
coronavirus. These primers are specic 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 incubaon, 43 μl Master Mix composed
of Primer F (20 pmol), Primer R (20 pmol), and
soluons of the Promega M8305 kit and the Promega
A3500 cDNA kit was treated with 7 μl RNA and DMSO
mixture. It was amplied 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 reacon, Go Taq Flexi DNA
Polymerase, 5X buer green exi color, MgCl2 (25
mM) and dNTP. It was amplied 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 amplicaon products, 1.5%
agarose gel containing ethidium bromide was
prepared. The PCR products were run at 100 V for 30-
45 minutes and the amplied DNA bands were
controlled under UV light.
Stascal analysis: We used the chi-square test (χ2)
for the stascal analysis of the diagnosc tests. We
recorded p<0.05 as stascally signicant.
Results
We examined 96 diarrheic fecal samples in total for
BCoV and BRV by rapid diagnosc test and RT-PCR
method. The collecve results of the study are
presented in Table 1. According to the results, the
rapid diagnosc test revealed 15 samples as BRV
posive (15.62%) and 1 sample as BCoV posive
(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 posive
by RT-PCR method. However, only one sample was
found to be BRV and BCoV posive with the rapid
diagnosc test.
Figure 1: Distribuon of the samples of calve feces by
age (days)
This sample showed a quite strong DNA band
appearance aer 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 posive by the
rapid test kit, the rates of posivity were 67%, 24%,
0% and 13% respecvely. Only 4% of the 6-15 days old
calves were found to be BCoV posive. For the 0-5, 6-
15, 16-20 and 21-30 days old calves found to be BRV
posive by the RT-PCR method, the rates of posivity
were 67%, 31%, 6% and 13% respecvely. In terms of
BCoV, while RT-PCR found no posive samples in the 0
-5 days old group, the other groups found to be 34%,
13% and 2% posive respecvely (Figure 4).
Figure 2: Electrophoresis image of the BRV (1062 bp)
posive samples, DNA Ladder (100 bp Fermentas), PC
(Posive Control), NC (Negave Control), DNA bands
of the samples; 27, 28, 12, 13, 14, 15, 16 and 17
Figure 3: Electrophoresis image of the BCoV (730 bp)
posive samples; DNA Ladder (100 bp Fermentas), PC
(Posive Control), NC (Negave 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
infecon is important for the control and eradicaon
of the disease in newborn animals in cale 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 eecve
test techniques in veterinary diagnosc laboratories.
Among these methods, isolang the RNA of the virus
and converng it into DNA (cDNA) and mulplying the
cDNAs by using specic primers (RT-PCR) has the
highest sensivity and originality. However, as these
techniques can only be applied under laboratory
condions and require me, clinical veterinarians
need rapid test kits to diagnose the infecon under
eld condions. These rapid test kits are important in
terms of determining the treatment process and
avoiding wrong anbioc 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 sensivity and
specicity of these rapid test kits. In recent years, the
rapid immunochromatographic tests, which are more
advantageous under led condions, it has become
possible to diagnose dierent 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 distribuon of the BRV and BCoV
posive calves by age (days)
Many invesgators 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 invesgators
more oen 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 oce condions which can be
found in each private clinic and even under eld
condions (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
invesgators (Klein et al., 2009) found the sensivity
of the rapid test kit for BRV as 71.9% and the
specicity for the same as 95.3%, and its sensivity as
60% for BCoV and its specicity as 96.4% for the same.
In their study comparing the commercial rapid test
kits with the mulplex PCR method, Cho et al. (2012)
found the sensivity 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 sensivity. In a study
Table 2. Sensivity, Specicity, PPV and NPV rates of the Rapid Test Kit versus RT-PCR in terms of BRV/BCoV
Reference Test Sensivity (%) Specicity (%) PPV (%) NPV (%)
BRV Rapid Test
BRV RT-PCR
83 100 100 96
BCoV Rapid Test
BCoV RT-PCR
7.6 100 100 88
PPV: Posive Predicve Value, NPV: Negave Predicve Value
Table 3. Stascal comparison of the posive results
by the tests applied
Rapid Test Kit (n) RT-PCR (n)
BRV Posive 15/96a 18/96a
BCoV Posive 1/96b 13/96a
BRV-BCoV Posive 0/96b 4/96b
a, b: Dierent leers within the same column are sta-
scally dierent. (p<0.05) a is stascally 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 sensivity of the rapid test kit as 32.7% for
BRV and as 28.2% for BCoV in comparison to RT-PCR
technique. The invesgators reported that the
sensivity and specicity 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
eological 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-posive
and 1.04% (1/96) to be BCoV-posive. The same
samples were tested using RT-PCR method and the
posivity rates for BRV and BCoV were found 18.75%
(18/96) and 13.5% (13/96), respecvely. A
combinaon of BRV and BCoV infecons was detected
in 4% of the diarrheic feces (4/96).The results
obtained in this study were found to be compable to
those of Altug et al. (2013). Besides, in comparison to
RT-PCR technique, the sensivity of the
immunochromatographic rapid test kits for BRV was
83% and the specicity of the same was 100%, its
sensivity for BCoV was 7.6% and specicity for the
same was 100%. The results for bovine rotaviruses
were found to be compable with those of (Klein et
al., 2009), who have previously contrasted the
immunochromatographic rapid test kits to RT-PCR in
terms of sensivity and specicity, 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 signicantly lower than
those of many other invesgators (Klein et al. 2009;
Cho et al., 2012; Izzo et al., 2012) who have studied
the same subject maer. 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 parcles are low.
The rapid immunochromatographic diagnosc
method is based on the aachment by the agent
within the sample dropped on the test stripe to the
conjugated specic anbodies. Therefore, it is
essenal to carry out the sampling during the peak
me of virus shedding. It is necessary to collect the
samples within 72 hours aer 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 diagnosc
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 idened both BRV and BCoV by RT-
PCR in four samples. Only one sample was found
posive in terms of BoCV using the rapid test kit. The
electrophoresis images from the RT-PCR diagnosis of
this posive sample presented/showed a stronger
DNA stripe image in comparison to the other posive
samples. This indicates that the rapid test kit
determines posive 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
infecon-posive results by RT-PCR for these age
ranges, we see that the highest level of BRV-posivity
was found as 67% in calves of 0-5 days of age. This
rate was idened 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 invesgaons by RT-PCR for
BCoV, we idened no posivity 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 idened 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 situaon can be associated with
the colostrum that calves receive from their mothers.
Alkan (1998) has reported that one of the most
important factors aecng the average infecon 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 specic
anbodies in the second week aer birth, but
anbodies specic to coronavirus reached signicantly
high levels in the third week. Contemplang on the
fact that the coronavirus anbodies 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
smulated as they shed the virus through their feces
on the day of giving birth, inducing the mammary
gland to secrete Ig anbody. Therefore, the total rate
of BRV-posivity 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, respecvely. With regards
to BCoV, the posivity rate in the rst 3 weeks is 26%
while it decreases to as low as 2% in the fourth week.
The distribuon of posivity by the age groups
idened 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 smulated the maternal immunity,
and aected the posivity rate by age specied in the
study. Although this study idenes a low level of
sensivity 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 anbiocs.
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 eecve method. This might
lead to an inaccurate interpretaon of the disease.
The presence of subclinical carrier animals is another
important issue to bear in mind while evaluang 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 soluon, molecular methods such as RT-
PCR play an essenal role in idencaon of these
animals. İt is possible to idenfy even one virus
parcle in the feces by RT-PCR (Klein et al. 2009).
Thus, veterinarians will be able to interpret the
disease accurately, and to take such protecve
measures as vaccinaon. It is of paramount
importance to idenfy the eld strains of infecons
such as bovine rotavirus and bovine coronavirus
present in Turkey. Apart from the group A rotavirus
idened in this study, idenfying the G and P type
rotavirus strains present in Turkey is of signicant
importance for the eecveness of vaccinaons. The
results of this study indicates that the rapid test kits
used by veterinarians under eld condions to
diagnose the diseases quickly can be benecial, but a
careful interpretaon is advisable since the sensivity
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 eecvely
and to seek more permanent soluons, 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
Aich, P., Heather, L., Wilson, Radley, S., Kaushik, T.,
Andy, A., Poer Lorne, A., & Griebel P. (2007).
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rotavirus and coronavirus. Journal of General
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Uyunmaz Saklı G. et al., 2019/ Journal of Istanbul Veterinary Sciences. Volume 3, Issue 3, pp: 57-63
... Diarrhea of especially with infectious origin (bacterial, viral and parasitic) occurs in the neonatal period, is commonly caused by E. coli, cryptosporidium, rotavirus and coronaviruses. Diarrhea may also develop due to non-infectious causes including alimentary, predisposing and environmental factors [3]. Among the infectious factors, Group A rotaviruses (RVA) are the leading cause of acute viral gastroenteritis in young animals as well as in children worldwide [4,5]. ...
... Detection of the disease factor in newborn calves in herds is an important situation in preventing economic losses and disease control. The detection of rotaviruses can be achieved by various methods such as virus isolation in cell culture, immunochromatographic test kits, polyacrylamide gel electrophoresis, fluorescent antibody assay, ELISA, RT-PCR and electron microscopy (EM) [7,16], though sharing different sensitivities [3,7,16,18]. Despite various results were reported, it is quite difficult to isolate and propagate bovine rotaviruses from the native specimens using cell culture systems [16,19]. ...
... As well as the applied protocol and selected cell line, the sampling time is one of the important criteria in virus isolation from stool samples. The chance of virus isolation is high in the samples taken during the onset and peak period of clinical findings [3]. In the present study, samples were freshly collected from the clinical cases, quickly transferred to laboratory and inoculated into cell culture without a freeze-storage period. ...
Article
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Background Neonatal calf diarrhea, which is the most common cause in calf deaths, leads to significant economic losses in dairy farming around the world. Diarrhea develops due to infectious and non-infectious reasons. Group A Rotaviruses (RVA) are the leading and predisposing factor for acute neonatal gastroenteritis.Methods and resultsIn this study, 20 diarrheic fecal samples were collected from one farm in Balıkesir province of Turkey. During virus isolation, a total of 2 stool samples were detected to produce cytopathogenic effects in MA-104 cell line. The two samples (RV-36, RV-38) were tested positive with antigen ELISA kits detecting RVA antigens. In order to detect the presence of rotavirus viral nucleic acid in cell supernatants, VP6 gene region-specific RT-PCR test was performed and the samples RV-36 and RV-38 were positive for RVA viral nucleic acid. By RT-PCR using genotype specific primers, both the isolates RV-36 and RV-38 formed amplicons compatible with G10 and P[11] genotypes of RVA. RVA nucleic acids segments were also visualized by poliacrilamide gel electrophoresis (PAGE) method. The phylogenetic tree constructed according to the VP6 gene region showed that these isolates were in the Rotavirus A group and in the I2 cluster same as other bovine and some human RVA isolates.Conclusion Succesful isolation of RVA G10P[11] was echieved in the cattle farm. As rotaviruses play the most important role in the etiology of diarrhea in newborn calves respected genotype G10P[11] should be considered in selection of the vaccines applied to the dams. Those isolates can be further evaluated as vaccine candidate.
... E. coli causes diarrhea in calves younger than 7 days of age and mostly in the 1st 12 h of their lives [15]. While BRV generally causes diarrhea in calves aged 2-21 days, BCoV is detected in calves with diarrhea aged 5-30 days [8,28]. In Turkey, it was reported BRV from 16 using PAGE method of the samples collected from 89 diarrheal calves between the ages of 1 and 30 days in Van province between 2002 and 2003, and reported BCoV from 1 sample using indirect ELISA [13]. ...
... Some researchers detected BRV in 6 calves, BCoV in 3, and E. coli K99 in 2 calves in the 0-7 day age range, and BRV and BCoV were detected in 1 animal each in the 8-14 age range, BRV was detected in only 1 animal in the age range of 15-21 days, while BRV and E. coli K99 were detected in 1 animal in the age range of 22-28 days by the rapid diagnostic kit [5]. However, the presence of BRV, 67% was detected in calves aged 0-5 days, 31% was found in calves aged 6-15 days, 6% was found in calves aged 16-21 days, and 13% was detected in calves aged 21-30 days with RT-PCR method [28]. In the same study, while BCoV was not detected in calves aged 0-5 days, they determined the presence of BCoV in 34% of calves aged 6-15 days, 13% in calves aged 16-20 days, and 2% in calves aged 21-30 days. ...
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Background: Neonatal calf diarrhea is one of the most important problems of calf breeding in the world. It causes serious economic losses by causing deaths in calves in the 1 st 10 days of their lives. Although many factors cause calf diarrhea, Escherichia coli (E. coli), Salmonella spp., Campylobacter spp., viral agents such as Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) and parasitic agents such as Cryptosporidium spp., Eimeria spp. and Toxocara spp. frequently plays a role in calf diarrhea. In addition to these infectious factors, the formation of diarrhea has been linked to a number of other factors, including unfavorable barn conditions, mass rearing, inadequate cleaning and disinfection of barn tools, delaying the feeding of colostrum to newborn calves, failing to do so, and failing to disinfect the umbilical cord after delivery. The identification of the microorganisms and virulence factors responsible for diarrhea in newborn calves will direct the development of preventative measures and control strategies. The present study aimed to determine various virulence factors (Stx1, Stx2, STa, eaeA, K99 and F41) of Escherichia coli isolated from fecal samples and to investigate the prevalence of BRV, BCoV and E. coli K99 agents which play a role in the etiology of neonatal calf diarrhea in Burdur province. Materials, Methods & Results: This study was carried out in 75 different cattle farms with diarrhea problem in Burdur province and its districts. Rectal swab samples were taken from 90 calves with diarrhea between 0-4 weeks of age, using 2 sterile swabs from each animal. The swabs are used for the detection of BRV, BCoV and E. coli K99 by direct ELISA, and the isolation of E. coli. The swabs were cultured on blood agar including 7% sheep blood and MacConkey agar. The isolated bacteria were identified by conventional bacteriological culture methods such as Gram staining, triple tube method, oxidase. The bacteria isolated and identified as E. coli were stored at-20ºC by using. Enzyme linked immunobinding assay (ELISA) was positive in 45 of the calf fecal samples. BRV was detected in 28 (31.11%) of samples, BCoV in 15 (16.67%) and E. coli K99 in 11 (12.22%) samples by ELISA. While BRV and BCoV were detected together in 5.56% of the samples, BRV and E. coli K99 were detected in 1.11% of samples, BCoV, E. coli K99 were detected in 1.11% of samples and all three infections were detected together in 1.11% of samples. The virulence factors of E. coli was investigated by polymerase chain reaction (PCR) and Stx1, K99, F41 and eaeA virulence genes were determined in 2, 5, 3, 4 of samples, respectively. K99 and F41 antigens were detected together only in 2 of the E. coli isolates. The according to ages, it was determined that the highest BRV was detected between 1-7 days of age and 8-15 days of age. While E. coli was detected in 5 of 1-day-old calves, BCoV was also detected in 1 of these calves. Discussion: In this study, it has been determined that neonatal calf diarrhea is mostly caused by BRV in Burdur province, followed by BCoV and E. coli. Even though E. coli was recovered from the samples, the inability to extract virulence factors suggests that additional virulence factors might potentially be involved in the infection. It was concluded that the determination of virulence factors of E. coli isolates can be a guide in the preparation of protection and control strategies in calf diarrhea.
... Traditional diagnostic methods involving RNA extraction and conversion to cDNA are labor-intensive and time-consuming. However, rapid test kits can swiftly and accurately detect prevalent enteropathogens associated with diarrhea in newborn calves [11]. In this study, all 19 positive samples tested using a rapid test kit confirmed rotavirus infection. ...
... 4-surface, extract the brilliant liquid from the top with a dropper, and then add 5 drops to the S-marked sample hole. 5-After reading the test card's result after[10][11][12][13][14][15] minutes at room temperature the result is no longer valid.Real time-PCR Fecal suspension was prepared in DEPC water to 1:4 dilution then clarified at 5000 rpm\15 minutes at 4°C and the supernatant submitted to RNA extraction. Viral RNA was extracted from stool samples by using AccuZolTM Total RNA extraction kit (Bioneer, Korea) and done according to company instructions. ...
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... These procedures are time consuming and require experienced technician. On other hand, the rapid S&C Biotech Bovine Rotavirus Antigen Rapid Test Kits is very helpful and practical for veterinarians in field as this (Sakli et al., 2019). ...
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The bovine rotavirus (BRoV) is one of the major causes of illness and death in newborn calves. The study's objective was to pinpoint the haemato-biochemical alterations, other risk factors, and molecular manifestations associated with BRoV infections in dairy cow calves in in Jhelum district, Pakistan. From July 2020 to June 2021, a total of 200 faeces samples were taken from neonate cow calves under 28 daysold that had a history of diarrhea and dysentery. Prior to further polymerase chain reaction processing, samples were initially screened using S&C Biotech Bovine Rotavirus Antigen Rapid Test Kits. For the haemato-biochemical study, blood samples were obtained from calves infected with BRoV. On a questionnaire form, information was gathered for the analysis of the various risk factors linked to the occurrence of BRoV infection. The occurrence of BRoV infection while utilizing diagnostic screening kits was 26% (52/200), and when using RT-PCR, it was 21.5% (43/200). BRoV infection was significantly (p≤0.05) influenced by breed, age, sex, vomiting, prior history of diarrhea, bodily conditions, food type, colostrum feeding, deworming history, living environment, interaction with other animals, and season. Hematological and biochemical markers showed significant (p≤0.05) alterations. Mean corpuscular volume, basophils and lymphocytes were decreased significantly (p≤0.05 while mean corpuscular hemoglobin, total leukocyte count, TEC, white blood cells count, red blood cell and Monocytes were increased significantly (p≤0.05). Similarly, among biochemical parameters, Potassium was non-significantly (p>0.05) increased, while Sodium, Calcium, copper and iron were significantly (p≤0.000) decreased. Itwas concluded that assumed risk factors were contributed to the BRoV infection, and infected calves showed haemato-biochemical changes. Keywords: neonate calves, rotavirus, diagnostic test, Pakistan
... In the present study, rotavirus was found in 20.3% of the diarrheic calves which falls within the range (10.4-53%) of infections reported elsewhere in Turkey [35,36,37,38]. Although the prevalence of rotavirus infection varies depending on factors such as the frequency of farm hygiene and protection and control measures, the detection of the disease in one of every five calves in the study group in this region indicates that the NCD disease is a disease that requires precautions. ...
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This study reports potential causes of diarrhea in neonatal calves, leading to calf mortality, from the selected population of the three Provinces of Turkey. A total of 300 fecal samples were collected purposively from diarrheic neonatal calves distributed to the three age groups (1–14 days, 15–29 days, and 30–90 days), from Konya, Karaman, and Aksaray Provinces of Turkey. The fecal specimens were examined for the existence of Cryptosporidium spp., rotavirus, coronavirus, and Escherichia coli by commercially available capture direct enzyme linked immunosorbent assay (ELISA) kit. The oocysts and coproantigens of Cryptosporidium were identified in 109 (36.3%) and 156 (52%) of the 300 calves, respectively. While, rotavirus, E. coli and coronavirus antigens were detected (P<0.05) in 57 (19%), 17 (5.6%) and 6 (2%) calves, respectively. Mixed infection of the study pathogens has also been found in this report. These results provide a baseline information on the frequent causes of neonatal calf diarrhea in the studied Provinces which can be used to develop a prophylaxis plan.
... These viruses can be identified using the cell-line culture method or the polymerase chain reaction or rapid detection kits tests. Rapid disease diagnosis using a Rapid Diagnosis kit is very useful and practical for veterinarians in the field because it provides quick results and takes less time (Sakli et al., 2019). There is no cure for viral infections; instead, supportive care is provided to correct electrolyte imbalances, metabolic acidosis, and dehydration (Fox, 2015). ...
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Bovine rotaviruses cause loss of calves and cause great financial losses to breeders. Bovine rotaviruses, which are classified in the Reovirales order, Sedoreoviridae family and Rotavirus genus, are mostly classified as G and P genotypes according to VP7 and VP4 gene regions. In addition, 10 different species (group A-J) have been identified according to genetic and antigenic properties of another major antigen, VP6. Group A rotaviruses are the most common cause of diarrhea in calves, while group B and C infections are also known. For the protection of calves, rotavirus screening should be performed on a herd basis and the infection status of cattle should be revealed. For this purpose, stool samples of 100 calves with diarrhea symptoms in the inventory of Atatürk University, Faculty of Veterinary Medicine, Department of Virology were used. Polyacrylamide gel electrophoresis (PAGE), which allows the examination of segments of the genome, was used to check for the presence of the virus. Nucleic acid extraction was performed on the stool samples before electrophoresis and then extracts were loaded into the prepared polyacrylamide gel and run. The samples were stained with silver nitrate stain, segment patterns were determined and the presence of rotavirus was analyzed. While 27 of the analyzed samples were positive, 5 samples were suspicious and 68 samples were negative. The segment pattern of the positive samples was compatible with group A and all of them were classified in this group. Although they were in the same group, it was determined that the positive samples had 3 different electrophoretypes. As a result, it was determined that rotaviruses still have an important role in the etiology of calf diarrhea. Besides, the detected rotaviruses showed variation, although they were in group A, and breeders in the region should pay attention to control and hygiene measures.
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Rotavirus is a type of virus that primarily affects infants and young children, causing gastroenteritis, which is inflammation of the stomach and intestines. It is one of the leading reasons for server diarrhea worldwide,especially in developing lands. The virus is highly contagious and can spread through the fecal-oral route, primarily through contaminated food, water, and surfaces. The introduction of the rotavirus vaccines has had a significant impact on reducing the burden of rotavirus-related illnesses. Many countries have incorporated rotavirus vaccination into their national immunization programs. The vaccines havesignificantly reduced rotavirus-related hospitalizations, deaths, and overall disease burden in countries where vaccination coverage is high. Here, we will discuss the recent developments in epidemiology, diagnosis, and treatments of rotavirus-induced gastroenteritis. Moreover, suggestions to prevent incidences of infection have been elegantly discussed.
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