Diagnosis of Viral Agents Associated with Neonatal Calf Diarrhea G. Marsolois, R. Assaf, C. Montpetit and P. Marois* ABSTRACT During this study, 134 samples have been examined for the detection of the viruses associated with neonatal calf diarrhea. The presence of Nebraska viruses (rotavirus and coronavirus) has been demonstrated by using the electron microscope and the fluorescent antibody techniques while the presence of other viruses has been detected by the observation of a cytopathic effect on monolayer cells of calf testis. The Nebraska viruses have been demonstrated in 107 (80%) out of 134 field case specimens. An association of rotaviruses and coronaviruses was found in 58 cases (54%) whilst the coronaviruses and the rotavirus were found singly in 34 cases (53%) and in 15 cases (14%) respectively. Four bovine virus diarrhea viruses, two infectious bovine rhinotracheitis viruses and two enteroviruses have also been isolated in the preceding 107 Nebraska positive specimens. For the detection of the Nebraska viruses, the fluorescent antibody techniques were more sensitive than the electron microscopy. However, those two techniques must be used simultaneously for a better detection of a greatest possible number of cases.
RASUME Cette etude portait sur l'examen de 134 echantillons et visait a deceler les virus impliques dans la diarrhee neo-natale du veau. On demontra la presence des virus du Nebraska (rotavirus et coronavirus) a I'aide de la *Ministere
1'Agriculture
de du Quebec, Centre de recherche en medecine veterinaire, Institut Armand-Frappier, Case Postale 100, Ville de Laval, Quebec H7N 4Z3 (Marsolais, Assaf et Montpetit) et Centre de recherche en medecine vetirinaire, Institut Armand-Frappier, Case Postale 100, Ville de Laval, Quebec H7N 4Z3 (Marois). This research was supported by grant IMH 74-586 Conseil des recherches et services agricoles du Quebec. Submitted June 24, 1977.
168
microscopie electronique et de l'immunofluorescence; la production d'un effet cytopathogene sur feuillet monocellulaire de testicules de veau permit par ailleurs de deceler les autres virus. On demontra la presence des virus du Nebraska dans 107 (80%) des 134 echantillons provenant de cas cliniques. On constata la presence simultanee des virus rota et corona dans 58 eehantillons (54%); on recouvra par ailleurs seulement des corona ou des rotavirus dans respectivement 34 (53%) et 15 (14%) cas. Parmi les 107 echantillons positifs prce'dents, quatre recelaient aussi le virus de la diarrhee a virus bovine, deux, celui de la rhino-tracheite infectieuse bovine, et deux, un enterovirus. La technique de l'immunofluorescence s'est averee plus efficace que la microscopique electronique, pour la detection des virus du Nebraska. I1 convient cependant d'utiliser simultanement ces deux techniques, afin d'ameliorer les chances de deceler les virus dans les echantillons positifs.
INTRODUCTION Acute neonatal calf diarrhea (NCD) is a disease associated with the presence of various agents, i.e. bacteria, viruses and parasites acting individually or in association (9). Mebus et al reported the presence of a reovirus-like agent (3, 7, 12) and of a coronavirus-like agent (8, 11) from affected calves and has focused the attention on the role of viruses implicated in this pathological condition. The cultivation of these viruses is rather difficult but their presence can however be demonstrated from infected specimens by using the electron microscopy (EM) and fluorescent antibody techniques (FAT) (2, 5, 6). This investigation has been undertaken in order to find the variety of viruses which
Can. J. comp. Med.
could be associated with field cases of NCD and also to compare the reliability of the two previous techniques for the detection of rotaviruses (4) and coronaviruses (10).
MATERIALS AND METHODS SPECIMENS
Diarrheic fecal samples were collected directly from calves within the first five to six hours after the onset of diarrhea. Segments of the middle and lower areas of jejunum were removed from the calves sacrificed within two days after the appearance of diarrhea. All specimens were frozen, shipped to the laboratory on dry ice and stored at -70°C. Upon arrival, a segment of the intestine was kept frozen for future detection of coronavirueses by FAT. Epithelial smears prepared from another sergment of intestine were set aside for the detection of rotaviruses by the same technique mentioned above. Intestinal epithelium was also collected for the detection of viruses by EM and for isolation of viruses in tissue culture. This simplified technique was chosen in order to facilitate routine EM examinations of field cases. ELECTRON MICROSCOPY
The fecal material and intestinal epithelium were suspended in two or three parts of physiological saline and homogenized by pipetting. After five to ten minutes of sedimentation, a small part of the supernatant was taken and two grids were prepared for negative contrast electron microscopy. FLUORESCENT ANTIBODY TECHNIQUE
Frozen sections of 6 p from the jejunum were placed on slides previously coated with 1% gelatine, then dried for 15 minutes. These sections as well as the fecal smears were then fixed in acetone at -20°C for ten minutes, rinsed in a phosphate buffer at pH 7.2, dried and subse-
Volume 42
-
April, 1978
quently covered with the monospecific labeled antibodies'. They were then incubated for 30 minutes in a moist chamber, rinsed three times in the same buffer, dried, mounted in a buffered glycerol solution and then examined using ultraviolet illumination. VIRUS ISOLATION
Virus isolation was attempted from intestinal scrapings suspended 1/10 in a mixture of Earle's salt solution containing 1.3% lactalbumine hydrolysate, 600 units of penicillin and 300 ,ug of streptomycin. Ten 11 x 150 mm round tubes and five Leighton tubes containing confluent primary or secondary fetal calf testis monolayer cells were inoculated with 0.2 ml of a suspension of each sample. After two hours incubation at 37°C the inoculum was removed, the cells rinsed three times with P.B.S. and a maintenance medium of Earle's balanced salt solution supplemented with 2% fetal calf serum was added. The cultures were maintained at 37°C for a week and examined daily for the presence of cytopathic effects (CPE). A second passage was made in the absence of CPE. Fluid from each tube having CPE was examined by EM for a preliminary identification of the virus and then followed by appropriate serological tests for a final identification. The cell cultures in Leighton tubes were also incubated for four to five days. The coverslips were then removed, fixed in acetone at -20°C, stained with bovine virus diarrhea (BVD) conjugate2 and examined to detect the presence of noncytopathic strain of this virus.
RESULTS Out of the 134 cases submitted, materials from 111 samples were examined by EM and FAT while 23 other samples were examined by EM only. iRotavirus
and coronavirus conjugates, Norden Laboratories, Lincoln, Nebraska 68501. 2Colorado Serum Company Laboratories, 4956 York Street, Denver, Colorado 80216.
Fig. 1. Electron photomicrograph of rotavirus particules from fecal material of neonatal calf diarrhea. Bar = 100 nm.
virus was the only virus identified in the 33 remaining cases. Bovine virus diarrhea virus was isolated from four specimens, infectious bovine rhinotracheitis virus (IBR) from two specimens and enterovirus from two specimens. One or more of these viruses were found associated with a rotavirus and/or coronavirus infection. In Table II, comparative FAT and EM tests applied for the detection of rotaviruses show that there was agreement of results in 27 cases. Fourteen other viruses were detected only by EM and 32 other by FAT. It was also found that the FAT and EM technique when used for the detection of the coronaviruses were in agreement in 52 cases. However, EM permitted the detection of this virus in three cases which were not observed by FAT whilst the FAT detected infection b,y this virus in 14 cases diagnosed by EM. Furthermore, coronaviruses were seen by EM examination in the 23 cases where intestinal sections were not available.
Fig. 2. Electron photomicrograph of coronavirus particules from fecal material of neonatal calf diarrhea.
DISCUSSION
The simplified EM technique used in this work enabled rapid detection of the rotavirus and coronavirus (Figs. 1 and 2). Viruses were demonstrated in 107 cases and results are summarized in Table I. Rotavirus and coronavirus were found in association in 52 cases. The rotavirus occurred alone in 15 cases while the corona-
Isolation of the virus in cell culture remains the ultimate proof of its association with disease. However, since this technique is time consuming, expensive and also tedious for the routine detection of neonatal calf diarrhea viruses (NDCV) some other techniques should be used for laboratory diagnosis or confirmation. In this work EM along with FAT has been used because of their relative simplicity, rapidity and also to compare their efficiency when used in routine examination of pathological specimens. The techniques used in this work have been purposely simplified in order to decrease the time and expenses involved in the detection of NCDV. The demonstration of coronaviruses and rotaviruses in 80% of NCD substantiates the use of these two techniques. These two agents were found in association in 54% of the cases whilst the coronaviruses and the rotaviruses were found singly in 32% and 14% respectively.
Bar = 100 nm.
TABLE I. Viruses Detected from 134 Field Cases of Neonatal Calf Diarrhea
Rotarivus and coronavirus.............. Coronavirus......................... Rotavirus............................ Rotarivus, coronavirus and togavirusa.... Rotavirus, coronavirus and enterovirus... Rotavirus, coronavirus and herpesvirus. . Rotavirus, coronavirus, togavirus from the gut and herpesvirus from the liver Coronavirus and togavirus.............. Total of positives cases................. aBovine virus diarrhea
170
Can. J. comp. Med.
TABLE II. Comparative Results of Electron Microscopy and Fluorescent Antibody Tests for the Detection of Nebraska Calf Diarrhea Viruses Rotavirus Fluorescent Number Antibody Test of Cases 27 + 14 32 + - 61 Total 134 aNE = not examined
Electron Microscopy + +
From the results obtained it was observed that FAT could not detect the presence of rotaviruses from 14 cases and the coronaviruses in three cases. Similarly, the EM did not detect the presence of rotaviruses in 32 cases and of coronaviruses in 14 cases which were observed by the use of FAT. From this data it is clear that either technique when used alone is not as efficient as when they are applied simultaneously. Consequently, these two techniques should be used to obtain a reliable diagnosis of the viral nature of NCD.
Recent developments in EM techniques could improve the results obtained with this method of examination. These improvements consist of concentration of the virus particles in the fecal material (2, 5) and immunoelectromicroscopy (1, 6). These techniques are now used in our laboratory in order to determine their comparative efficiency for the detection of the NCDV.
This investigation gives also some enlightenment on the association of other viruses such as IBR, BVD and enteroviruses with calf rotavirus and coronavirus. These other viruses were involved in 7% of the cases and were always found in association with the rotavirus and/or the coronavirus. The importance of these agents in the aetiology of NCD does not appear to be important, however, one can foresee that their presence could be responsible for the failure of the vaccines to protect all animals.
Volume 42 - April, 1978
Coronavirus Electron Fluorescent Microscopy Antibody Test
+ + +
+ + NEa
Total
-
Number of Cases 52 3 14 23 42 134
ACKNOWLEDGMENTS The authors wish to thank Misses R. Jette, G. Godin and Mr. S. Leclerc for their excellent technical assistance. REFERENCES 1. BRIDGER, J. C. and G. N. WOODE. Neonatal calf diarrhea: Identification of a reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy. Br. vet. J. 131: 528-535. 1975. 2. ENGLAND, J. J., C. S. FRYE and E. A. ENRIGHT. Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea. Cornell Vet. 66: 172-181. 1976. 3. FERNELIUS, A. L., A. E. RITCHIE, L. G. CLASSICK, 0. J. 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. 4. FLEWETT, T. H., A. S. BRYDEN and H. DAVIS. Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet 2: 61-63. 1974. 5. LABORATORY METHODS FOR DETECTING CALF DIARRHEA VIRUS. Norden Laboratories, Lincoln, Nebraska. 1973. 6. McNULTY, M. S., G. M. ALLAN, W. L. CURRAN and J. B. McFERRAN. Comparison of methods for diagnosis of rotavirus infection of calves. Vet. Rec. 98: 463-464. 1976. 7. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. H. TWIEHAUS. Calf diarrhea (scours): reproduces with a virus from a field outbreak. Univ. Nebr. Agric. Exp. Stn. Res. Bul. 233: 1.16. 1969. 8. MEBUS, C. A., E. L. STAIR, M. B. RHODES and M. J. TWIEHAUS. Neonatal calf diarrhea: propagation, attenuation and characteristics of coronavirus-like agent. Am. J. vet. Res. 34: 145-150. 1973. 9. MORIN, M., S. LARIVIkRE and R. LALLIER. Pathological and microbiological observations made on spontaneous e'ases of acute neonatal calf diarrhea. Can. J. comp. Med. 40: 228-240. 1976. 10. SHARPEE, R. L., C. A. MEBUS and E. P. BASS. Characterization of a calf diarrheal coronavirus. Am. J. vet. Res. 37: 1031-1041. 1976. 11. STAIR, E. L., M. B. RHODES, R. G. WHITE and C. A. MEBUS. Neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent. Am. J. vet. Res. 33: 1147-1156. 1972. 12. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 195-202. 1971.
171
RASUME Cette etude portait sur l'examen de 134 echantillons et visait a deceler les virus impliques dans la diarrhee neo-natale du veau. On demontra la presence des virus du Nebraska (rotavirus et coronavirus) a I'aide de la *Ministere
1'Agriculture
de du Quebec, Centre de recherche en medecine veterinaire, Institut Armand-Frappier, Case Postale 100, Ville de Laval, Quebec H7N 4Z3 (Marsolais, Assaf et Montpetit) et Centre de recherche en medecine vetirinaire, Institut Armand-Frappier, Case Postale 100, Ville de Laval, Quebec H7N 4Z3 (Marois). This research was supported by grant IMH 74-586 Conseil des recherches et services agricoles du Quebec. Submitted June 24, 1977.
168
microscopie electronique et de l'immunofluorescence; la production d'un effet cytopathogene sur feuillet monocellulaire de testicules de veau permit par ailleurs de deceler les autres virus. On demontra la presence des virus du Nebraska dans 107 (80%) des 134 echantillons provenant de cas cliniques. On constata la presence simultanee des virus rota et corona dans 58 eehantillons (54%); on recouvra par ailleurs seulement des corona ou des rotavirus dans respectivement 34 (53%) et 15 (14%) cas. Parmi les 107 echantillons positifs prce'dents, quatre recelaient aussi le virus de la diarrhee a virus bovine, deux, celui de la rhino-tracheite infectieuse bovine, et deux, un enterovirus. La technique de l'immunofluorescence s'est averee plus efficace que la microscopique electronique, pour la detection des virus du Nebraska. I1 convient cependant d'utiliser simultanement ces deux techniques, afin d'ameliorer les chances de deceler les virus dans les echantillons positifs.
INTRODUCTION Acute neonatal calf diarrhea (NCD) is a disease associated with the presence of various agents, i.e. bacteria, viruses and parasites acting individually or in association (9). Mebus et al reported the presence of a reovirus-like agent (3, 7, 12) and of a coronavirus-like agent (8, 11) from affected calves and has focused the attention on the role of viruses implicated in this pathological condition. The cultivation of these viruses is rather difficult but their presence can however be demonstrated from infected specimens by using the electron microscopy (EM) and fluorescent antibody techniques (FAT) (2, 5, 6). This investigation has been undertaken in order to find the variety of viruses which
Can. J. comp. Med.
could be associated with field cases of NCD and also to compare the reliability of the two previous techniques for the detection of rotaviruses (4) and coronaviruses (10).
MATERIALS AND METHODS SPECIMENS
Diarrheic fecal samples were collected directly from calves within the first five to six hours after the onset of diarrhea. Segments of the middle and lower areas of jejunum were removed from the calves sacrificed within two days after the appearance of diarrhea. All specimens were frozen, shipped to the laboratory on dry ice and stored at -70°C. Upon arrival, a segment of the intestine was kept frozen for future detection of coronavirueses by FAT. Epithelial smears prepared from another sergment of intestine were set aside for the detection of rotaviruses by the same technique mentioned above. Intestinal epithelium was also collected for the detection of viruses by EM and for isolation of viruses in tissue culture. This simplified technique was chosen in order to facilitate routine EM examinations of field cases. ELECTRON MICROSCOPY
The fecal material and intestinal epithelium were suspended in two or three parts of physiological saline and homogenized by pipetting. After five to ten minutes of sedimentation, a small part of the supernatant was taken and two grids were prepared for negative contrast electron microscopy. FLUORESCENT ANTIBODY TECHNIQUE
Frozen sections of 6 p from the jejunum were placed on slides previously coated with 1% gelatine, then dried for 15 minutes. These sections as well as the fecal smears were then fixed in acetone at -20°C for ten minutes, rinsed in a phosphate buffer at pH 7.2, dried and subse-
Volume 42
-
April, 1978
quently covered with the monospecific labeled antibodies'. They were then incubated for 30 minutes in a moist chamber, rinsed three times in the same buffer, dried, mounted in a buffered glycerol solution and then examined using ultraviolet illumination. VIRUS ISOLATION
Virus isolation was attempted from intestinal scrapings suspended 1/10 in a mixture of Earle's salt solution containing 1.3% lactalbumine hydrolysate, 600 units of penicillin and 300 ,ug of streptomycin. Ten 11 x 150 mm round tubes and five Leighton tubes containing confluent primary or secondary fetal calf testis monolayer cells were inoculated with 0.2 ml of a suspension of each sample. After two hours incubation at 37°C the inoculum was removed, the cells rinsed three times with P.B.S. and a maintenance medium of Earle's balanced salt solution supplemented with 2% fetal calf serum was added. The cultures were maintained at 37°C for a week and examined daily for the presence of cytopathic effects (CPE). A second passage was made in the absence of CPE. Fluid from each tube having CPE was examined by EM for a preliminary identification of the virus and then followed by appropriate serological tests for a final identification. The cell cultures in Leighton tubes were also incubated for four to five days. The coverslips were then removed, fixed in acetone at -20°C, stained with bovine virus diarrhea (BVD) conjugate2 and examined to detect the presence of noncytopathic strain of this virus.
RESULTS Out of the 134 cases submitted, materials from 111 samples were examined by EM and FAT while 23 other samples were examined by EM only. iRotavirus
and coronavirus conjugates, Norden Laboratories, Lincoln, Nebraska 68501. 2Colorado Serum Company Laboratories, 4956 York Street, Denver, Colorado 80216.
Fig. 1. Electron photomicrograph of rotavirus particules from fecal material of neonatal calf diarrhea. Bar = 100 nm.
virus was the only virus identified in the 33 remaining cases. Bovine virus diarrhea virus was isolated from four specimens, infectious bovine rhinotracheitis virus (IBR) from two specimens and enterovirus from two specimens. One or more of these viruses were found associated with a rotavirus and/or coronavirus infection. In Table II, comparative FAT and EM tests applied for the detection of rotaviruses show that there was agreement of results in 27 cases. Fourteen other viruses were detected only by EM and 32 other by FAT. It was also found that the FAT and EM technique when used for the detection of the coronaviruses were in agreement in 52 cases. However, EM permitted the detection of this virus in three cases which were not observed by FAT whilst the FAT detected infection b,y this virus in 14 cases diagnosed by EM. Furthermore, coronaviruses were seen by EM examination in the 23 cases where intestinal sections were not available.
Fig. 2. Electron photomicrograph of coronavirus particules from fecal material of neonatal calf diarrhea.
DISCUSSION
The simplified EM technique used in this work enabled rapid detection of the rotavirus and coronavirus (Figs. 1 and 2). Viruses were demonstrated in 107 cases and results are summarized in Table I. Rotavirus and coronavirus were found in association in 52 cases. The rotavirus occurred alone in 15 cases while the corona-
Isolation of the virus in cell culture remains the ultimate proof of its association with disease. However, since this technique is time consuming, expensive and also tedious for the routine detection of neonatal calf diarrhea viruses (NDCV) some other techniques should be used for laboratory diagnosis or confirmation. In this work EM along with FAT has been used because of their relative simplicity, rapidity and also to compare their efficiency when used in routine examination of pathological specimens. The techniques used in this work have been purposely simplified in order to decrease the time and expenses involved in the detection of NCDV. The demonstration of coronaviruses and rotaviruses in 80% of NCD substantiates the use of these two techniques. These two agents were found in association in 54% of the cases whilst the coronaviruses and the rotaviruses were found singly in 32% and 14% respectively.
Bar = 100 nm.
TABLE I. Viruses Detected from 134 Field Cases of Neonatal Calf Diarrhea
Rotarivus and coronavirus.............. Coronavirus......................... Rotavirus............................ Rotarivus, coronavirus and togavirusa.... Rotavirus, coronavirus and enterovirus... Rotavirus, coronavirus and herpesvirus. . Rotavirus, coronavirus, togavirus from the gut and herpesvirus from the liver Coronavirus and togavirus.............. Total of positives cases................. aBovine virus diarrhea
170
Can. J. comp. Med.
TABLE II. Comparative Results of Electron Microscopy and Fluorescent Antibody Tests for the Detection of Nebraska Calf Diarrhea Viruses Rotavirus Fluorescent Number Antibody Test of Cases 27 + 14 32 + - 61 Total 134 aNE = not examined
Electron Microscopy + +
From the results obtained it was observed that FAT could not detect the presence of rotaviruses from 14 cases and the coronaviruses in three cases. Similarly, the EM did not detect the presence of rotaviruses in 32 cases and of coronaviruses in 14 cases which were observed by the use of FAT. From this data it is clear that either technique when used alone is not as efficient as when they are applied simultaneously. Consequently, these two techniques should be used to obtain a reliable diagnosis of the viral nature of NCD.
Recent developments in EM techniques could improve the results obtained with this method of examination. These improvements consist of concentration of the virus particles in the fecal material (2, 5) and immunoelectromicroscopy (1, 6). These techniques are now used in our laboratory in order to determine their comparative efficiency for the detection of the NCDV.
This investigation gives also some enlightenment on the association of other viruses such as IBR, BVD and enteroviruses with calf rotavirus and coronavirus. These other viruses were involved in 7% of the cases and were always found in association with the rotavirus and/or the coronavirus. The importance of these agents in the aetiology of NCD does not appear to be important, however, one can foresee that their presence could be responsible for the failure of the vaccines to protect all animals.
Volume 42 - April, 1978
Coronavirus Electron Fluorescent Microscopy Antibody Test
+ + +
+ + NEa
Total
-
Number of Cases 52 3 14 23 42 134
ACKNOWLEDGMENTS The authors wish to thank Misses R. Jette, G. Godin and Mr. S. Leclerc for their excellent technical assistance. REFERENCES 1. BRIDGER, J. C. and G. N. WOODE. Neonatal calf diarrhea: Identification of a reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy. Br. vet. J. 131: 528-535. 1975. 2. ENGLAND, J. J., C. S. FRYE and E. A. ENRIGHT. Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea. Cornell Vet. 66: 172-181. 1976. 3. FERNELIUS, A. L., A. E. RITCHIE, L. G. CLASSICK, 0. J. 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. 4. FLEWETT, T. H., A. S. BRYDEN and H. DAVIS. Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet 2: 61-63. 1974. 5. LABORATORY METHODS FOR DETECTING CALF DIARRHEA VIRUS. Norden Laboratories, Lincoln, Nebraska. 1973. 6. McNULTY, M. S., G. M. ALLAN, W. L. CURRAN and J. B. McFERRAN. Comparison of methods for diagnosis of rotavirus infection of calves. Vet. Rec. 98: 463-464. 1976. 7. MEBUS, C. A., N. R. UNDERDAHL, M. B. RHODES and M. H. TWIEHAUS. Calf diarrhea (scours): reproduces with a virus from a field outbreak. Univ. Nebr. Agric. Exp. Stn. Res. Bul. 233: 1.16. 1969. 8. MEBUS, C. A., E. L. STAIR, M. B. RHODES and M. J. TWIEHAUS. Neonatal calf diarrhea: propagation, attenuation and characteristics of coronavirus-like agent. Am. J. vet. Res. 34: 145-150. 1973. 9. MORIN, M., S. LARIVIkRE and R. LALLIER. Pathological and microbiological observations made on spontaneous e'ases of acute neonatal calf diarrhea. Can. J. comp. Med. 40: 228-240. 1976. 10. SHARPEE, R. L., C. A. MEBUS and E. P. BASS. Characterization of a calf diarrheal coronavirus. Am. J. vet. Res. 37: 1031-1041. 1976. 11. STAIR, E. L., M. B. RHODES, R. G. WHITE and C. A. MEBUS. Neonatal calf diarrhea: purification and electron microscopy of a coronavirus-like agent. Am. J. vet. Res. 33: 1147-1156. 1972. 12. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 195-202. 1971.
171
