Microbiol.
Immunol.
Vol. 22 (8), 499-503, 1978
Isolation of a Reovirus-Like Agent (Rotavirus) from Neonatal Calf Diarrhea in Japan Kunihiko SATO,Yuji INABA,Eiji TAKAHASHI, Yasuichiro ITO, Hiroshi KUROGI, Hiroomi AKASHI,Kazuyuki SATODA, Tsuneyoshi OMORI,and Minoru MATUMOTO National Institute of Animal Health, Tokyo, and Kitasato Institute, Tokyo (Received for publication, November 21, 1977)
Mebus and his associates (3, 8, 9) have demonstrated a reovirus-like agent to be a cause of neonatal calf diarrhea in the United States. Subsequent reports from various parts of the world have indicated a much wider distribution of this virus in the cattle population (1, 2, 7, 10, 12-14). In Japan, the infection has also been shown by serological tests to be widespread among cattle (5, 11). In the present study we could isolate the virus in primary bovine kidney (BK) cell cultures from an affected calf in an outbreak of neonatal calf diarrhea. Since the first report of Mebus et al (8) in the United States the isolation of the virus in tissue cultures has been reported from European countries (1, 7, 10). The name rotavirus has been proposed for this group of viruses to which this calf virus belongs (4). In the present paper we will refer the virus as calf rotavirus. An outbreak of neonatal calf diarrhea occurred at a breeding center in Shimane Prefecture over the period from January through March in 1977. Almost all the calves of Japanese black breed born at the center during this period were taken ill at one to 10 days of age with slight fever and watery yellowish diarrhea which severely dehydrated the calf. Of the 106 affected calves 44% died of dehydration. Diarrheal feces and intestinal contents was collected from an affected calf (Shimane No. 1) and prepared for demonstration of virus particles by electron microscopy. The mixture of feces and intestinal contents was homogenized to make a 10% suspension in PBS (0.15 M NaCl, m/150 phosphate buffer, pH 7.2). The suspension was clarified by centrifugation at 1,500 g for 20 min, and was centrifuged at 20,000 g for 30 min. The resulting supernatant fluid was successively filtered through membrane filters, 1,000, 800 and 650 nm in pore size (Toyo-Kagaku Co., Tokyo). The final filtrate was centrifuged at 100,000 g for 2 hr, and the pellet was dissolved in 0.1 volume of PBS. The suspension was then centrifuged at 20,000 g for 10 min. Electron microscopic examination of the supernatant fluid by the phosphotungstic negative staining technique revealed numerous viral particles, 6575 nm in diameter, morphologically similar to the reovirus-like particles described by Mebus et al (9) and other workers (1, 2, 12, 14) (Fig.1A). The final preparation, which was used for electron microscopic examination in the above experiment, was also tested for the presence of complement-fixing (CF) 499
500
K.
Fig.
1.
A:
Negatively
fluorescence changes ×100.D;
in in
stained
bovine
bovine Uninfected
viral
kidney
kidney
cells
cells
control
ET AL
particles
from
a fecal
infected
with
the
infected of
SATO
bovine
with kidney
the
isolated cells.
extract.•~250,000.
isolated
B:
virus.•~200.C: virus
Unstained,
3 days
ImmunoCytopathic
earlier.
Unstained,
×100.
antigen of calf rotavirus. Before CF testing the preparation was treated to remove anticomplementary activity by mixing with an equal volume of fluorocarbon (Daiflon-S3, Daikin Co., Osaka). After 10 min at room temperature the mixture was centrifuged and the water phase was used for CF test. The antiserum used for the test was prepared in rabbits with the Lincoln strain of calf rotavirus (8). The virus was grown in primary BK cell cultures (5,6). Clarified culture fluid from infected
NOTES
501
cells was centrifuged at 100,000 g for 2 hr. The pellet was dissolved in 0.01 volume of PBS. The suspension was mixed with a CsCl solution to a density of 1.36 g/ml and centrifuged in a Beckman SW 50.1 rotor at 100,000 g for 18 hr. Infectivity and hemagglutinating activity peaked at the same density of 1.36 g/ml, and the peak fractions were used for immunization after twofold dilution with PBS. Each rabbit received one intravenous dose of 5 ml of the virus suspension and 4 weeks later 3 weekly intramuscular doses of one ml each of equal-volume mixture of the virus suspension and Freund's complete adjuvant. Serum was obtained 2 weeks after the last dose. CF test was carried out as described previously (11). The antiserum was found to have a CF antibody titer of 1:1024 when tested with 4 units of a CF antigen prepared as described previously from culture fluid of BK cell cultures infected with the Lincoln strain (11). Using this antiserum diluted 1:256 the antigen prepared from the fecal specimen was tested for CF antigenicity and was found to have a CF antigen titer of 1:8. The pre-immunization serum was negative with either of these antigens. Attempts were made to isolate virus with the feces and intestinal contents from the calf Shimane No. 1. Primary BK cell cultures were prepared in 100 x 11 mm tubes. The growth medium used was LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate and 0.07% sodium bicarbonate) supplemented with 10% calf serum, and the maintenance medium was LE medium containing 0.1% yeast extract. Cultures inoculated with 0.1-ml volumes, after virus adsorption at 37 C for 2 hr, were washed three times with LE medium, fed with 0.5 ml of maintenance medium, and incubated at 37 C in a roller drum. The cultures were checked for virus growth at each passage level by the direct immunofluorescent technique with antiserum to calf rotavirus. For the test the cultures were washed with PBS and scraped of into PBS with a rubber policeman. The cells were then spun down and smeared on glass slides with a micropipet. The preparations were air-dried, fixed with acetone at -20 C for one hr, and stained at 37 C for one hr with fluorescein isothiocyanate-conjugate. The conjugate was prepared as described by Mebus et al (9) with the rabbit antiserum which was used in the above CF tests. Virus isolation was tried three times and all trials were successful. In the first attempt the mixture of the feces and intestinal contents was homogenized to form a 10% suspension in PBS. The suspension, after centrifugation to remove coarse debris, was centrifuged at 20,000 g for 30 min and the supernatant fluid was used for inoculation into tube cultures of BK cells. Each of serial decimal dilutions, 100-10-3, of the material was inoculated into 10 BK cultures. The inoculated cultures were incubated for 7 days and observed for any cytopathic effect with negative results. At the end of the incubation the cells from each group of tubes were pooled and tested for immunofluorescence. A few fluorescing cells were observed in the cultures inoculated with undiluted material. Further passages were made at intervals of 7 days with serial decimal dilutions, 100-10-3, of pooled culture fluid from the tubes inoculated with undiluted material. Fluorescing cells were readily observed in all the cultures at the second and further passages, whereas
502
K.
SATO
ET AL
cytopathic effect was first clearly noticed at the 7th passage. Further passages were readily accomplished. Immunofluorescence was first observed as perinuclear fine granules at 3 days post-inoculation and became diffuse in the cytoplasm as the incubation progressed (Fig.1B). The cytopathic effect appeared 2 to 3 days post-inoculation and was characterized by granulation, aggregation and eventual disintegration of the cells, leaving cell sheets of a moss-eaten appearance with many cells adhering to glass by the 5th day of incubation (Fig.1C, D). The infectious titer reached about 107 TCID50/ml although early passage cultures yielded low titers. In the second trial of virus isolation the pooled specimen of feces and intestinal contents was processed in the same manner as in the first experiment and the supernatant fluid obtained after centrifugation at 20,000 g for 30 min was further treated by vigorously shaking with an equal volume of trichlorotrifluoroethane (chilled at -20 C)(Kanto
Kagaku
Co ., Tokyo).
Thc material was then spun down
in a
refrigerated centrifuge at 3,000 g for 20 min and supernatant fluid was used for inoculation into BK cell cultures. Inoculation, passage and observation were done in the same manner as in the first experiment. Immunofluorescence was positive only in the cultures inoculated with undiluted material at the primary passage, and was positive upto 10-1 dilution at the second passage and upto 10-3 dilution at the further passages. A questionable cytopathic effect was noted at the third passage and it became clear at the 4th passage showing a titer of 104.5TCID50/ml or higher. Further passages were readily accomplished. In the third trial of virus isolation the intestinal contents from the calf Shimane No. 1 was used. The material was processed as in the preceding experiments, and the supernatant fluid obtained after centrifugation 20,000 g for 30 min was further centrifuged through discontinuous sucrose density gradient, 50, 30 and 10%, at 70,000 g for 18 hr. A band visible above the 50% layer was collected and centrifuged 100,000 g for 2 hr. The pellet was dissolved in 0.01 volume of PBS and used for inoculation into BK cell cultures after centrifugation at 3,000 g for 10 min. Inoculation, passage and observation were made in the same manner as in the preceding experiments. Cytopathic effect was not clear at the first passage but it became clear at the second passage, showing a titer of 104.0 TCID50/ml. Immunofluorescence was positive upto 10-2 at the first passage and upto 10-3 at the second passage. Further passages were readily accomplished. The demonstration of specific antigen of calf rotavirus in infected cells by the immunofluorescent staining indicated the isolated viruses to be calf rotavirus. This was further confirmed by the neutralization test with the rabbit antiserum to calf rotavirus which was used for immunofluorescent staining. The test was carried out by the serum dilution method using tube cultures of BK cells. Virus-serum mixtures were incubated at 37 C for one hr before inoculation into 2 BK cell cultures per serum dilution. The virus dose per tube was 100 TCID50. The antibody titer was expressed as the highest serum dilution which showed neutralization in at least one of the two tubes. The antibody titers obtained with the three isolated viruses showed no significant difference from the homologous titer of 1:8192 with the Lincoln strain. The preimmunization serum did not neutralize any of these viruses. More
NOTES
503
detailed comparison of the isolated viruses with the prototype Lincoln strain of calf rotavirus is needed for further confirmation of the identification of the isolated viruses. This is the first report of the isolation of calf rotavirus in Japan. REFERENCES
1) Bridger, J.C., and Woode, G.N. 1975. Neonatal calf diarrhea : Identification of a reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy. Brit. Vet. J. 131: 528-535. 2) Burgess, G.W., and Simpson, B.H. 1976. An orbi-like virus in the faeces of neonatal calves with diarrhea. New Zealand Vet. J. 24: 35-36. 3) Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.0., 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. 4) Flewett, T.H., Bryden, A.S., Davies, H., Woode, G.N., Bridger, J.C., and Derrick, J.M. 1974. Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet 2: 61-63. 5) Inaba, Y., Sato, K., Takahashi, E., Kurogi, H., Satoda, K., Omori, T., and Matumoto, M. 1977. Hemagglutination with Nebraska calf diarrhea virus. Microbiol. Immunol. 21: 531-534. 6) Kurogi, H., Inaba, Y., Takahashi, E., Sato, K., Goto, Y., Omori, T. 1976. Cytopathic effect of Nebraska calf diarrhea virus (Lincoln strain) on secondary bovine kidney cell monolayer. Nat. Inst. Anim. Hlth Quart. 16: 133-134. 7) McNulty, M.S., Allan, G.M., and McFerran, J.B. 1976. Isolation of a cytopathic calf rotavirus. Res. Vet. Sci. 21: 114-115. 8) Mebus, C.A., Kono, M., Underdahl, N.R., and Twiehaus, M.J. 1971. Cell culture propagation of neonatal calf diarrhea (scours) virus. Canad. Vet. J. 12: 69-72. 9) 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. 10) Meyling, A. 1974. Reo-like neonatal calf diarrhea (NCD) virus demonstrated in Denmark. Acta Vet. Scand. 15: 457-459. 11) Takahashi, E., Inaba, Y., Kurogi, H., Sato, K., Satoda, K., and Omori, T. 1977. Complement fixation test of Nebraska calf diarrhea virus with calf serum. Nat. Inst. Anim. Hlth Quart. 17: 33-38. 12) Turner, A.J., Caple, I.W., Craven, J.A., and Reinganum, C. 1973. Demonstration of virus particles in intestinal contents of calves with diarrhea. Austral. Vet. J. 49: 544. 13) White, R.G., Mebus, C.A., and Twiehaus, M.J. 1970. Incidence of herds infected with a neonatal calf diarrhea virus (NCDV). Vet. Med./Small Anim. Clin. 65: 487-490. 14) Woode, G.N., Bridger, J.C., Hall, G., and Dennis, M.J. 1974. The isolation of a reovirus-like agent associated with diarrhea in colostrum-deprived calves in Great Britain. Res. Vet. Sci. 16: 102-105. Requests for reprints should be addressed to Dr. Kunihiko Sato, National Institute of Animal Health, Kodaira, Tokyo 187, Japan.
Immunol.
Vol. 22 (8), 499-503, 1978
Isolation of a Reovirus-Like Agent (Rotavirus) from Neonatal Calf Diarrhea in Japan Kunihiko SATO,Yuji INABA,Eiji TAKAHASHI, Yasuichiro ITO, Hiroshi KUROGI, Hiroomi AKASHI,Kazuyuki SATODA, Tsuneyoshi OMORI,and Minoru MATUMOTO National Institute of Animal Health, Tokyo, and Kitasato Institute, Tokyo (Received for publication, November 21, 1977)
Mebus and his associates (3, 8, 9) have demonstrated a reovirus-like agent to be a cause of neonatal calf diarrhea in the United States. Subsequent reports from various parts of the world have indicated a much wider distribution of this virus in the cattle population (1, 2, 7, 10, 12-14). In Japan, the infection has also been shown by serological tests to be widespread among cattle (5, 11). In the present study we could isolate the virus in primary bovine kidney (BK) cell cultures from an affected calf in an outbreak of neonatal calf diarrhea. Since the first report of Mebus et al (8) in the United States the isolation of the virus in tissue cultures has been reported from European countries (1, 7, 10). The name rotavirus has been proposed for this group of viruses to which this calf virus belongs (4). In the present paper we will refer the virus as calf rotavirus. An outbreak of neonatal calf diarrhea occurred at a breeding center in Shimane Prefecture over the period from January through March in 1977. Almost all the calves of Japanese black breed born at the center during this period were taken ill at one to 10 days of age with slight fever and watery yellowish diarrhea which severely dehydrated the calf. Of the 106 affected calves 44% died of dehydration. Diarrheal feces and intestinal contents was collected from an affected calf (Shimane No. 1) and prepared for demonstration of virus particles by electron microscopy. The mixture of feces and intestinal contents was homogenized to make a 10% suspension in PBS (0.15 M NaCl, m/150 phosphate buffer, pH 7.2). The suspension was clarified by centrifugation at 1,500 g for 20 min, and was centrifuged at 20,000 g for 30 min. The resulting supernatant fluid was successively filtered through membrane filters, 1,000, 800 and 650 nm in pore size (Toyo-Kagaku Co., Tokyo). The final filtrate was centrifuged at 100,000 g for 2 hr, and the pellet was dissolved in 0.1 volume of PBS. The suspension was then centrifuged at 20,000 g for 10 min. Electron microscopic examination of the supernatant fluid by the phosphotungstic negative staining technique revealed numerous viral particles, 6575 nm in diameter, morphologically similar to the reovirus-like particles described by Mebus et al (9) and other workers (1, 2, 12, 14) (Fig.1A). The final preparation, which was used for electron microscopic examination in the above experiment, was also tested for the presence of complement-fixing (CF) 499
500
K.
Fig.
1.
A:
Negatively
fluorescence changes ×100.D;
in in
stained
bovine
bovine Uninfected
viral
kidney
kidney
cells
cells
control
ET AL
particles
from
a fecal
infected
with
the
infected of
SATO
bovine
with kidney
the
isolated cells.
extract.•~250,000.
isolated
B:
virus.•~200.C: virus
Unstained,
3 days
ImmunoCytopathic
earlier.
Unstained,
×100.
antigen of calf rotavirus. Before CF testing the preparation was treated to remove anticomplementary activity by mixing with an equal volume of fluorocarbon (Daiflon-S3, Daikin Co., Osaka). After 10 min at room temperature the mixture was centrifuged and the water phase was used for CF test. The antiserum used for the test was prepared in rabbits with the Lincoln strain of calf rotavirus (8). The virus was grown in primary BK cell cultures (5,6). Clarified culture fluid from infected
NOTES
501
cells was centrifuged at 100,000 g for 2 hr. The pellet was dissolved in 0.01 volume of PBS. The suspension was mixed with a CsCl solution to a density of 1.36 g/ml and centrifuged in a Beckman SW 50.1 rotor at 100,000 g for 18 hr. Infectivity and hemagglutinating activity peaked at the same density of 1.36 g/ml, and the peak fractions were used for immunization after twofold dilution with PBS. Each rabbit received one intravenous dose of 5 ml of the virus suspension and 4 weeks later 3 weekly intramuscular doses of one ml each of equal-volume mixture of the virus suspension and Freund's complete adjuvant. Serum was obtained 2 weeks after the last dose. CF test was carried out as described previously (11). The antiserum was found to have a CF antibody titer of 1:1024 when tested with 4 units of a CF antigen prepared as described previously from culture fluid of BK cell cultures infected with the Lincoln strain (11). Using this antiserum diluted 1:256 the antigen prepared from the fecal specimen was tested for CF antigenicity and was found to have a CF antigen titer of 1:8. The pre-immunization serum was negative with either of these antigens. Attempts were made to isolate virus with the feces and intestinal contents from the calf Shimane No. 1. Primary BK cell cultures were prepared in 100 x 11 mm tubes. The growth medium used was LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate and 0.07% sodium bicarbonate) supplemented with 10% calf serum, and the maintenance medium was LE medium containing 0.1% yeast extract. Cultures inoculated with 0.1-ml volumes, after virus adsorption at 37 C for 2 hr, were washed three times with LE medium, fed with 0.5 ml of maintenance medium, and incubated at 37 C in a roller drum. The cultures were checked for virus growth at each passage level by the direct immunofluorescent technique with antiserum to calf rotavirus. For the test the cultures were washed with PBS and scraped of into PBS with a rubber policeman. The cells were then spun down and smeared on glass slides with a micropipet. The preparations were air-dried, fixed with acetone at -20 C for one hr, and stained at 37 C for one hr with fluorescein isothiocyanate-conjugate. The conjugate was prepared as described by Mebus et al (9) with the rabbit antiserum which was used in the above CF tests. Virus isolation was tried three times and all trials were successful. In the first attempt the mixture of the feces and intestinal contents was homogenized to form a 10% suspension in PBS. The suspension, after centrifugation to remove coarse debris, was centrifuged at 20,000 g for 30 min and the supernatant fluid was used for inoculation into tube cultures of BK cells. Each of serial decimal dilutions, 100-10-3, of the material was inoculated into 10 BK cultures. The inoculated cultures were incubated for 7 days and observed for any cytopathic effect with negative results. At the end of the incubation the cells from each group of tubes were pooled and tested for immunofluorescence. A few fluorescing cells were observed in the cultures inoculated with undiluted material. Further passages were made at intervals of 7 days with serial decimal dilutions, 100-10-3, of pooled culture fluid from the tubes inoculated with undiluted material. Fluorescing cells were readily observed in all the cultures at the second and further passages, whereas
502
K.
SATO
ET AL
cytopathic effect was first clearly noticed at the 7th passage. Further passages were readily accomplished. Immunofluorescence was first observed as perinuclear fine granules at 3 days post-inoculation and became diffuse in the cytoplasm as the incubation progressed (Fig.1B). The cytopathic effect appeared 2 to 3 days post-inoculation and was characterized by granulation, aggregation and eventual disintegration of the cells, leaving cell sheets of a moss-eaten appearance with many cells adhering to glass by the 5th day of incubation (Fig.1C, D). The infectious titer reached about 107 TCID50/ml although early passage cultures yielded low titers. In the second trial of virus isolation the pooled specimen of feces and intestinal contents was processed in the same manner as in the first experiment and the supernatant fluid obtained after centrifugation at 20,000 g for 30 min was further treated by vigorously shaking with an equal volume of trichlorotrifluoroethane (chilled at -20 C)(Kanto
Kagaku
Co ., Tokyo).
Thc material was then spun down
in a
refrigerated centrifuge at 3,000 g for 20 min and supernatant fluid was used for inoculation into BK cell cultures. Inoculation, passage and observation were done in the same manner as in the first experiment. Immunofluorescence was positive only in the cultures inoculated with undiluted material at the primary passage, and was positive upto 10-1 dilution at the second passage and upto 10-3 dilution at the further passages. A questionable cytopathic effect was noted at the third passage and it became clear at the 4th passage showing a titer of 104.5TCID50/ml or higher. Further passages were readily accomplished. In the third trial of virus isolation the intestinal contents from the calf Shimane No. 1 was used. The material was processed as in the preceding experiments, and the supernatant fluid obtained after centrifugation 20,000 g for 30 min was further centrifuged through discontinuous sucrose density gradient, 50, 30 and 10%, at 70,000 g for 18 hr. A band visible above the 50% layer was collected and centrifuged 100,000 g for 2 hr. The pellet was dissolved in 0.01 volume of PBS and used for inoculation into BK cell cultures after centrifugation at 3,000 g for 10 min. Inoculation, passage and observation were made in the same manner as in the preceding experiments. Cytopathic effect was not clear at the first passage but it became clear at the second passage, showing a titer of 104.0 TCID50/ml. Immunofluorescence was positive upto 10-2 at the first passage and upto 10-3 at the second passage. Further passages were readily accomplished. The demonstration of specific antigen of calf rotavirus in infected cells by the immunofluorescent staining indicated the isolated viruses to be calf rotavirus. This was further confirmed by the neutralization test with the rabbit antiserum to calf rotavirus which was used for immunofluorescent staining. The test was carried out by the serum dilution method using tube cultures of BK cells. Virus-serum mixtures were incubated at 37 C for one hr before inoculation into 2 BK cell cultures per serum dilution. The virus dose per tube was 100 TCID50. The antibody titer was expressed as the highest serum dilution which showed neutralization in at least one of the two tubes. The antibody titers obtained with the three isolated viruses showed no significant difference from the homologous titer of 1:8192 with the Lincoln strain. The preimmunization serum did not neutralize any of these viruses. More
NOTES
503
detailed comparison of the isolated viruses with the prototype Lincoln strain of calf rotavirus is needed for further confirmation of the identification of the isolated viruses. This is the first report of the isolation of calf rotavirus in Japan. REFERENCES
1) Bridger, J.C., and Woode, G.N. 1975. Neonatal calf diarrhea : Identification of a reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy. Brit. Vet. J. 131: 528-535. 2) Burgess, G.W., and Simpson, B.H. 1976. An orbi-like virus in the faeces of neonatal calves with diarrhea. New Zealand Vet. J. 24: 35-36. 3) Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.0., 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. 4) Flewett, T.H., Bryden, A.S., Davies, H., Woode, G.N., Bridger, J.C., and Derrick, J.M. 1974. Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet 2: 61-63. 5) Inaba, Y., Sato, K., Takahashi, E., Kurogi, H., Satoda, K., Omori, T., and Matumoto, M. 1977. Hemagglutination with Nebraska calf diarrhea virus. Microbiol. Immunol. 21: 531-534. 6) Kurogi, H., Inaba, Y., Takahashi, E., Sato, K., Goto, Y., Omori, T. 1976. Cytopathic effect of Nebraska calf diarrhea virus (Lincoln strain) on secondary bovine kidney cell monolayer. Nat. Inst. Anim. Hlth Quart. 16: 133-134. 7) McNulty, M.S., Allan, G.M., and McFerran, J.B. 1976. Isolation of a cytopathic calf rotavirus. Res. Vet. Sci. 21: 114-115. 8) Mebus, C.A., Kono, M., Underdahl, N.R., and Twiehaus, M.J. 1971. Cell culture propagation of neonatal calf diarrhea (scours) virus. Canad. Vet. J. 12: 69-72. 9) 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. 10) Meyling, A. 1974. Reo-like neonatal calf diarrhea (NCD) virus demonstrated in Denmark. Acta Vet. Scand. 15: 457-459. 11) Takahashi, E., Inaba, Y., Kurogi, H., Sato, K., Satoda, K., and Omori, T. 1977. Complement fixation test of Nebraska calf diarrhea virus with calf serum. Nat. Inst. Anim. Hlth Quart. 17: 33-38. 12) Turner, A.J., Caple, I.W., Craven, J.A., and Reinganum, C. 1973. Demonstration of virus particles in intestinal contents of calves with diarrhea. Austral. Vet. J. 49: 544. 13) White, R.G., Mebus, C.A., and Twiehaus, M.J. 1970. Incidence of herds infected with a neonatal calf diarrhea virus (NCDV). Vet. Med./Small Anim. Clin. 65: 487-490. 14) Woode, G.N., Bridger, J.C., Hall, G., and Dennis, M.J. 1974. The isolation of a reovirus-like agent associated with diarrhea in colostrum-deprived calves in Great Britain. Res. Vet. Sci. 16: 102-105. Requests for reprints should be addressed to Dr. Kunihiko Sato, National Institute of Animal Health, Kodaira, Tokyo 187, Japan.
