Evaluation of relationship between Rotavirus and Coronavirus infections with calf diarrhea by capture ELISA

ORIGINAL ARTICLE
Evaluation of relationship between Rotavirus
and Coronavirus infections with calf diarrhea
by capture ELISA
Ashraf Mayameei & Gholamreza Mohammadi &
Samane Yavari & Ehsan Afshari & Arash Omidi
Received: 28 May 2009 /Accepted: 24 September 2009
# Springer-Verlag London Limited 2009
Abstract Rotavirus and Coronavirus are two main causes
of severe diarrhea in human infants and many animal
species worldwide. Calves up to 3 months old can be
affected by these viruses. The importance of these viruses
in cattle industry is due to substantial economic loss,
treatment costs, and reduced growth rates in beef and dairy
calves. Mixed infections caused by Rotavirus and Corona-
virus can lead to severe form of diarrhea. In present case-
control study, 261 samples of healthy and diarrheic calves
from farms around Mashhad were collected. These samples
were tested by antigen-capture enzyme-linked immunosor-
bent assay (Bio-X diagnosis kit). The results showed that
the prevalence of Rotavirus and Coronavirus infection in
diarrheic calves is 26.98% and 3.17%, respectively. Also,
there was no significant relationship between infection with
Rotavirus, Coronavirus, and diarrhea in the studied
population.
Keywords Rotavirus .Coronavirus . Calf diarrhea .
Capture ELISA
Abbreviations
R Rotavirus
C Coronavirus
ELISA Enzyme-linked immunosorbent assay test
BRV Bovine rotavirus
BCV Bovine coronavirus
Sp Specification of ELISA test
Se Sensitivity of ELISA test
Tp True prevalence
Introduction
Calf diarrhea, one of the most widespread diseases, is a
complex syndrome with a complex etiopathogenesis,
causing important economic losses due to morbidity and
mortality, treatment costs, and reduced growth rates in
affected calves (Garaicoechea et al. 2006; Reidy et al.
2006). The etiology of this syndrome involves infectious
agents (viruses, bacteria, and protozoa) and also non-
infectious factors such as herd management, host nutrition-
al, and immunological condition which affect the outcome
of the disease (Brussow et al. 1992; Maes et al. 2003;
Garaicoechea et al. 2006).
Group A Rotaviruses, as the member of Reoviridae
family, have clearly been established as causing significant
diarrheal disease in infants and in the young of various
A. Mayameei (*)
Department of Pathobiology, School of Veterinary Medicine,
Ferdowsi University,
Mashhad, Iran
e-mail: mayamee@ferdowsi.um.ac.ir
G. Mohammadi : E. Afshari
Department of Clinical Studies, School of Veterinary Medicine,
Ferdowsi University,
Mashhad, Iran
G. Mohammadi
e-mail: gmohamad@ferdowsi.um.ac.ir
S. Yavari
Graduated DVM student, School of Veterinary Medicine,
Ferdowsi University,
Mashhad, Iran
e-mail: dr81_yavari@yahoo.com
A. Omidi
Department of Animal Sciences, School of Agriculture,
Birjand University,
Birjand, Iran
e-mail: Arashomidi2@gmail.com
Comp Clin Pathol
DOI 10.1007/s00580-009-0920-x

mammalian and avian species (Brussow et al. 1992;
Murphy et al. 1999; Flint et al. 2000; Estes and Kapikian
2001).Group A bovine rotavirus (BRV) is considered the
major cause of severe diarrhea in calves worldwide
(Garaicoechea et al. 2006). Coronaviruses as the member
of Coronaviridae family are associated with economically
important diseases in cattle, poultry, pigs, and cats (Lai et al.
2007; Acheson 2007). Bovine coronavirus (BCV) causes
diarrhea in both dairy and beef calves worldwide (Radostitis
et al. 2007). Since diarrhea in calf and other animals is a
great problem which involves economic losses, several
diagnostic methods are used to detect enteropathogenic
agents. Diagnosis is done through collecting feces of animals
suffering from diarrhea by a rectal swab or collecting
intestinal contents (Castro and Heuschele 1992) and using
laboratory diagnostic tests such as direct electron-
microscopic test, Enzyme-linked immunosorbent assay
(ELISA), latex agglutination, polyacrylamide gel electropho-
resis, reverse transcription polymerase chain reaction, and
also immuno-electron microscopy (Parwani et al. 1992;
Radostitis et al. 2007). Among these methods, ELISA using
monoclonal antibody, as a sensitive, fast, and inexpensive
method could be used for the simultaneous detection of
bovine coronavirus, bovine rotavirus group A, and also other
enteropathogenic factors in the feces of diarrheic calves
(Crouch et al. 1984; Radostitis et al. 2007). In this study, to
examine the possible involvement of BRV and BCV in calf
diarrhea, a case-control study by using capture ELISA, was
performed in dairy farms around Mashhad, Iran.
Materials and methods
A total of 261 fecal samples from diarrheic (126 samples)
and healthy (135 samples) calves of up to 3 months of age
were collected from some herds around Mashhad, Iran. The
collected samples were kept at −20°C until analyzing. A
commercial indirect antigen-capture ELISA kit (Duo
Digestive kit (Rotavirus and Coronavirus), Bio K083,
Bio-X Diagnostics, Belgium) employing specific mono-
clonal antibodies was used to detect BRV and BCV in
fecal samples. The ELISA procedure was performed
according to the manufacturer instruction. After adding
stop solution (1 M phosphoric acid), the optical density
(OD) of each well was measured at 450 nm. Calculating
the net OD of each sample and interpreting the results was
performed as described by manufacturer instruction.
Briefly, the limit of positivity of each sample was 0.15.
Any sample yielding a net OD value greater than or equal
to 0.15, was considered positive, and less than 0.15,
considered as negative.
The analysis of the results was done with Statistical
Package for the Social Sciences software. To evaluate the
relationship between two variables: disease and risk factors
(the presence of BRV and BCV in calf fecal samples) the
chi-square test was applied.
Results
In this study, a total of 261 samples, 126 from diarrheic and
135 from healthy calves, were analyzed. Rotavirus antigen
was detected in 56 and Coronavirus was detected in eight of
the total 261 samples (Table 1). The results showed that
according to ELISA test, the apparent prevalence of BRVand
BCV infection in diarrheic calves, was 26.98% (34:126) and
3.17% (4:126), respectively, and also the apparent prevalence
of Rotavirus and Coronavirus infection in healthy calves was
17.03% (23:135) and 2.96% (4:135), respectively.
By considering 98% sensitivity and 100% specificity of
ELISA kit for Rotavirus and 95% sensitivity and 90%
specificity of ELISA kit for Coronavirus, the true prevalence
of BRV and BCV in diarrheic and healthy calves was
estimated using the “Rogan and Gladen” method (Thrusfield
2005).
Trueprevalence ¼
apparentprevalenceþ sp� 1ð Þ
seþ sp� 1ð Þ
In diarrhetic calvesð Þ TpRota ¼
26:98þ 99� 1
98þ 100� 1
¼ 0:64 ¼ 64%
In diarrhetic calvesð Þ TpCorona ¼
3:17þ 90� 1
95þ 90� 1
¼ 0:50 ¼ 50%
In healthy calvesð Þ TpRota ¼
17:03þ 100� 1
98þ 100� 1
¼ 0:59 ¼ 59%
In healthy calvesð ÞTpCorona ¼
2:96þ 90� 1
95þ 90� 1
¼ 0:50 ¼ 50%
According to the results of statistical analysis (chi-
square), in spite of higher prevalence of Rotavirus and
Coronavirus infection in diarrheic calves, no significant
Calves Fecal sample BRV BCV
Positive Negative Positive Negative
With diarrhea 126 34 92 4 122
Healthy 135 23 112 4 131
Total 261 57 204 8 253
Table 1 The ELISA results for
BRV and BCV antigens in the
fecal samples collected from the
healthy and diarrheic calves
BRV bovine rotavirus, BCV
bovine coronavirus
Comp Clin Pathol

relationship was established based on comparing healthy
and diarrheic calves (P>0.05).
Tables 2 and 3 show the frequency and prevalence of
BRV and BCV infection in different ages. Since, the cases
of Rotavirus infection were more; it was statistically
analyzed in different ages. There was not any significant
relationship between Rotavirus infection and disease in
different ages of calves in studied population (P>0.05).
Discussion
Due to spread of intestinal infections outbreak all over the
world and the out coming mortality, these infections along
with severe diarrhea in many young animals and children
are of great importance (Gumusova et al. 2007). In young
calves, diarrhea is considered as one of the most important
diseases, because the resulting economic loss following
mortality, treatment costs, and decrease of growth rate
would be detrimental (Maes et al. 2003). Indeed, long-term
effects of diarrhea on the health and efficiency of calves
cured from clinical courses of the disease may cause more
economic loss (Fuente et al. 1998). Rotavirus, Coronavirus,
enterotoxigenic Escherichia coli, and Cryptosporidium
parvum are four major pathogens accompanied by diarrhea
in young calves around the world. These organisms are
responsible for most of the intestinal infections (75–90%)
in young calves in the world. Different species of
Salmonellae are also of importance in dairy calves
(Athanassious et al. 1994; Fuente et al. 1998; Radostitis et
al. 2007). To increase the production of each cattle without
increasing the number of live stocks, it is important to
detect causative factors of diarrhea in calves so as to use,
based on this information, the effective preventive methods
for decreasing the mortality of calves in the first months of
their life (Perez et al. 1998). In this study, along with
determining the BRV and BCV prevalence in diarrheic
calves, a case-control study was performed to examine
possible involvement of BRV and BCV in diarrhea in some
dairy farms around Mashhad, Iran. Many other studies
reported the prevalence of BRVand BCV in scouring calves
over the world. The presence of BRV in six and BCV in
two samples out of nine fecal samples from diarrheic calves
with 10–60 days of age was reported in Sao Paulo, Brazil
(Brandao et al. 2007). The other study in Turkey showed
the presence of BRV antigen and BCV antigen in diarrheic
calves, 41.17% and 1.96%, respectively. In healthy calves,
BRV was detected in 4.08% and BCV was not detected
(Gumusova et al. 2007). In a case-control study in Costa
Rica on dairy and beef calves younger than 3 months, by
using dot-ELISA test, Rotavirus was detected in 7% and
2% of scouring and control calves, respectively, and
Coronavirus in 9% and 1%, respectively. In 25% of
diarrheic calves and 21% of healthy ones, there were not any
infectious factors and viruses were found in infected calves
younger than 1 month more predominantly (Perez et al. 1998).
In Spain, by using a commercial ELISA kit, Rotavirus was
detected in 42.7% and Coronavirus was found in 7.3% of
fecal samples from diarrheic calves. Mixed infection with
Rotavirus and Coronavirus was found in 5.1% of the total
samples of feces, among which, in 2.3% of fecal samples,
the only enteropathogens found were Rotavirus and Coro-
Table 2 The ELISA results for BCV and BRV and mixed infection with both BRV and BCV in healthy and diarrheic calves according to age
Age Healthy calves Diarrheic calves
No of
samples
BRV (%) BCV (%) Mixed BRV
and BCV (%)
No of
samples
BRV (%) BCV (%) Mixed BRV
and BCV (%)
1 week 34 8 (23) 0 0 27 6 (22) 0 0
2 weeks 28 7 (25) 1 (3.5) 1 (3.5) 47 17 (36) 3 (6.3) 1
3 weeks 18 3 (16) 2 (11) 0 15 7 (46) 1 (6.6) 0
4 weeks 18 1 (5.5) 0 0 21 2 (9.5) 0 0
Above1 month 37 4 (10) 1 (2.7) 1 (2.7) 16 2 (12.5) 0 0
BRV bovine rotavirus, BCV bovine coronavirus
Age Prevalence of BRV (%) Prevalence of BCV(%) Prevalence ofmix(%)
1 week 23 – –
2 weeks 32 5 2
3 weeks 30 9 –
4 weeks 8 – –
Above 1 month 11 2 1
Table 3 The prevalence of
BRV and BCV infections and
mixed infection in all of the 261
fecal samples
BRV bovine rotavirus, BCV
bovine coronavirus
Comp Clin Pathol

navirus (Fuente et al. 1998). In another study on 218 fecal
samples from diarrheic dairy calves, 1–30 days old, rotaviral
infection was detected in 46.2%, 45.6%, 33.8%, and 48.3%
of the calves in the respective age groups of 1–7, 8–14, 15–
21, and 22–30 days. There was no significant differences in
the detection rate of rotaviral infection among calves on the
different age groups (Garcia et al. 2000).The other study
reported the presence of BRV in 10% of fecal samples of
dairy calves younger than 3 months in Costa Rica (Hird et al.
1990). In a study in Switzerland, the prevalence of bovine
rotavirus and Coronavirus in infected calves with age of 1–
21 days, were 58.7% and 7.8%, respectively (Uhde et al.
2008). In Iran, the presence of Rotavirus, Coronavirus, and
other enteropathogens in feces of calves were studied in
farms in Tehran (Kargar et al. 1981; Keyvanfar et al. 2001),
Arak (Qaem Maghami et al. 2000), northwest of Iran (Khalili
et al. 2006), and Khouzestan (Mayameei et al. unpublished).
In the present study, by using antigen-capture ELISA test,
the prevalence of BRV in diarrheic and healthy fecal samples
was 26.98% and 17.03%, respectively, and the rate of
Coronavirus prevalence in diarrheic samples was 3.17% and
2.96% in healthy ones.
The determined rate of prevalence for Rotavirus in
diarrheic fecal samples in this study (26.98%) is rather
consistent with the results of Kargar Moakher (31.74%),
Qaem Maghami (34%), and Keyvanfar studies (28.8%); but
in studies by Hird (10%), Perez(7%), Gumosuva (41.7%),
Foente (42.7%), Garcia(43.65%), Uhde (58.7%), and
Mayameei (48%), there was significant difference in
determined rate of prevalence for Rotavirus in diarrheic
samples. The determined rate of prevalence for Coronavirus
in diarrheic feces in this study (3.17%), in some cases, has
considerable difference with studies by Gumosuva (1.96%),
Perez (9%), Foente (7.3%), Garcia (20.4%), Uhde (7.8%),
Qaem Maghami (34%), and Khalili (12.03%). The number
of samples and the range of age for the studied calves,
sensitivity of the employing detection method, season of
sampling, management and hygienic situation of the ranches,
nutritional situation of calves, rainfall and humidity rate, and
other factors were not identical in these studies, which all of
them can influence on the results. Specially, in cold seasons,
increase of pollution and presence of sensitive calves and
crowding would cause the prevalence rate of rotaviral and
coronaviral infection to increase. The range of age in which
sampling is performed and also the kind of husbandry (dairy
or beef) surely affect the prevalence. In this study, although
infection with Rotavirus and Coronavirus in diarrheic calves
was more than that of healthy ones, there was no significant
relationship between BRV and BCV infection with diarrhea
based on statistical analysis. So the role of other enter-
opathogens in the occurrence of calf diarrhea could be
considered in this study. It is also possible that some healthy
calves, which were recognized positive for Rotavirus and
Coronavirus in ELISA test, had been in the beginning of the
disease process without any clinical signs of diarrhea.
Although, according to the short incubation period of
diarrhea caused by BRV and BCV, this would be an unlikely
event. By considering the prevalence of BRV and BCV in
diarrheic calves in different age groups (Table 2), it is
concluded that the rate of BRV infection in third week is
higher than the other age groups and the lower rate of BRV
infection is related to age older than 1 month. This could be
attributed to the presence of colostrum antibodies and strong
local immunity, particularly in the first week and almost the
second week which decrease the occurrence of rotaviral
diarrhea in the first 2 weeks. After decreasing of local
passive immunity and the occurrence of natural rotaviral
infections in the third and fourth weeks of life and
subsequent activation of local host immunity, the rate of
viral infections drop in calves older than 1 month.
As a result, it is suggested that along with management
practices and promoting the immunity level in calves to
decrease the rate of infection by BRV and BCV, it is
necessary to analyze the fecal samples for the presence of
other enteropathogens such as E. coli and Cryptosporidium.
References
Acheson NH (2007) Fundamentals of molecular virology. Wiley, New
York, pp 202–211
Athanassious R, Marsolais G, Assaf R, Dea S, Descoteaux JP, Dulude
S, Montpetit C (1994) Detection of bovine coronavirus and type
A Rotavirus in neonatal calf diarrhea and winter dysentery of
cattle in Quebec: evaluation of three diagnostic methods. Can Vet
J 35:163–169
Brandao PE, Villarreal LYB, de Souza SLP, Richtzenhain LJ, Jerez JA
(2007) Mixed infections by bovine coronavirus, Rotavirus and
Cryptosporidium parvum in an outbreak of neonatal diarrhea in
beef cattle. Arq Inst Biol Sao Paulo 74:33–34
Brussow H, Nakagomi O, Gerna G, Eichhorn W (1992) Isolation of
an avian-like group A Rotavirus from a calf with diarrhea. J Clin
Microbiol 30:67–73
Castro AE, Heuschele WP (1992) Veterinary diagnostic virology.
Mosby-Year Book, St. Louis, pp 92–96
Crouch CF, Raybould TGF, Acres SD (1984) Monoclonal
antibody capture enzyme linked immunosorbent assay for
detection of bovine enteric coronavirus. J Clin Microbiol 19
(3):388–393
Estes MK, Kapikian AZ (2007) Rotaviruses. In: Knipe DM, Howley
PM (eds) Fields virology. Lippincott Wiliams and Wilkins,
Philadelphia, pp 1917–1974
Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM (2000)
Principles of virology, molecular biology, pathogenesis and
control. ASM Press, Washington, D.C., p 663
Fuente R, Garcia A, Ruiz-Santa-Quiteria JA, Luzon M, Cid D, Garcia
S, Orden J, Gomez-Bautista M (1998) Proportional morbidity
rates of enteropathogens among diarrheic dairy calves in Central
Spain. Prev Vet Med 36:145–152
Garaicoechea L, Bok K, Jones LR, Combessies G, Odeon A,
Fernandez F, Parreno V (2006) Molecular characterization of
bovine rotavirus circulating in beef and dairy herds in
Comp Clin Pathol

Argentina during a 10-year period (1994–2003). Vet Microbiol
118:1–11
Garcia A, Ruiz-Santa-Quiteria JA, Orden JA, Cid D, Sanz R,
Gomes-Bautista M, Fuente R (2000) Rotavirus and concurrent
infections with other enteropathogens in neonatal diarrheic dairy
calves in Spain. Comp Immunol Microbiol Infect Dis 23:175–
183
Gumusova SO, Yazici Z, Albayrak H, Meral Y (2007) Rotavirus and
Coronavirus prevalence in healthy calves and calves with
diarrhea. Med Wet 63(1):62–64
Hird D, Perez E, Caballero M, Rodriguez L, Velazquez J (1990)
Identification of selected disease agents from calves on Costa
Rican tropical cloud-forest dairy farms. Prev Vet Med 9:221–
231
Kargar R, Yoosefi Y, Shahrabadi MS, Khodashenas M, Heidarzade B
(1981) Diarrhea in calves: diagnosis and incidence around
Tehran. Archive de I'institut Razi 32:91–99
Keyvanfar H, Ghorbanpour M, Seifi Abad Shapouri MR (2001) A
survey on prevalence of rotaviral diarrhea in dairy calves in
Tehran region and determination of serotypes. J Vet Med
University of Tehran 56:3–11
Khalili M, Morshedi A, Keyvanfar H, Hemmatzadeh F (2006)
Detection of bovine coronavirus by RT-PCR in field study.
Veterinarski Arh 76(4):291–296
Lai MMC, Perlman S, Anderson LJ (2007) Coronaviridae. In: Knipe
DM, Howley PM (eds) Fields virology. Lippimcott Wiliams and
Wilkins, Philadelphia, pp 1307–1333
Maes RK, Grooms DL, Wise AG, Han C, Ciesicki V, Hanson L,
Vickers MJ, Kanitz C, Holland R (2003) Evaluation of a human
group A Rotavirus assay for on-site detection of bovine rotavirus.
J Clin Microbiol 41:290–294
Murphy, F.A., Gibbs, E. Paul J., Horzinek, M.C., Studdert, M.J.
(1999) Veterinary Virology 3rd edition. New York: Academic
Press. 402-404, 495-500
Parwani AV, Rosen BI, Flores J, McCrae MA, Gorziglia M, Saif LJ
(1992) Detection and differentiation of bovine group A Rotavirus
serotypes using polymerase chain reaction-generated probes to
the VP7 gene. Jour Vet Diag Med 4:148–158
Perez E, Kummeling A, Janssen M, Jimenez C, Alvarado R, Caballero
M, Donado P, Dwiner R (1998) Infectious agents associated with
diarrhea of calves in the canton of Tilaran, Costa Rica. Prev Vet
Med 33:195–205
Radostitis OM, Gay CC, Hinchcliff KW, Constable PD (2007)
Veterinary Medicine, A textbook of the diseases of cattle, horses,
sheep, pigs, and goats, 10th edn. Saunders-Elsevier, Philadelphia,
pp 1286–1296
Reidy N, Lennon G, Fanning S, Power E, O'Shea H (2006) Molecular
characterization and analysis of bovine rotavirus strains circulat-
ing in Ireland 2002–2004. Vet Microbiol 117:242–247
Thrusfield M (2005) Veterinary epidemiology, 3rd edn. Blackwell
Science, Oxford, pp 281–285
Uhde F, Kaufmann T, Sager H, Albini S, Zanoni R, Schelling E, Meylan
M (2008) Prevalence of four enteropathogens in the young
diarrheic dairy calves in Switzerland. Vet Rec 163(12):362–6
Comp Clin Pathol