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Journal of Clinical Microbiology, May 1998, p. 1266-1270, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Bovine Torovirus in Fecal Specimens of
Calves with Diarrhea from Ontario Farms
Lynn
Duckmanton,1,2
Susy
Carman,3
Éva
Nagy,4 and
Martin
Petric1,2,5,*
Department of Microbiology and Medical
Genetics1 and
Department of Pathobiology
and Laboratory Medicine,5 The University of
Toronto, and
Division of Microbiology, The Hospital for Sick
Children,2 Toronto, and
Animal Health
Laboratory3 and
Department of
Pathobiology,4 University of Guelph, Guelph,
Ontario, Canada
Received 2 October 1997/Returned for modification 15 January
1998/Accepted 18 February 1998
 |
ABSTRACT |
Breda virus (BRV), a member of the genus Torovirus, is
an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in
both Iowa and Ohio and in several areas of Europe. However, this virus
has been reported only once in Canada. Therefore, a study was performed
to determine the extent to which bovine torovirus is present in calves
with diarrhea from farms in southern Ontario. A total of 118 fecal
samples from symptomatic calves and 43 control specimens from
asymptomatic calves were examined by electron microscopy (EM) and
reverse transcription-PCR (RT-PCR) for the presence of torovirus.
Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%)
by RT-PCR with primers designed in the conserved 3' end of the
torovirus genome. By EM, torovirus particles were observed in 37 of the
118 specimens (31.4%). All but one of these samples were also positive
by RT-PCR. The incidence of torovirus in the asymptomatic control
specimens by RT-PCR was only 11.6%. To establish the identity of the
particles observed in the diarrheic specimens, five of the amplicons
from samples positive by both RT-PCR and EM were cloned and sequenced.
Nucleotide sequence analysis revealed that the bovine torovirus found
in southern Ontario manifests between 96 and 97% sequence identity to
the BRV type 1 strain found in Iowa. This study shows that bovine
torovirus is a common virus in the fecal specimens of calves with
diarrhea from farms in southern Ontario and thus may be an important
pathogen of cattle.
| |
INTRODUCTION |
The etiology of infectious diarrhea
in calves has been attributed to rotavirus, coronavirus, calicivirus,
parvovirus, and astrovirus (14-16, 22), agents of defined
morphology that can be readily detected by electron microscopy (EM).
Breda virus (BRV), a member of the genus Torovirus, was
first associated with enteritis of calves in 1982 (25). This
agent has been relatively infrequently reported because it is more
difficult to recognize by EM and because, unlike the torovirus
prototype, Berne virus (BEV), it cannot as yet be grown in cell
culture, which has precluded the development of routine immunospecific
diagnostic tests. However, the partial sequencing of the 3' end of the
BRV genome (11) has allowed for the application of reverse
transcription-PCR (RT-PCR) for the diagnosis of bovine torovirus
infections.
Prospective studies in The Netherlands, in which viruses were examined
in symptomatic and asymptomatic calves by enzyme-linked immunosorbent
assay (ELISA), demonstrated that torovirus was present in 6.4% of
calves with diarrhea compared with only 1.7% of asymptomatic controls.
In contrast, rotavirus was found in 37.4% of symptomatic animals and
13.9% of controls in this study (12). Epidemiologic studies
have shown that bovine torovirus is widespread in The Netherlands
(10), Germany and Switzerland (19), the United Kingdom (2), and the United States (17, 23, 26),
with approximately 90% of dairy cattle being seropositive. In one
study from Belgium, bovine toroviruses were found to play a role in respiratory, digestive, and reproductive disorders of cattle
(18). In a recent study from Saskatchewan, Canada, BRV-like
particles were detected in 42 of 221 fecal or intestinal specimens of
symptomatic calves (7).
The aim of the present study was to determine the incidence of
torovirus excretion in calves with diarrhea from farms in southern Ontario by RT-PCR and EM and to compare this with the excretion of
other enteric pathogens including bovine rotaviruses and coronaviruses.
| |
MATERIALS AND METHODS |
Specimens.
A total of 118 stool specimens from calves with
diarrhea and 43 specimens from asymptomatic calves were obtained from
the Animal Health Laboratory (AHL) in Guelph, Ontario, Canada. These specimens were submitted to the AHL by veterinarians in the region of
southern Ontario between April 1995 and March 1997. The majority of the
calves were between 2 and 60 days old at the time of specimen collection. Since testing for torovirus was performed retrospectively, the diarrheic specimens had all previously been tested at the AHL for
bovine coronavirus by an in-house ELISA system (1, 3), for
bovine type A rotavirus by latex agglutination (Microgen Bioproducts
Ltd., Camberley, United Kingdom), and for bovine viral diarrhea virus
(BVDV) by isolation in cell culture (4). Furthermore, all
specimens were coded, examined for the presence of viruses by
negative-contrast EM, and tested for bovine torovirus by RT-PCR in a
blinded fashion.
EM.
Fecal specimens were diluted with an equal volume of 1%
(wt/vol) ammonium acetate and clarified by centrifugation at 9,000 × g for 15 min at 4°C. The supernatant was transferred to
a new tube and centrifuged at 12,000 × g for 15 min at
4°C. Each sample was applied to a 400-mesh grid precoated with
polyvinyl formal and carbon. The grids were stained for 1 min with 2%
phosphotungstic acid (pH 7.0) and examined by negative-contrast EM with
a Philips EM 300 microscope, at a magnification of ×50,000
(13).
RNA extraction.
Fecal specimens were diluted in an equal
volume (wt/vol) of phosphate-buffered saline and clarified by
centrifugation at 9,000 × g for 15 min at 4°C. The
supernatant was transferred to a new tube and centrifuged at
12,000 × g for 15 min at 4°C. In a separate room,
with dedicated micropipetters and aerosol-resistant tips, viral RNA was
extracted from 100 µl of the partially purified supernatant with
TRIzol reagent (Gibco BRL, Burlington, Ontario, Canada) according to
the manufacturer's protocol. Each RNA pellet was resuspended in 10 µl of DNase-free, RNase-free double-distilled water
(ddH2O) (5 prime-3 prime Inc., Boulder, Colo.) and stored at 
80°C.
RT-PCR.
All specimens were tested for the presence of
torovirus RNA by RT-PCR. For each run of specimens assayed, a negative
control (ddH2O) and a control RNA extracted from a stool
specimen shown to contain only bovine rotavirus by EM were included.
RNA extracted from a BRV type 1 (BRV-1) (Iowa strain)-positive stool
specimen (obtained from G. Woode, Texas A&M University) was tested as a positive control in every third RT-PCR assay due to the limited amount
of control stool available. Oligonucleotide primers (General Synthesis
and Diagnostics, Toronto, Ontario, Canada) were designed from the 3'
end of the BEV genome (DDBJ accession no. D00563). The sense primer
(5'TAATGGCACTGAAGACTC3') and the antisense primer (5'ACATAACATCTTACATGG3') bracketed a genome fragment of 219 bases, which included the 3' end of the N protein coding region and
most of the 3' noncoding region upstream of the poly(A) tail.
The RT reaction and PCR mixtures were set up in an isolated room, with
dedicated micropipetters and aerosol-resistant tips. Reactions were
then performed in another room designated for PCR amplification. For
the RT reaction, a 10-µl RNA aliquot was incubated for 5 min at
65°C and added to 10 µl of the RT mixture containing 5 mM
MgCl2; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.25 mM (each) dATP, dCTP, dGTP, and dTTP (Promega, Madison, Wis.); 2.6 µM random hexamer primers; 20 U of RNase Guard (Gibco BRL); and 50 U of Moloney
murine leukemia virus reverse transcriptase (Gibco BRL). The reaction
mixture was overlaid with sterile mineral oil and incubated at room
temperature for 10 min. The RT reaction was performed in a Perkin-Elmer
(Mississauga, Ontario, Canada) 480 thermal cycler at 42°C for 30 min
and 99°C for 5 min, and then the reaction mixture was held at 5°C
for 5 min.
For the PCR, 10 µl of the RT reaction was added to the PCR mixture
containing 1 mM MgCl2, 8 mM Tris-HCl (pH 8.3), 40 mM KCl, 2.5 U of Amplitaq DNA polymerase (Perkin-Elmer Cetus and Applied Biosystems Inc.), and 50 pmol of each primer. The total volume of the
PCR was 50 µl. The reaction mixture was overlaid with sterile mineral
oil and amplified in a Perkin-Elmer 480 thermal cycler with an initial
denaturation at 94°C for 2 min, followed by 35 cycles consisting of
denaturation at 95°C for 40 s, annealing at 50°C for 1 min,
and extension at 72°C for 1 min 30 s. Reaction mixtures were
then incubated at 72°C for 10 min and held at 4°C. Products were
analyzed by electrophoresis through a 1.2% agarose gel containing
ethidium bromide and viewed under a UV transilluminator.
Cloning and DNA sequencing.
Selected PCR products were
purified by the Wizard PCR Preps DNA purification system (Promega),
cloned into a pCR-Script Amp SK+ cloning vector, and
transformed into Epicurian coli XL1-blue MRF' Kan supercompetent cells
(pCR-Script Amp SK+ cloning kit; Stratagene, La Jolla,
Calif.) as per the manufacturer's recommendations. Clones were
screened by PCR with the same primers as described above. Plasmids
containing inserts were purified from broth cultures with the Wizard
Miniprep DNA purification system (Promega) and sequenced with the
fmol DNA sequencing system (Promega) according to the
manufacturer's recommendations. Sequence data were analyzed with the
computer program GCG, version 8 (Genetics Computer Group, Inc.,
Madison, Wis.). As an additional precaution against plasmid
contamination of PCRs, all cloning and sequencing assays were
undertaken only after the RT-PCR experiments on stool samples had been
completed.
Statistical analysis.
The chi-square test was used to
establish statistical significance.
| |
RESULTS |
EM.
Of the 118 diarrheic specimens that were examined, 37 (31.4%) were found to be positive for bovine torovirus by
negative-contrast EM. In contrast, torovirus particles were detected in
only 2 of the 43 asymptomatic control specimens examined (Table
1).
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TABLE 1.
EM and RT-PCR results for the detection of torovirus in
stool specimens from diarrheic and asymptomatic calves in
southern Ontarioa
|
|
The torovirus particles detected by EM were morphologically similar to
the BRV previously reported in the stool specimens
of diarrheic calves
in Iowa and Ohio (
26). These particles measured
between 100 and 120 nm at their largest diameter, and they exhibited
torus-,
crescent-, and rod-shaped conformations. The particles
were clearly
enveloped and bore a fringe of peplomers, each measuring
approximately
10 nm in length (Fig.
1).
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FIG. 1.
Electron micrograph of a calf fecal specimen showing
torus- and crescent-shaped bovine torovirus particles. Bar, 100 nm.
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|
RT-PCR.
By RT-PCR analysis, a DNA fragment of 219 bases was
detected in 43 of the 118 (36.4%) specimens from symptomatic calves.
Of these 43 samples, 36 were shown to contain torovirus particles by
EM. Only one diarrheic specimen that was negative by RT-PCR was found
to contain torovirus by EM (Table 1). Repeat testing was performed on
all discordant samples to affirm the initial RT-PCR results.
Among the 43 asymptomatic control samples tested, only 5 (11.6%) were
positive for torovirus by RT-PCR, and 2 of these were
also positive by
EM. All 38 specimens that were negative by RT-PCR
were also negative by
EM (Table
1). Therefore, there was a significantly
greater number of
torovirus-positive specimens in the symptomatic
calf population than in
the asymptomatic calves (
P = 0.0023).
None of the
negative control and rotavirus control samples gave
a positive result
by RT-PCR. Figure
2 shows representative
amplification
products after agarose gel electrophoresis.
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FIG. 2.
Gel electrophoresis of RT-PCR products from five
torovirus-positive fecal specimens. Lanes marked and R
represent a negative control (ddH2O) and a bovine rotavirus
sample, respectively. A 100-bp ladder was used as the molecular size
marker.
|
|
Other viruses.
Of the 118 specimens from diarrheic calves, 29 were positive for viruses other than bovine torovirus, including
coronavirus, rotavirus, BVDV, and small round-structured viruses
(SRSVs) as seen by EM. Of the 43 specimens that were positive for
torovirus by RT-PCR, 10 were also positive for another virus. Of these, five had torovirus and rotavirus, two were positive for torovirus and
coronavirus, one had torovirus and BVDV, and two were found to contain
a mixed infection of torovirus, rotavirus, and coronavirus (Table
2).
In addition, 19 of the 75 diarrheic specimens that were negative for
torovirus by RT-PCR were positive for other viruses.
Of these, three
had rotavirus, five had coronavirus, three were
positive for BVDV, one
contained SRSV, and seven had mixed infections
of either rotavirus and
coronavirus or rotavirus and BVDV (Table
2). There was no significant
difference between torovirus-positive
diarrheic specimens and
torovirus-negative diarrheic specimens
for the presence of other
viruses in the stools (
P = 0.8).
Only three of the asymptomatic control samples that were negative for
torovirus by both EM and RT-PCR contained other viruses,
including two
specimens with rotavirus and one specimen with coronavirus
as seen by
EM.
Cloning and sequencing of torovirus-positive RT-PCR products.
To confirm that the 219-base product obtained by RT-PCR was indeed
bovine torovirus and to determine the degree of heterogeneity among
samples, amplicons obtained from five different specimens were cloned
and sequenced. Clones were screened by PCR with the same primers, and
at least two clones per sample were found to contain the 219-base
fragment. These clones were isolated and sequenced, and their
nucleotide sequences were compared to the 3' regions of the BRV-1 and
BEV genomes (Fig. 3). The nucleotide sequences of each of the five torovirus-positive isolates were found to
be between 96 and 97% identical to BRV-1 and between 95 and 96%
identical to BEV in this area. The nucleotide substitutions were
interspersed throughout the 219-base fragment. None of these substitutions caused changes in the predicted amino acid sequence of
the BRV-1 nucleocapsid (N) protein, and substitutions at nucleotide positions 23 and 25 caused only one amino acid change, from glutamine to lysine, in the predicted amino acid sequence of the BEV N protein.
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FIG. 3.
Alignment of the nucleotide sequence from the 3' end of
the BRV-1 (Iowa strain) genome with those of five bovine
torovirus-positive samples from diarrheic calves in southern Ontario (a
to e) and the BEV genome. Nucleotides identical to the consensus
sequence are shown as dots.
|
|
| |
DISCUSSION |
Although bovine torovirus has been established as a widespread
agent of diarrhea in calves, its prevalence has been investigated to
only a limited extent, largely because it cannot be grown in cell
culture. This inability to isolate the virus has precluded the
large-scale preparation of reference antisera and antigens for the
development of commercial immunospecific diagnostic tests such as ELISA
and latex agglutination, which are currently used to diagnose
coronavirus and type A rotavirus infections (3, 8, 9).
Consequently, the etiology of a substantial proportion of viral
diarrhea in calves due to BRV may not be diagnosed, which impacts on
the establishment of containment measures and the potential incentive
towards the development of vaccines.
The RT-PCR was successfully applied to the diagnosis of torovirus in
fecal specimens of calves in this study. While molecular approaches
such as hybridization with cDNA probes have been previously reported
(11), our findings show that RT-PCR is a useful method for
routine diagnosis of torovirus. The assay compared favorably with
direct EM and was more sensitive in that it detected tonovirus in 6 additional specimens beyond the 37 detected by EM alone. It is unlikely
that these specimens represent false positives because of the
exhaustive precautions taken to control for contamination of the RT-PCR
assay. Only one specimen that was positive by EM was negative by
RT-PCR. This may have been due to nonspecific inhibitors present in the
stool, as has been shown previously for other gastroenteritis viruses
(20). Due to the limited amounts of BRV-1 control stool
available, the presence of inhibitors could not be verified by spiking
an aliquot of the discordant sample with a known amount of BRV-1
control specimen and repeating the RT-PCR assay.
In this study, bovine torovirus was present significantly more
frequently (36.4%) in calves with diarrhea than in the asymptomatic controls (11.6%; P = 0.0023). Thus, bovine torovirus
is associated with the symptoms of enteritis in these symptomatic
cases. The excretion of torovirus, as well as other viruses including
rotavirus and coronavirus, in asymptomatic animals has been previously
reported (6), and our findings are consistent with these
observations.
In a number of cases, other viruses were also present along with
torovirus. That is, 10 of 43 diarrheic specimens that were positive for
bovine torovirus by RT-PCR were also shown to contain other viruses.
Nevertheless, the incidence of torovirus alone in the diarrheic
specimens (27.9%) was still significantly greater than the presence of
bovine torovirus in asymptomatic calves (11.6%; P = 0.016). However, it remains impossible to determine whether bovine
torovirus was the primary cause of diarrhea in the symptomatic calves
who had concomitant infections with other viral agents of enteritis.
This phenomenon of mixed infection was also observed among the
diarrheic specimens that were negative by EM and RT-PCR which contained
other viruses and has previously been reported in other studies of
animals and humans (14, 15, 21, 24).
This study has added to our understanding of the epidemiology of
torovirus enteritis by demonstrating that, in keeping with previous
observations from North America (7, 17, 23, 26) and Europe
(2, 10, 18, 19), it is also prevalent in the southern
Ontario cattle population. Moreover, bovine torovirus was shown to be a
common pathogen in the stools of symptomatic calves, exceeding the
prevalence of bovine rotavirus and bovine coronavirus in this study.
This was also the case in the Saskatchewan study, in which 19% of
symptomatic specimens were found to contain BRV-like particles, whereas
only 9.5% were positive for rotavirus as detected by EM
(7).
Sequence analysis of a subset of the torovirus-positive RT-PCR
amplicons confirmed that the particles detected in the fecal specimens
of calves in southern Ontario are related to the torovirus prototype,
BEV, and to BRV-1, since their sequences demonstrated between 95 and
97% sequence identity in the 3' noncoding region of the genomes of
these viruses (11). There was also a small yet defined
amount of genomic heterogeneity among the bovine torovirus clones
despite the conserved nature of the 3' end of the torovirus genome.
This could be interpreted as evidence that genetic variation may exist
among toroviruses isolated from different outbreaks. A more complete
analysis of the sequences of specific viral genes, especially the
peplomer and hemagglutinin-esterase genes, the latter of which has
recently been described for BRV (5), is needed to address
this hypothesis.
| |
ACKNOWLEDGMENT |
This research was supported by a grant from the Medical Research
Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, The Hospital for Sick Children, 555 University Ave.,
Toronto, Ontario, Canada M5G 1X8. Phone: (416) 813-6111. Fax: (416)
813-5993. E-mail: martin.petric{at}mailhub.sickkids.on.ca.
| |
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Journal of Clinical Microbiology, May 1998, p. 1266-1270, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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[Abstract]
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Abstract
Introduction
