Microbiol. Immunol. Vol. 21 (9), 531-534, 1977
Hemagglutination
with
Nebraska
Calf
Diarrhea
Virus
Y. INABA, K. SATO, E. TAKAHASHI,H. KUROGI, K. SATODA,T. OMORI, and M. MATUMOTO NationalInstituteof AnimalHealth, Tokyo,and KitasatoInstitute,Tokyo (Received for publication, April 19, 1977)
Nebraska calf diarrhea (NCD) virus or reovirus-like agent of calf diarrhea has been shown to be a primary etiological agent of neonatal calf diarrhea by Mebus and his associates (1, 4, 5). Recently Spence et al (6) prepared hemagglutinin from African green-monkey kidney cells infected with NCD virus. This antigen agglutinated human group O cells. We also examined hemagglutination (HA) with NCD virus grown in calf kidney (BK) cells and found that the virus agglutinated erythrocytes of human and some other animals. This paper describes our observations on the HA with this virus and its inhibition (HI) by specific antiserum. Lincoln strain of NCD virus (4) maintained by serial passage in BK cells, was supplied by Dr. C.A. Mebus, University of Nebraska, Lincoln, Nebraska, and used in the present study after several passages in BK cells in our laboratory. BK cell cultures were prepared as described previously (2). The growth medium used was LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate) supplemented with 10% calf serum. Primary BK cell monolayers prepared in 500-ml bottles were inoculated with 0.1 TCID50 per cell of virus and incubated at 37 C for 2 days with LE medium containing 0.1 % yeast extract. As described later, in some preliminary experiments, culture fluid harvested from infected cells was used as HA antigen after centrifugation to remove coarse debris. In subsequent experiments, however, HA antigens concentrated from infected culture fluid were used. HA and HI tests were carried out by the microtiter method. The diluent used was VBS (veronal-buffered saline, pH 7.0, containing 0.1 % bovine serum albumin and 0.001% gelatin), unless otherwise stated. Blood was obtained in Alsever's solution and stored at 4 C. Erythrocytes from horse, cattle, sheep and goat were used as 0.3% suspensions, and those from other species as 0.5% suspensions. In HA tests, serial 2-fold dilutions of HA antigen were prepared in 0.05-ml amounts, and mixed with 0.025 ml of an erythrocyte suspension. The mixtures were incubated at 37 C for 1 hr before the results were read, unless otherwise stated. The HA titer was expressed as the reciprocal of the highest antigen dilution showing complete HA. For HI tests, 0.2 ml of the serum was inactivated by heating at 56 C for 30 min, diluted 5-fold, mixed with 0.2 ml of packed erythrocytes and incubated at 37 C for 1 hr. After removal of the cells by centrifugation, the supernatant fluid was mixed with 0.5 ml of 25% kaolin, incubated at room temperature for 1 hr, and centrifuged to remove kaolin. The resulting supernatant fluid was taken as the 10-fold dilution 531
Y.
532 of
the
serum.
dilutions and
Eight
of
the
mixed
with
incubated as
at
the
found
to
be
also
done 1 hr
fuged
at
original 0.001
followed g
M Tris,
erythrocytes
30
pH
to were
with
8%
buffer,
and
the
saline
at
4
M NaCl,
prepared
were
low.
To
cellular
confirm In-
in
pH
Concentration
The
cells were
debris,
suspended
pellets
(0.15
BK
animals
ultracentrifugation.
7.0). glycol
C.
then
expressed
from other
remove
polyethylene
resulting
was
1 hr,
HI.
were
by
2-fold for
were
titer
some
obtained
pellets
incubation
min,
antigens at
human,
Table
Table
the
HI
harvested
and
serial
at
room
material were
0.001
1/50
original was
temperature
was
then
suspended M CaCl2,
the
was
in 0.001
centri1/50
the
M MgCl2,
7.0).
of species of the
and
of
mixtures
complete
antigens
phosphate
fluid
of Tris-buffered
concentrated
a variety
2 hr,
The
fluid
titers
centrifugation
overnight
for
HA
ml
temperature
The
read.
human
low-speed
g for
0.025
room
showing
from the
and at
supernatant
HA
culture by
8,000 •~
were
dilution
with
M NaCl, M/150
stirring
volume
The from
by
serum
ml
suspension.
concentrated after
(0.15
0.025
incubated
results
although
100,000 •~
PBS
the
erythrocytes
prepared
in
mixed, erythrocyte
and
virus,
fluid,
at of
an
highest
agglutinated,
culture
volume
of
ET AL
antigen
experiments
we
centrifuged
HA were
1 hr,
the
NCD
finding,
fectious
for
of
with
ml
C for
preliminary
infected
this
37
of
serum
0.025
reciprocal In
units
treated
INABA
1.
2.
4
thus C,
cattle,
HA
HI
titers
and
NT
room
temperature
horse,
sheep,
at 37 C with
titers
tested and
goat,
erythrocytes
of antisera from with NCD virus
37 swine,
from
rabbits
for C.
HA HA
guinea
various
with
erythrocytes
was
observed
pig,
goose,
species
hyperimmunized
with chicken
NOTES
533
Fig. 1. Production of serum HI and NT antibodies in a calf infected orally with 400 ml of an NCD virus suspension (104.5 TCID50/ml). HI titers were determined with human and bovine erythrocytes which gave the same results.
Fig. 2.
HI and NT antibody
titers of individual
adult cattle.
and pigeon, whereas no HA was observed with rabbit, hamster, rat or mouse erythrocytes. The different temperatures employed for the tests showed little difference in HA titer with any of these HA-positive cell types except horse, guinea pig and chicken cells which gave much lower or no HA titer at 4 C. These experiments were carried out using VBS as the diluent. However, when PBS or 0.15 M NaCl solution was used, no HA was observed at any of these temperatures with any of the cells. On the basis of these results VBS was used as the diluent and tests were incubated at 37 C
534
Y. INABA
ET AL
in the standard method described earlier. HA titers obtained by this method varied considerably among the species as well as among different samples of a species (Table 1). The HA of NCD virus was inhibited by antisera to NCD virus (Table 2). The antisera used were prepared in rabbits, which were immunized by 4 intramuscular doses of partially purified virus mixed with complete Freund adjuvant. Sera were obtained 2 weeks after the last dose. HI titers were determined with both human (O) and bovine erythrocytes, and practically the same titers were obtained with these two types of erythrocytes. These sera were also titrated for neutralizing (NT) antibody to NCD virus. NT tests were carried out in tube cultures of BK cells as described previously (3). The antisera had high HI titers as well as NT titers, whereas the sera before immunization were invariably negative for HI and NT antibodies. The proportional production of HI antibody as well as NT antibody to NCD virus was demonstrated in a calf infected orally with the virus (Fig. 1). In this experiment HI titers were also determined with both human and bovine erythrocytes and the same titers were obtained. A correlation diagram was obtained with NT and HI antibody titers of individual sera from 39 apparently healthy adult cattle in Japan (Fig. 2). HI titers were determined with bovine erythrocytes and they were closely correlated with NT titers (r=0.6829). The HA and HI tests developed in this study seem very useful and will find a wide application in studies on the virus and its disease. REFERENCES
1)
2) 3) 4) 5) 6)
Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.O., and Mebus, C.A. 1972. Cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in Nebraska. Arch. Ges. Virusforsch. 37: 114-130. Kurogi, H., Inaba, Y., Goto, Y., Takahashi, A., Sato, K., Omori, T., and Matumoto, M. 1974. Isolation of rhinovirus from cattle in outbreaks of acute respiratory disease. Arch. Ges. Virusforsch. 44: 215-226. Kurogi, H., Inaba, Y., Takahashi, E., Sato, K., Goto, Y., and Omori, T. 1976. Cytopathic effect of Nebraska calf diarrhea virus (Lincoln Strain) on secondary bovine kidney cell monolayer. Natl. Inst. Anim. Health Quart. 16: 133-134. Mebus, C.A., Kono, M., Underdahl, N.R., and Twiehaus, M.J. 1971. Cell culture propagation of neonatal calf diarrhea (scours) virus. Can. Vet. J. 12: 69-72. Mebus, C.A., Underdahl, N.R., Rhodes, M.B., and Twiehaus, M.J. 1969. Calf diarrhea (scours) : Reproduced with a virus from a field outbreak. Univ. of Nebraska Agr. Exp. Stat. Res. Bull. No. 233, p. 2-15. Spence, L., Fauvel, M., Petro, R., and Bloch, S. 1976. Haemagglutinin from rotavirus. Lancet ii: 1023.
Requests for reprints should be addressed to Dr. Y. Inaba, National Institute of Animal Health, Kodaira, Tokyo 187, Japan.
Hemagglutination
with
Nebraska
Calf
Diarrhea
Virus
Y. INABA, K. SATO, E. TAKAHASHI,H. KUROGI, K. SATODA,T. OMORI, and M. MATUMOTO NationalInstituteof AnimalHealth, Tokyo,and KitasatoInstitute,Tokyo (Received for publication, April 19, 1977)
Nebraska calf diarrhea (NCD) virus or reovirus-like agent of calf diarrhea has been shown to be a primary etiological agent of neonatal calf diarrhea by Mebus and his associates (1, 4, 5). Recently Spence et al (6) prepared hemagglutinin from African green-monkey kidney cells infected with NCD virus. This antigen agglutinated human group O cells. We also examined hemagglutination (HA) with NCD virus grown in calf kidney (BK) cells and found that the virus agglutinated erythrocytes of human and some other animals. This paper describes our observations on the HA with this virus and its inhibition (HI) by specific antiserum. Lincoln strain of NCD virus (4) maintained by serial passage in BK cells, was supplied by Dr. C.A. Mebus, University of Nebraska, Lincoln, Nebraska, and used in the present study after several passages in BK cells in our laboratory. BK cell cultures were prepared as described previously (2). The growth medium used was LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate) supplemented with 10% calf serum. Primary BK cell monolayers prepared in 500-ml bottles were inoculated with 0.1 TCID50 per cell of virus and incubated at 37 C for 2 days with LE medium containing 0.1 % yeast extract. As described later, in some preliminary experiments, culture fluid harvested from infected cells was used as HA antigen after centrifugation to remove coarse debris. In subsequent experiments, however, HA antigens concentrated from infected culture fluid were used. HA and HI tests were carried out by the microtiter method. The diluent used was VBS (veronal-buffered saline, pH 7.0, containing 0.1 % bovine serum albumin and 0.001% gelatin), unless otherwise stated. Blood was obtained in Alsever's solution and stored at 4 C. Erythrocytes from horse, cattle, sheep and goat were used as 0.3% suspensions, and those from other species as 0.5% suspensions. In HA tests, serial 2-fold dilutions of HA antigen were prepared in 0.05-ml amounts, and mixed with 0.025 ml of an erythrocyte suspension. The mixtures were incubated at 37 C for 1 hr before the results were read, unless otherwise stated. The HA titer was expressed as the reciprocal of the highest antigen dilution showing complete HA. For HI tests, 0.2 ml of the serum was inactivated by heating at 56 C for 30 min, diluted 5-fold, mixed with 0.2 ml of packed erythrocytes and incubated at 37 C for 1 hr. After removal of the cells by centrifugation, the supernatant fluid was mixed with 0.5 ml of 25% kaolin, incubated at room temperature for 1 hr, and centrifuged to remove kaolin. The resulting supernatant fluid was taken as the 10-fold dilution 531
Y.
532 of
the
serum.
dilutions and
Eight
of
the
mixed
with
incubated as
at
the
found
to
be
also
done 1 hr
fuged
at
original 0.001
followed g
M Tris,
erythrocytes
30
pH
to were
with
8%
buffer,
and
the
saline
at
4
M NaCl,
prepared
were
low.
To
cellular
confirm In-
in
pH
Concentration
The
cells were
debris,
suspended
pellets
(0.15
BK
animals
ultracentrifugation.
7.0). glycol
C.
then
expressed
from other
remove
polyethylene
resulting
was
1 hr,
HI.
were
by
2-fold for
were
titer
some
obtained
pellets
incubation
min,
antigens at
human,
Table
Table
the
HI
harvested
and
serial
at
room
material were
0.001
1/50
original was
temperature
was
then
suspended M CaCl2,
the
was
in 0.001
centri1/50
the
M MgCl2,
7.0).
of species of the
and
of
mixtures
complete
antigens
phosphate
fluid
of Tris-buffered
concentrated
a variety
2 hr,
The
fluid
titers
centrifugation
overnight
for
HA
ml
temperature
The
read.
human
low-speed
g for
0.025
room
showing
from the
and at
supernatant
HA
culture by
8,000 •~
were
dilution
with
M NaCl, M/150
stirring
volume
The from
by
serum
ml
suspension.
concentrated after
(0.15
0.025
incubated
results
although
100,000 •~
PBS
the
erythrocytes
prepared
in
mixed, erythrocyte
and
virus,
fluid,
at of
an
highest
agglutinated,
culture
volume
of
ET AL
antigen
experiments
we
centrifuged
HA were
1 hr,
the
NCD
finding,
fectious
for
of
with
ml
C for
preliminary
infected
this
37
of
serum
0.025
reciprocal In
units
treated
INABA
1.
2.
4
thus C,
cattle,
HA
HI
titers
and
NT
room
temperature
horse,
sheep,
at 37 C with
titers
tested and
goat,
erythrocytes
of antisera from with NCD virus
37 swine,
from
rabbits
for C.
HA HA
guinea
various
with
erythrocytes
was
observed
pig,
goose,
species
hyperimmunized
with chicken
NOTES
533
Fig. 1. Production of serum HI and NT antibodies in a calf infected orally with 400 ml of an NCD virus suspension (104.5 TCID50/ml). HI titers were determined with human and bovine erythrocytes which gave the same results.
Fig. 2.
HI and NT antibody
titers of individual
adult cattle.
and pigeon, whereas no HA was observed with rabbit, hamster, rat or mouse erythrocytes. The different temperatures employed for the tests showed little difference in HA titer with any of these HA-positive cell types except horse, guinea pig and chicken cells which gave much lower or no HA titer at 4 C. These experiments were carried out using VBS as the diluent. However, when PBS or 0.15 M NaCl solution was used, no HA was observed at any of these temperatures with any of the cells. On the basis of these results VBS was used as the diluent and tests were incubated at 37 C
534
Y. INABA
ET AL
in the standard method described earlier. HA titers obtained by this method varied considerably among the species as well as among different samples of a species (Table 1). The HA of NCD virus was inhibited by antisera to NCD virus (Table 2). The antisera used were prepared in rabbits, which were immunized by 4 intramuscular doses of partially purified virus mixed with complete Freund adjuvant. Sera were obtained 2 weeks after the last dose. HI titers were determined with both human (O) and bovine erythrocytes, and practically the same titers were obtained with these two types of erythrocytes. These sera were also titrated for neutralizing (NT) antibody to NCD virus. NT tests were carried out in tube cultures of BK cells as described previously (3). The antisera had high HI titers as well as NT titers, whereas the sera before immunization were invariably negative for HI and NT antibodies. The proportional production of HI antibody as well as NT antibody to NCD virus was demonstrated in a calf infected orally with the virus (Fig. 1). In this experiment HI titers were also determined with both human and bovine erythrocytes and the same titers were obtained. A correlation diagram was obtained with NT and HI antibody titers of individual sera from 39 apparently healthy adult cattle in Japan (Fig. 2). HI titers were determined with bovine erythrocytes and they were closely correlated with NT titers (r=0.6829). The HA and HI tests developed in this study seem very useful and will find a wide application in studies on the virus and its disease. REFERENCES
1)
2) 3) 4) 5) 6)
Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.O., and Mebus, C.A. 1972. Cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in Nebraska. Arch. Ges. Virusforsch. 37: 114-130. Kurogi, H., Inaba, Y., Goto, Y., Takahashi, A., Sato, K., Omori, T., and Matumoto, M. 1974. Isolation of rhinovirus from cattle in outbreaks of acute respiratory disease. Arch. Ges. Virusforsch. 44: 215-226. Kurogi, H., Inaba, Y., Takahashi, E., Sato, K., Goto, Y., and Omori, T. 1976. Cytopathic effect of Nebraska calf diarrhea virus (Lincoln Strain) on secondary bovine kidney cell monolayer. Natl. Inst. Anim. Health Quart. 16: 133-134. Mebus, C.A., Kono, M., Underdahl, N.R., and Twiehaus, M.J. 1971. Cell culture propagation of neonatal calf diarrhea (scours) virus. Can. Vet. J. 12: 69-72. Mebus, C.A., Underdahl, N.R., Rhodes, M.B., and Twiehaus, M.J. 1969. Calf diarrhea (scours) : Reproduced with a virus from a field outbreak. Univ. of Nebraska Agr. Exp. Stat. Res. Bull. No. 233, p. 2-15. Spence, L., Fauvel, M., Petro, R., and Bloch, S. 1976. Haemagglutinin from rotavirus. Lancet ii: 1023.
Requests for reprints should be addressed to Dr. Y. Inaba, National Institute of Animal Health, Kodaira, Tokyo 187, Japan.
