Propagation of the Rotavirus of Neonatal Calf Diarrhea in Fetal Intestinal Cell Cultures K. A. Mohammed and J. R. Saunders* ABSTRACT
and experiences in our laboratory led us to the conclusion that the virus could not be consistently detected in BFK cells on the Bovine fetal intestinal cells were success- basis of cytopathic effect (CPE). Therefully propagated in monolayer cultures for up fore, we attempted the in vitro propagation to 21 passages. Infection of these cells with of those cells naturally infected, namely the the rotavirus of neonatal calf diarrhea resulted intestinal epithelial cells, on the premise in a cytopathic effect that was more obvious that they might prove to be more susceptthan in infected bovine fetal kidney cells. ible to the virus. Ten bovine feti, approximately two to six months old, were collected from a local abattoir' for this study. Small segments (5 cm length approximately) of the jejunum and ileum were dissected out and cut RESUME open with scissors. Batches of bovine fetal intestinal (BFI) cells were prepared in two Les auteurs ont effectue 21 passages suc- ways, the first being a standard trypsinizacessifs et fructueux de cellules intestinales de tion method (0.25% trypsin, two cycles of foetus bovin, en culture tissulaire. L'infection 15 minutes at 370C) the other by washing de ces cellules par le rotavirus de la diarrhee the intestinal segments for a half hour at neo-natale du veau resulta en un effet cytopa- 37°C in phosphate buffered saline (PBS) thogene plus evident qu'avec des cellules re- agitated gently by a magnetic stirrer. Cells, nales de foetus bovin. detached by either treatment, were filtered through gauze and centrifuged at 1000 x g for 20 minutes. The cells were resuspended in growth medium and seeded at the rate of A virus designated initially as neonatal 0.5 to 1.0 million cells (viable count by calf diarrhea (NCD) virus was isolated in methylene blue staining) per ml in falcon Nebraska in 1968 and reported as the pri- flasks2 subsequently incubated in 5%O C02 mary etiological agent of some outbreaks at 37°C. The growth medium used was of this disease (1). In infected calves, viral MEM3 (Eagle's base) containing 10% heat antigens were demonstrated in villous epi- inactivated fetal calf serum, 1% nonessenthelial cells of the jejunum by a direct fluo- tial amino acids4 and 100 units of penicillin rescent antibody test (FAT) applied to and 100 pg of streptomycin per ml. The frozen sections of fecal smears (1). Studies growth medium was changed every two of the morphology and physicochemical days. When the cells were confluent they properties of NCD virus indicated that it were versenized and subcultured in a split was reovirus-like (2). With some difficulty ratio of 1:2. The virus used to infect the cells was the virus was adapted to several cell culture systems (3), bovine fetal kidney (BFK) Scourvax-reo vaccine virus (Norden Labcells being the most satisfactory (4). A re- oratories). An inoculum of 0.5 to 2.0 ml of view of published papers on the propagation of NCD virus (3, 4), personal communication with other research workers UIntercontinental Packers, Saskatoon, Saskatchewan. *Department of Veterinary Microbiology, Western College of Veterinary Medicine, Universitv of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. Submitted April 28, 1976.
226
2Falcon Products, Becton, Dickinson and Company, Canada, Ltd., Mississauga, Ontario. 3Grand Island Biological Company. Micro-Can Research Ltd., Calgary, Alberta. 4Grand Island Biological Company. Micro-Can Research Ltd., Calgary, Alberta.
Can. J. comp. Med.
freshly reconstituted vaccine virus was introduced onto the confluent monolayers (previously washed three times with MEM medium) and allowed to adsorb for one to two hours prior to the addition of maintenance medium (MEM containing 0.5% lactalbumin hydrolysate,5 0.1 % yeast extract,6 1 S, nonessential amino acids and 100 units penicillin and 100 ,ug streptomycin per ml).
The criteria which were considered for
replication of the virus in the BFI cells were: (a) the demonstration of a CPE, (b) a positive direct FAT and (c) the demonstration of inclusion bodies in the cytoplasm of cells stained with hematoxylin and eosin (H & E). CPE was first observed in infected cells at 24-48 hours postinoculation. Affected cells became elongated and crescent shaped and before detaching from the plastic were attached only by a single cytoplasmic strand (Fig. 2). Specific, granular, cytoplasmic fluorescence by direct FAT was also observed by 24-48 hours postinoculation. The elongated crescent-like cells were the ones which showed maximum fluorescence Fig. 3, control Fig. 4). By H and E staining in this same time period postinfection, numerous cells containing cytoplasmic eosinophilic inclusions and va-
Fig. 1. Uninfected BFI monolayer. Unstained X40.
BFI cells were prepared from only three feti by the trypsinization procedure and in only one case did the cells attach to the plastic, divide and produce a confluent monolayer. These cells were of fibroblastic morphology and consequently were not tested for susceptibility to the NCD rotavirus. BFI cells were prepared from all ten feti by the PBS washing procedure. In the case of four feti (two to four months approximate gestational age) confluent monolayers of epithelial cells were obtained in seven to 14 days but in the case of the remaining six feti (four to six months gestational age) the cells failed to grow. When these primary cultures were versenized and subcultured they produced confluent monolayers (Fig. 1) in one to two days. The cells were passaged a maximum of 21 times, after which they degenerated or failed to grow. In the initial passages the BFI cells were predominantly epithelial with only a few fibroblastic cells. At about the tenth passage and subsequently the fibroblastic cells outnumbered the epithelial cells. 5Fisher Scientific Company, Ltd., Edmonton, Alberta. 6CanLab (Canadian Laboratory Supplies), Winnipeg, Manitoba.
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Fig. 2. CPE 48 hours after inoculation with vaccine strain of NCD rotavirus. Arrow indicates elongated cell detached from monolayer. X100.
Fig. 3. Smear prepared 48 hours after inoculat.on with vaccine strain of NCD rotavirus. Arrow points to a cell containing granular cytoplasmic fluorescence. FA stain. X160.
227
cucles were seen (Fig. 5, control Fig. 6), similar to that previously described for BFK cells infected with high passage strains of NCD rotavirus (4). For compairison, BFK cells (primary to fifth passage level) were infected in the same way as for BFI cells but did not show CPE or specific cytoplasmic fluorescence to the same degree. Therefore, the BFI cells had some advantages over BFK cells for the detection and study of the rotavirus of neonatal calf diarrhea.
Fig. 4. Uninfected BFI
smear.
FA stain. X160.
ACKNOWLEDGMENTS This study was supported by a grant (3-306-524) from Agriculture Canada to the Deans of Agriculture and Veterinary Medicine. The authors wish to thank Dr. C. A. Mebus, University of Nebraska, Lincoln, Nebraska for providing a low, calf passage strain of rotavirus and Norden Laboratories, Lincoln, Nebraska for providing the Scourvax vaccine and the reovirus FA conjugate. We thank, also, Mrs;. A. Z. Batten for able technical assistance.
REFERENCES
Fig. 5. CPE 48 hours after inoculation with vaccine strain of NCD rotavirus. Arrows indicate: (a) cell containing cytoplasmic inclusions and (b) cell containing cytoplasmic vacuoles. H & E stain. X160.
1. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. J. TWIEHAUS. Calf diarrhea (Scours): Reproduced with a virus from a field outbreak. The Agric. Exp. Station. Research Bull. No. 233. College of Agriculture, University of Nebraska, Lincoln, Nebraska. 1969. 2. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 196-202. 1971. 3. FERNELIUS, A. L., A. E. RITCHIE. L. G. CLASSICK, J. 0. NORMAN and C. A. MEBUS. 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. 1972. Cell culture 4. WELCH, A. B. and M. J. TWIEHAUS. studies of a neonatal calf diarrhea virus. Can. J. 1973. comp. Med. 37: 287-294.
ADDENDUM
Fig. 6. Uninfected BFI monolayer. H & E stain. XIGO.
228
Subsequent to the above studies, an NCD rotavirus at the third calf-passage level was obtained (from Dr. C. A. Mebus, University of Nebraska) and after three further passages in this laboratory in colostrum deprived, newborn calves was inoculated onto third passage BFI cells.
Can. J. comp. Med.
The inoculum (0.5 ml) was low passage, stock virus, prepared from frozen fecal samples (sixth calf-passage) by: (a) thawing and diluting in ten volumes of PBS, (b) low speed (3,000 X g) and high speed (55,000 X g) centrifugation, (c) resuspending the virus pellet in PBS and sonicating, (d) diluting the suspension in PBS to give a concentration of virus 10 to 20 times that of the original fecal sample, (e) clarifying by low speed centrifugation (3,000 X g), (f) filtering the supernatant through a 0.45 ,u millipore filter and (g) storing at -70°C. The BFI cultures were then examined
daily for three days, at which time they frozen and thawed three times and 0.5 ml used to inoculate fresh BFI monolayers. This procedure was then repeated to give a total of three passages of the virus in BFI cells. This low, calf-passage rotavirus, in comparison with the vaccine virus, produced a lesser CPE with positive fluorescence in 48 to 72 hours in first passage in BFI cells. The CPE and fluorescence was further decreased in the second passage and in the third passage only a few fluorescing cells, (without a CPE) were seen. were
VETERINARY PATHOLOGIST Connaught Laboratories Limited requires a veterinary pathologist certified
or eligible for certification by the Board of the American College of Veterinary Pathologists. Responsibilities will include providing gross and histological interpretation on tissues of animals from safety testing of biologics both in process and at the final quality control level; functioning as a research team member in the development and evaluation of new products and monitoring of disease status of all animals used in the production and testing of the company's human and veterinary biological products. The position is available immediately. Salary negotiable. All enquiries should be directed to: Dr. A. Fletch, Director Animal Resources Division CONNAUGHT LABORATORIES LTD. 1755 Steeles Avenue West Willowdale, Ontario M2N 5T8 Telephone: (416) 667-2870
Volume 41
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April, 1977
229
and experiences in our laboratory led us to the conclusion that the virus could not be consistently detected in BFK cells on the Bovine fetal intestinal cells were success- basis of cytopathic effect (CPE). Therefully propagated in monolayer cultures for up fore, we attempted the in vitro propagation to 21 passages. Infection of these cells with of those cells naturally infected, namely the the rotavirus of neonatal calf diarrhea resulted intestinal epithelial cells, on the premise in a cytopathic effect that was more obvious that they might prove to be more susceptthan in infected bovine fetal kidney cells. ible to the virus. Ten bovine feti, approximately two to six months old, were collected from a local abattoir' for this study. Small segments (5 cm length approximately) of the jejunum and ileum were dissected out and cut RESUME open with scissors. Batches of bovine fetal intestinal (BFI) cells were prepared in two Les auteurs ont effectue 21 passages suc- ways, the first being a standard trypsinizacessifs et fructueux de cellules intestinales de tion method (0.25% trypsin, two cycles of foetus bovin, en culture tissulaire. L'infection 15 minutes at 370C) the other by washing de ces cellules par le rotavirus de la diarrhee the intestinal segments for a half hour at neo-natale du veau resulta en un effet cytopa- 37°C in phosphate buffered saline (PBS) thogene plus evident qu'avec des cellules re- agitated gently by a magnetic stirrer. Cells, nales de foetus bovin. detached by either treatment, were filtered through gauze and centrifuged at 1000 x g for 20 minutes. The cells were resuspended in growth medium and seeded at the rate of A virus designated initially as neonatal 0.5 to 1.0 million cells (viable count by calf diarrhea (NCD) virus was isolated in methylene blue staining) per ml in falcon Nebraska in 1968 and reported as the pri- flasks2 subsequently incubated in 5%O C02 mary etiological agent of some outbreaks at 37°C. The growth medium used was of this disease (1). In infected calves, viral MEM3 (Eagle's base) containing 10% heat antigens were demonstrated in villous epi- inactivated fetal calf serum, 1% nonessenthelial cells of the jejunum by a direct fluo- tial amino acids4 and 100 units of penicillin rescent antibody test (FAT) applied to and 100 pg of streptomycin per ml. The frozen sections of fecal smears (1). Studies growth medium was changed every two of the morphology and physicochemical days. When the cells were confluent they properties of NCD virus indicated that it were versenized and subcultured in a split was reovirus-like (2). With some difficulty ratio of 1:2. The virus used to infect the cells was the virus was adapted to several cell culture systems (3), bovine fetal kidney (BFK) Scourvax-reo vaccine virus (Norden Labcells being the most satisfactory (4). A re- oratories). An inoculum of 0.5 to 2.0 ml of view of published papers on the propagation of NCD virus (3, 4), personal communication with other research workers UIntercontinental Packers, Saskatoon, Saskatchewan. *Department of Veterinary Microbiology, Western College of Veterinary Medicine, Universitv of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO. Submitted April 28, 1976.
226
2Falcon Products, Becton, Dickinson and Company, Canada, Ltd., Mississauga, Ontario. 3Grand Island Biological Company. Micro-Can Research Ltd., Calgary, Alberta. 4Grand Island Biological Company. Micro-Can Research Ltd., Calgary, Alberta.
Can. J. comp. Med.
freshly reconstituted vaccine virus was introduced onto the confluent monolayers (previously washed three times with MEM medium) and allowed to adsorb for one to two hours prior to the addition of maintenance medium (MEM containing 0.5% lactalbumin hydrolysate,5 0.1 % yeast extract,6 1 S, nonessential amino acids and 100 units penicillin and 100 ,ug streptomycin per ml).
The criteria which were considered for
replication of the virus in the BFI cells were: (a) the demonstration of a CPE, (b) a positive direct FAT and (c) the demonstration of inclusion bodies in the cytoplasm of cells stained with hematoxylin and eosin (H & E). CPE was first observed in infected cells at 24-48 hours postinoculation. Affected cells became elongated and crescent shaped and before detaching from the plastic were attached only by a single cytoplasmic strand (Fig. 2). Specific, granular, cytoplasmic fluorescence by direct FAT was also observed by 24-48 hours postinoculation. The elongated crescent-like cells were the ones which showed maximum fluorescence Fig. 3, control Fig. 4). By H and E staining in this same time period postinfection, numerous cells containing cytoplasmic eosinophilic inclusions and va-
Fig. 1. Uninfected BFI monolayer. Unstained X40.
BFI cells were prepared from only three feti by the trypsinization procedure and in only one case did the cells attach to the plastic, divide and produce a confluent monolayer. These cells were of fibroblastic morphology and consequently were not tested for susceptibility to the NCD rotavirus. BFI cells were prepared from all ten feti by the PBS washing procedure. In the case of four feti (two to four months approximate gestational age) confluent monolayers of epithelial cells were obtained in seven to 14 days but in the case of the remaining six feti (four to six months gestational age) the cells failed to grow. When these primary cultures were versenized and subcultured they produced confluent monolayers (Fig. 1) in one to two days. The cells were passaged a maximum of 21 times, after which they degenerated or failed to grow. In the initial passages the BFI cells were predominantly epithelial with only a few fibroblastic cells. At about the tenth passage and subsequently the fibroblastic cells outnumbered the epithelial cells. 5Fisher Scientific Company, Ltd., Edmonton, Alberta. 6CanLab (Canadian Laboratory Supplies), Winnipeg, Manitoba.
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April, 1977
Fig. 2. CPE 48 hours after inoculation with vaccine strain of NCD rotavirus. Arrow indicates elongated cell detached from monolayer. X100.
Fig. 3. Smear prepared 48 hours after inoculat.on with vaccine strain of NCD rotavirus. Arrow points to a cell containing granular cytoplasmic fluorescence. FA stain. X160.
227
cucles were seen (Fig. 5, control Fig. 6), similar to that previously described for BFK cells infected with high passage strains of NCD rotavirus (4). For compairison, BFK cells (primary to fifth passage level) were infected in the same way as for BFI cells but did not show CPE or specific cytoplasmic fluorescence to the same degree. Therefore, the BFI cells had some advantages over BFK cells for the detection and study of the rotavirus of neonatal calf diarrhea.
Fig. 4. Uninfected BFI
smear.
FA stain. X160.
ACKNOWLEDGMENTS This study was supported by a grant (3-306-524) from Agriculture Canada to the Deans of Agriculture and Veterinary Medicine. The authors wish to thank Dr. C. A. Mebus, University of Nebraska, Lincoln, Nebraska for providing a low, calf passage strain of rotavirus and Norden Laboratories, Lincoln, Nebraska for providing the Scourvax vaccine and the reovirus FA conjugate. We thank, also, Mrs;. A. Z. Batten for able technical assistance.
REFERENCES
Fig. 5. CPE 48 hours after inoculation with vaccine strain of NCD rotavirus. Arrows indicate: (a) cell containing cytoplasmic inclusions and (b) cell containing cytoplasmic vacuoles. H & E stain. X160.
1. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. J. TWIEHAUS. Calf diarrhea (Scours): Reproduced with a virus from a field outbreak. The Agric. Exp. Station. Research Bull. No. 233. College of Agriculture, University of Nebraska, Lincoln, Nebraska. 1969. 2. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 196-202. 1971. 3. FERNELIUS, A. L., A. E. RITCHIE. L. G. CLASSICK, J. 0. NORMAN and C. A. MEBUS. 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. 1972. Cell culture 4. WELCH, A. B. and M. J. TWIEHAUS. studies of a neonatal calf diarrhea virus. Can. J. 1973. comp. Med. 37: 287-294.
ADDENDUM
Fig. 6. Uninfected BFI monolayer. H & E stain. XIGO.
228
Subsequent to the above studies, an NCD rotavirus at the third calf-passage level was obtained (from Dr. C. A. Mebus, University of Nebraska) and after three further passages in this laboratory in colostrum deprived, newborn calves was inoculated onto third passage BFI cells.
Can. J. comp. Med.
The inoculum (0.5 ml) was low passage, stock virus, prepared from frozen fecal samples (sixth calf-passage) by: (a) thawing and diluting in ten volumes of PBS, (b) low speed (3,000 X g) and high speed (55,000 X g) centrifugation, (c) resuspending the virus pellet in PBS and sonicating, (d) diluting the suspension in PBS to give a concentration of virus 10 to 20 times that of the original fecal sample, (e) clarifying by low speed centrifugation (3,000 X g), (f) filtering the supernatant through a 0.45 ,u millipore filter and (g) storing at -70°C. The BFI cultures were then examined
daily for three days, at which time they frozen and thawed three times and 0.5 ml used to inoculate fresh BFI monolayers. This procedure was then repeated to give a total of three passages of the virus in BFI cells. This low, calf-passage rotavirus, in comparison with the vaccine virus, produced a lesser CPE with positive fluorescence in 48 to 72 hours in first passage in BFI cells. The CPE and fluorescence was further decreased in the second passage and in the third passage only a few fluorescing cells, (without a CPE) were seen. were
VETERINARY PATHOLOGIST Connaught Laboratories Limited requires a veterinary pathologist certified
or eligible for certification by the Board of the American College of Veterinary Pathologists. Responsibilities will include providing gross and histological interpretation on tissues of animals from safety testing of biologics both in process and at the final quality control level; functioning as a research team member in the development and evaluation of new products and monitoring of disease status of all animals used in the production and testing of the company's human and veterinary biological products. The position is available immediately. Salary negotiable. All enquiries should be directed to: Dr. A. Fletch, Director Animal Resources Division CONNAUGHT LABORATORIES LTD. 1755 Steeles Avenue West Willowdale, Ontario M2N 5T8 Telephone: (416) 667-2870
Volume 41
-
April, 1977
229
