ELISA and RT-PCR Based Detection of Bovine Coronavirus in Northern India
M. Yaqoob Wani
Bovine coronavirus (BCoV) is widespread in cattle population, resulting in
heavy economic losses to both dairy and beef industry throughout the world.
The syndromes associated with BCoV include winter dysentery in adult dairy cattle
and respiratory and intestinal tract infections in young calves. The virus has
specific tropism for intestinal and pulmonary epithelial cells. Reports regarding
prevalence and molecular detection of BCoV from India are scarce. In this study,
101 fecal samples were collected from clinical cases of diarrheic calves from
North Indian region covering three dairy farms of Uttar Pradesh and dead calves
of post mortem facility of Indian Veterinary Research Institute. Fecal samples
from all the cases were screened for the presence of BCoV by commercially available
ELISA kit. Furthermore, all samples were subjected to RT-PCR for detection and
confirmation BCoV. RT-PCR was carried out using two different sets of primers
to amplify the conserved nucleocapsid (N) gene of the virus targeting a 407
and 730 bp fragments. An incidence rate of more than 14% (15/101) was observed
with ELISA and about 20% (20/101) by RT-PCR. The present report is first in
its nature regarding the detection of BCoV at molecular level in India. In conclusion,
RT-PCR was found more sensitive than commercial ELISA kit for detecting BCoV
in fecal samples. Further extensive epidemiological studies are suggested for
the virus in the country to know the magnitude of BCoV infection in dairy calves
along with isolation of viral strains and to investigate their antigenic and
Received: May 05, 2012;
Accepted: July 17, 2012;
Published: September 15, 2012
Diarrhea remains an important cause of illness and death in young calves and
the economic losses associated with the disease include decreased performance,
high morbidity and mortality and the expenses of medication and labor to treat
the sick animals. It has been recognized as one of the main six causes of all
deaths from infectious diseases, thus is of much concern regarding animal health
and for dairy industry (Kapikian, 1996; Murray
and Lopez, 1997; Dhama et al., 2009). The
major infectious agents associated calf diarrhea include Rotavirus, Coronavirus,
Cryptosporidium parvum, enterotoxigenic Escherichia coli and Salmonella
spp., which are collectively responsible for 75-95% of infection in neonatal
calves worldwide (Moon et al., 1978; Snodgrass
et al., 1986; Bendali et al., 1999;
Hoet et al., 2003; Gumusova
et al., 2007; Dhama et al., 2009;
Malik et al., 2012). Among all these pathogenic
agents Rotaviruses are the leading cause and Coronaviruses are a major contributor
of calf diarrhea (Gumusova et al., 2007; Uhde
et al., 2008; Dhama et al., 2009).
The infections associated with bovine coronaviruses (BCoV) are more severe because
they affect both the small and large intestines (Gunn et
al., 2009; Boileau and Kapil, 2010). The other
syndromes associated with BCoV include Winter Dysentery (WD) in adult dairy
cattle and respiratory tract infections in calves and feedlot cattle (Saif
et al., 1991; Storz et al., 2000;
Bovine coronavirus, first reported by Mebus et al.
(1972), is a single-stranded, non-segmented, RNA virus of 32 kb genome and
belongs to group 2 corona viruses of the family Coronaviridae, order Nidovirales
(De Vries et al., 1997; Van
Regenmortel et al., 2000). The virus affects both gastrointestinal
and respiratory tracts (Clark, 1993). Generally it affects
calves ranging in age from day 1 to 3 months and diarrhea typically occurs between
2-8 weeks of age (Mebus et al., 1973; Langpap
et al., 1979; Mostel and Burki, 1987; Boileau
and Kapil, 2010). The primary sites of BCoV replication are epithelial cells
in the respiratory tract, enterocytes in the distal ileum and colon, thus virus
being excreted with the respective excretions. In early years, the association
of BCoV with pneumoenteritis had been investigated by many works indicating
its importance (Thomas et al., 1982; Reynolds
et al., 1985, 1986; Saif
et al., 1986; Heckert et al., 1989,
1991). Recent reports indicate that it is second only
to bovine herpesvirus affecting the respiratory system and the serologic incidence
suggests that most cattle become exposed to BCoV during their lifetime (Fukai
et al., 1998; Boileau and Kapil, 2010). Bovine
Coronavirus has worldwide distribution and being reported from several countries
(Jeong et al., 2005; Khalili
and Morshedi, 2006; Takiuchi et al., 2006;
Traven et al., 2006; Gumusova
et al., 2007; Park et al., 2007; Boileau
and Kapil, 2010; Dash et al., 2012). However,
epidemiological data on BCoV in India is scarce. Therefore, the aim of the present
study was to screen the fecal samples for BCoV collected from clinical cases
and dead calves with the history of diarrhea from North Indian region covering
three dairy farms of Uttar Pradesh and post mortem facility of Indian Veterinary
Research Institute, respectively. ELISA being a sensitive method for antigen
detection was used for determining BCoV incidence. Furthermore, virus confirmation
was done at genomic level by the molecular method of RT-PCR and its sensitivity
was compared with that of ELISA for detecting virus in clinical samples.
MATERIALS AND METHODS
Collection and processing of faecal samples: During the period from September 2009 to March 2010, faecal samples were collected from 101 calves of below 3 months of age, reported to be clinical cases with the history of diarrhea, from two dairy farms of Bareilly district (Military Dairy Farm, 38; and LPM farm, IVRI, 30), one dairy farm of Raebareli district (22) of Uttar Pradesh State and from 11 spontaneous dead calf cases from the post-mortem (PM) facility, Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly (U.P.).
All the fecal samples were obtained in sterile collection vials and kept at
-20°C till use. Fecal suspensions (v/v; 50%, watery feces; 20%, loose feces;
or 10%, normal feces) were prepared with ultra-pure water treated with 0.1%
diethyl-pyrocarbonate (DEPC water).
|| Oligonucleotide primers used in the present study for amplification
of N gene of BCoV
Samples were centrifuged at 14000 x g for 10 min at 4°C and supernatants
were collected and used for screening of BCoV by ELISA and RT-PCR assays. Generally,
the processing of fecal samples was done within 24 h of collection of samples.
Screening of fecal samples: All the 101 fecal samples were subjected to ELISA for detecting the presence of BCoV employing commercially available ELISA kit (Bio-X Diagnostics, Belgium kit-BIO K 151) and following the protocol as per the manufacturers instructions. The results were interpreted by using the OD values obtained at 450 nm using ELISA Reader (BioTec, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR primers: Two distinct sets of established primers were used for confirmation
of BCoV (Tsunemitsu et al., 1999; Cho
et al., 2001) (Table 1). The RT-PCR was performed
on all the 101 faecal samples from broth live and dead diarrheic calves. Both
the sets of primers were used to amplify the N gene which is the conserved region
in the genome of Mebus strain virus.
RNA extraction: Viral RNA was extracted from the clinical samples with TRIzol LS reagent (Invitrogen, USA) as per the manufacturers instructions.
RT-PCR: cDNA was prepared from the extracted viral RNA by reverse transcription
reaction and used for PCR amplification of the N gene generating expected amplicons
of 407 and 730 bp sizes, as per protocol described previously by Tsunemitsu
et al. (1999) and Cho et al. (2001),
respectively. The amplified products were visualized on 1.2% agarose gel (Amresco,
USA) stained with ethidium bromide (0.5 μg mL-1).
Screening of faecal samples by ELISA: The screening of fecal samples, collected from diarrheic calves below three months of age from Northern state of India-Uttar Pradesh, by commercially available ELISA kit indicated high prevalence of BCoV. Out of the total of 101 fecal samples including 90 diarrhoeic samples from clinical cases and 11 intestinal contents from dead calves, 15 (14.85%) samples were found positive for BCoV (Table 2). The BCoV incidence rate in clinical cases in Military dairy farm and LPM farm, IVRI, both in Bareilly district was found to be 15.79% (6/38) and 10% (3/30), respectively. The overall prevalence of BCoV in Bareilly district was found to be 13.23% (9/68) and 27.27% (3/11) in clinical cases and dead cases, respectively. In dairy farm of Raebareli district, 13.64% (3/22) virus incidence rate was recorded (Table 2).
Detection of BCoV by RT-PCR: Standardized RT-PCR was applied for detection and confirmation of BCoV in all the 101 fecal samples collected from clinical cases of diarrheic and dead calves (Fig. 1, 2).
||RT-PCR detection of BCoV, 407 bp amplified product of N gene
using established primer, Lane M: DNA ladder of 100 bp, Lane 1-7: RT-PCR
amplified products (407 bp) of BCoV N gene in faecal samples
||RT-PCR detection of BCoV, 407 bp and 730 bp amplified product
of N gene using established primer, Lane M: DNA ladder of 100 bp, Lane 2:
Control, Lane 1 and 7: RT-PCR amplified products (407 bp) and 730 bp of
BCoV N gene in diarrhea faecal samples, Lane 3 to 6: RT-PCR amplified products
(407 bp) of BCoV N gene in post mortem faecal samples
Out of total 101 samples screened by RT-PCR, 20 (19.80%) samples were found positive for BCoV. The virus incidence rate in clinical cases at military dairy farm and LPM farm, IVRI, both in Bareilly district was found to be 21.05% (8/38) and 16.67% (5/30), respectively, with an overall prevalence of 19.11% (13/68). In dead cases from Post Mortem Facility, IVRI, Bareilly, the incidence rate of 27.27% (3/11) was observed. At dairy farm of Raebareli district, an incidence rate of 18.18% (4/22) was recorded by RT-PCR (Table 2).
|| Comparison between ELISA and RT-PCR for the detection of
BCoV infections in the faecal samples
Neonatal calf mortality is one of the most common animal health concerns for
dairy industry. The increased mortality in young calves during early days of
life, adversely affects the economic stability of most of the livestock farming
ventures. It has been estimated that neonatal diseases resulting in calf mortality
reduce farm net profit by 38% (Martin and Wiggins, 1973;
Khan and Khan, 1991). Among the various neonatal diseases
of calves, diarrhea is of major health concern causing high economical losses.
Diarrhea affects young calves at an age when they have immature immune status,
lacks specific antibody, illustrate high metabolism with added stresses imposed
by weaning and sometimes deprivation of immune colostrum feeding. Among the
various etiological agents of calf diarrhea, viral pathogens viz., rotavirus
and coronavirus are the most important ones (Jeong et
al., 2005; Gumusova et al., 2007; Uhde
et al., 2008; Dhama et al., 2009;
Boileau and Kapil, 2010; Malik et
al., 2012). However, the pathology of BCoV associated diarrhea is often
more severe compared to rotavirus and others and it often results in mucohemorrhagic
enterocolitis (Gunn et al., 2009; Boileau
and Kapil, 2010). The reports regarding Coronavirus prevalence in dairy
calves of India are limited; therefore, the present study was carried out to
investigate the incidences of BCoV in dairy farms of Northern Indian state employing
ELISA kit and RT-PCR detection of the virus. In the present study, screening
of the fecal samples was carried out both on live and dead animals during the
winter months because BCoV is more stable during colder climates due to its
enveloped nature (Evermann and Benfield, 2001) and is
reported to cause winter dysentery in adult cattle during winter season (Cho
et al., 2000; Boileau and Kapil, 2010).
In the present study, a total of 101 clinical samples comprising of 90 fecal
samples from calves with clinical presentation of diarrhea and intestinal contents
of 11 dead cases were screened for BCoV antigen by commercially available ELISA
kit. The enzyme linked immunosorbent assay, ELISA was used owing to the facts
that most of the other immunodiagnostic tests developed for detection of enteric
pathogen have either low sensitivity and/or specificity. ELISA also has ability
to detect enteric pathogens even at low concentrations (Selim
et al., 1991; Kelkar et al., 2004).
The antigen capture ELISA type is regarded as diagnostic test of choice for
BCoV detection (Boileau and Kapil, 2010). The prevalence
rate, as tested by commercially available ELISA kit, at Raebareli and Bareilly
districts of Uttar Pradesh state was recorded for bovine coronavirus as 13.64
and 13.23%, respectively, in clinical cases and 27.27% in dead cases from Bareilly
region. The overall prevalence of BCoV in both Raebareli and Bareilly district
as detected by ELISA was 14.85%, which is similar with earlier reported virus
prevalence of 14% (Reynolds et al., 1986), 20%
(Bordas et al., 1985) and 20% (Vanamayya,
1990). However, Dash et al. (2012) recently
reported a comparatively lower BCoV prevalence rate of 4.76% in Mathura and
its adjacent regions of Uttar Pradesh. In our study, a higher incidence of 27.27%
was recorded in dead cases from post mortem facility indicating that BCoV plays
an important role in causing the calf diarrhea and the associated mortality
as suggested by several workers (Rai and Singh, 1983,
1986; Khan and Khan, 1991; Boileau
and Kapil, 2010). The pathological evaluation of these dead calves also
confirmed that BCoV affects multiple organ systems (Hansa,
In the present investigation, molecular tool of RT-PCR was also employed further
for rapid detection and confirmation of BCoV by targeting N gene. RT-PCR was
standardized selecting viral N gene based primers as it is conserved among BCoV
strains. The expected specific viral amplicons with sizes of 407 and 730 bp
were obtained confirming virus detection in clinical samples and were in agreement
with earlier reports (Tsunemitsu et al., 1999;
Cho et al., 2001; Khalili
and Morshedi, 2006). Out of total 101 samples, 20 (19.80%) were found positive
for BCoV as compared to 15 (14.85%) samples found positive by ELISA. It is of
significant importance to mention that all the ELISA tested fecal samples were
found positive by RT-PCR also. It is expected that RT-PCR assays can be used
for sensitive detection of BCoV in clinical samples as RT-PCR may detect BCoV-positive
animals that might otherwise be classified as BCoV-negative by ELISA or other
methods. The results support that RT-PCR is highly applicable in detecting BCoV
in clinically diarrheic calves and is more sensitive than ELISA for detecting
BCoV in fecal samples. A rapid, sensitive, confirmatory and timely detection
of BCoV infection by RT-PCR in calves, detecting even subclinical cases or within
their early or late course of illness or after re-infection when low level of
virus is being shed from intestines into the feces, is an essential approach
concerning epidemiological surveillance as it allows for the application of
suitable preventative measures prior to the emergence of diarrhea on a farm
(Cho et al., 2001; Boileau
and Kapil, 2010). To the best of our knowledge this report is first in its
nature regarding the molecular detection and screening of fecal samples for
BCoV by RT-PCR in India. However, RT-PCR needs to be applied at a large scale
for studying the BCoV detection both in diarrheic calves (clinical cases) as
well as in non-diarrheic calves (subclinical infections) so as to know the magnitude
of BCoV infection and its molecular epidemiology, thereby adding more information
to the available data on this virus.
It is to be kept in mind that BCoV genome being the longest (approximately
32,000 RNA bases) among animal viruses, the virus has chance of further evolution.
It is still unclear whether isolates/strains of BCoV having different tropism
for various organs like affecting the respiratory and enteric system can be
distinguished antigenically by ELISA (Boileau and Kapil,
2010). Nowadays, the molecular tools and techniques are being widely used
in animal disease diagnosis including PCR and allied techniques viz., PCR-RE/RFLP,
RT-PCR, Q-PCR, RRT-PCR, real-time PCR, LAMP, nucleotide sequencing and phylogenetic
analysis. These have strengthened detection of animal pathogens in terms of
reliability and rapidity and also for characterizing and monitoring various
pathogens, which are altogether very helpful for an effective disease control
programme. For BCoV detection and characterization, several workers have recently
developed and applied molecular diagnostics viz., RT-PCR, nested PCR, multiplex
semi-nested RT-PCR, TaqMan-based real-time RT-PCR, SYBR Green real time
RT-PCR (Cho et al., 2001, 2010;
Escutenaire et al., 2007; Park
et al., 2007; Decaro et al., 2008;
Klein et al., 2009; Asano
et al., 2010). Techniques like multiplex real-time PCR assay have
the advantage of simultaneous detection and quantification of major pathogens
causing calf diarrhea.
BCoV is prevalent and likely endemic in northern India and has high correlation with neonatal calf diarrhea and associated mortality. For screening of large number of fecal samples ELISA was found very convenient; however, RT-PCR was found to be more sensitive than ELISA for BCoV detection. It is suggested that more investigations should to be carried out for BCoV detection in Indian dairy herds covering different geographical regions and with large number of samples, which would highlight the epidemiological significance of this important virus and would be helpful for devising appropriate and timely prevention and control measures, thereby reducing economical losses. For this purpose, the use of molecular based diagnostic methods needs to be exploited to their full potential for continuous virus monitoring and surveillance. Further studies are also needed to isolate BCoV strains in India and to characterize them at genomic levels investigating their antigenic and genetic properties.
Authors are thankful to National Agricultural Innovation Project (NAIP) Project, ICAR, New Delhi for financial support and Director, IVRI for providing necessary research facilities to carry out the present research work.
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