NOTES
Use of Electron Microscopy and an Enzyme-linked Immunosorbent Assay for the Detection of Rotaviruses in Neonatal Calf Diarrhea P. Payment, G. Marsolais, M. Trudel, M. Fauvel, L. Lamontagne, R. Assaf and P. Marois* ABSTRACT Electron microscopy and an enzyme-linked immunosorbent assay were compared for the diagnosis of rotaviruses associated with neonatal calf diarrhea. One hundred percent correlation was observed when 125 samples were tested by both techniques. Both techniques were equally efficient in detecting rotaviruses. The enzyme-linked immunosorbent assay was more sensitive but being specific could not detect other viruses.
RESUME Cette experience consistait a comparer l'efficacite de l'immuno-electromicroscopie a celle d'une epreuve immuno-enzymatique, pour la demonstration des rotavirus associes a la diarrhee neo-natale du veau. L'examen de 125 echantillons, par ces deux techniques, revela qu'elles possedaient la meme efficacite pour la mise en evidence des rotavirus. En depit d'une sensibilite beaucoup plus grande, la technique immuno-enzymatique ne permit pas de deceler plus de rotavirus que l'immuno-electromicroscopie; sa grande specificite l'empecha en outre de detecter les autres virus. *Centre de recherche en virologie (Payment, Trudel, Fauvel) et Ministere de l'Agriculture, Centre de recherche en medecine veterinaire (Marsolais, Lamontagne, Assaf, Marois), Institut Armand-Frappier, C.P. 100, Ville de Laval, Quebec H7N 4Z3. Submitted October 4, 1978.
328
The enzyme-linked immunosorbent assay (ELISA) has been described as a very sensitive and specific assay (3) and used to detect rotaviruses in human (8) or animal (2) stools. Recently (5) we reported a comparison between immunofluorescence (IF) and electron-microscopy (EM) and concluded that both tests had to be used to detect a larger number of positive cases. As we recently have used the more sensitive technique of immune-electron microscopy (IEM) we also developed ELISA for rotavirus and compared both techniques. Fecal samples and intestinal samples were described earlier (5). Immune-electron-microscopy (IEM) (1) was done using bovine or rabbit antisera. For ELISA, specific antisera were prepared in rabbits and guinea pigs using bovine rotavirus grown in tissue culture and purified in density gradient
(4).
The ELISA test was performed as described by Ellens and de Leeuw (2). The specific rabbit gamma globulin at 5 jug/ml were used to coat the polystyrene plates. The guinea pig specific sera and a nonimmune serum were used at 1:1000 to detect the bound antigens. The bound guinea pig IgG were detected using a commercial conjugate (peroxidase conjugated goat IgG anti-guinea pig IgG).' The substrate was 5-amino-salicylic acid at 2 mg/ml, pH 6.0 and 0.005% hydrogen peroxide. After 60 min at room temperature the reactions were graduated visually from negative to 4. 1Cat. no. 61-208-1, Miles Laboratories, Elkhart, Indiana.
Can. J. comp. Med.
Preliminary use of ELISA for diagnostic purposes involved the serial dilution of the fecal samples to be analysed (Fig. 1). Positive reactions from positive samples were always strong and some stool samples reacted up to a dilution of 1:50,000. Further work was performed on only three dilutions. That is 1:20, 1:40 and 1:80. One hundred and twenty-five samples were tested under code by IEM and ELISA. The results are reported in Table I. All samples negative for rotavirus by IEM were also negative by ELISA. Nine samples, positive for rotavirus by ELISA contained rotavirus alone or with coronavirus or picornavirus by IEM. Two samples found positive by 20 40 80 20 40
80 160 320 640 12802560 5120
1~ 000qooOO 0 is0 000000 4 0@ OoQ00 0@
ELISA and negative by IEM were found positive upon retesting. The ELISA test is very specific and highly sensitive. However, high sensitivity does not result in a higher number of positive samples when compared with IEM and the presence of other viruses is not detected. When several viruses can be implicated as the etiological agent, as is the case in gastroenteritis (5, 6, 7), ELISA is not adequate. The very sensitive IEM technique using pooled sera as the source of multispecific antibodies is certainly much more efficient in detecting viruses in pathological sanrples. The ELISA test can be adequately used more properly in serology to test for specific antibodies and in some specific cases where only one virus is searched for.
2
ANIN @ 00oft, 0
3
5
6
P0s
ACKNOWLEDGMENTS The authors wish to acknowledge the skillful technical assistance of Celine Tremblay and the support of the Conseil des Recherches et Services agricoles du Quebec by their grant IAF-78-780.
NEG NON-IMMAUNE
IMMAUNE GUINEA PIG SERA 1:2000
Fig. 1. ELISA diagrnostic results with six bovine samples, a positive and a negative control. All samples were reacted against an immune and a nonimmune guinea pig serum to detect nonspecific reactions. The positive control and stools 2. 5 and 6 are positive. A slight nonapecific reaction is observed with sample 5 and the positive control.
TABLE I. Detection of Viruses in Stool or rissue Samples by Electron Microscopy and the ELISA-Rota Assay
Viruses Observed by EM None 2a/ 85
ELISA-Rota (No. Positive/No
Coronaviruses Rotaviruses Rotaviruses coronaviruses Rotaviruses picomnaviruses
Samplesa)
0 /31 6 /6 2/ 2 1 / 1 11 /125 -These samples upon reexamination were found positive by IEM
Volume 43 - July, 1979
REFERENCES 1. ANDERSON, N. and F. W. DOANE. Specific identification of enteroviruses by immunoelectron microscopy using a sei um-in-agar diffusion method. Can. J. Microbiol. 19: 585-589. 1973. 2. ELLENS D. J. and P. W. de LEEUW. Enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves. J. clin. Microbiol. 6: 530-532. 1977. 3. ENGVALL, E. Quantitative enzyme immunoassay (ELISA) in microbiology. Med. Virol. 55: 193-200. 1977. 4. FAUVEL, M., L. SPENCE, L. A. BABIUK, R. PETRO and S. BLOCH. Hemagglutination and hemagglutination-inhibition studies with a strain of nebraska calf diarrhea virus (bovine rotavirus). Intervirology 9: 95-105. 1978. 5. MARSOLAIS, G., R. ASSAF, C. MONTPETIT and P. MAROIS. Diagnosis of viral agents associated with neonatal calf diar rhea. Can. J. comp. Med. 42: 168171. 1978. 6. 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. 7. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 195202. 1971. 8. YOLKEN, R. H., H. W. KIM, T. CLEM, R. G. WYATT, R. M. CHANOCK, A. R. KALICA and A. Z. KAPIKIAN. Enzyme-linked immunosorbent assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis. Lancet 1: 263-267. 1977.
329
Use of Electron Microscopy and an Enzyme-linked Immunosorbent Assay for the Detection of Rotaviruses in Neonatal Calf Diarrhea P. Payment, G. Marsolais, M. Trudel, M. Fauvel, L. Lamontagne, R. Assaf and P. Marois* ABSTRACT Electron microscopy and an enzyme-linked immunosorbent assay were compared for the diagnosis of rotaviruses associated with neonatal calf diarrhea. One hundred percent correlation was observed when 125 samples were tested by both techniques. Both techniques were equally efficient in detecting rotaviruses. The enzyme-linked immunosorbent assay was more sensitive but being specific could not detect other viruses.
RESUME Cette experience consistait a comparer l'efficacite de l'immuno-electromicroscopie a celle d'une epreuve immuno-enzymatique, pour la demonstration des rotavirus associes a la diarrhee neo-natale du veau. L'examen de 125 echantillons, par ces deux techniques, revela qu'elles possedaient la meme efficacite pour la mise en evidence des rotavirus. En depit d'une sensibilite beaucoup plus grande, la technique immuno-enzymatique ne permit pas de deceler plus de rotavirus que l'immuno-electromicroscopie; sa grande specificite l'empecha en outre de detecter les autres virus. *Centre de recherche en virologie (Payment, Trudel, Fauvel) et Ministere de l'Agriculture, Centre de recherche en medecine veterinaire (Marsolais, Lamontagne, Assaf, Marois), Institut Armand-Frappier, C.P. 100, Ville de Laval, Quebec H7N 4Z3. Submitted October 4, 1978.
328
The enzyme-linked immunosorbent assay (ELISA) has been described as a very sensitive and specific assay (3) and used to detect rotaviruses in human (8) or animal (2) stools. Recently (5) we reported a comparison between immunofluorescence (IF) and electron-microscopy (EM) and concluded that both tests had to be used to detect a larger number of positive cases. As we recently have used the more sensitive technique of immune-electron microscopy (IEM) we also developed ELISA for rotavirus and compared both techniques. Fecal samples and intestinal samples were described earlier (5). Immune-electron-microscopy (IEM) (1) was done using bovine or rabbit antisera. For ELISA, specific antisera were prepared in rabbits and guinea pigs using bovine rotavirus grown in tissue culture and purified in density gradient
(4).
The ELISA test was performed as described by Ellens and de Leeuw (2). The specific rabbit gamma globulin at 5 jug/ml were used to coat the polystyrene plates. The guinea pig specific sera and a nonimmune serum were used at 1:1000 to detect the bound antigens. The bound guinea pig IgG were detected using a commercial conjugate (peroxidase conjugated goat IgG anti-guinea pig IgG).' The substrate was 5-amino-salicylic acid at 2 mg/ml, pH 6.0 and 0.005% hydrogen peroxide. After 60 min at room temperature the reactions were graduated visually from negative to 4. 1Cat. no. 61-208-1, Miles Laboratories, Elkhart, Indiana.
Can. J. comp. Med.
Preliminary use of ELISA for diagnostic purposes involved the serial dilution of the fecal samples to be analysed (Fig. 1). Positive reactions from positive samples were always strong and some stool samples reacted up to a dilution of 1:50,000. Further work was performed on only three dilutions. That is 1:20, 1:40 and 1:80. One hundred and twenty-five samples were tested under code by IEM and ELISA. The results are reported in Table I. All samples negative for rotavirus by IEM were also negative by ELISA. Nine samples, positive for rotavirus by ELISA contained rotavirus alone or with coronavirus or picornavirus by IEM. Two samples found positive by 20 40 80 20 40
80 160 320 640 12802560 5120
1~ 000qooOO 0 is0 000000 4 0@ OoQ00 0@
ELISA and negative by IEM were found positive upon retesting. The ELISA test is very specific and highly sensitive. However, high sensitivity does not result in a higher number of positive samples when compared with IEM and the presence of other viruses is not detected. When several viruses can be implicated as the etiological agent, as is the case in gastroenteritis (5, 6, 7), ELISA is not adequate. The very sensitive IEM technique using pooled sera as the source of multispecific antibodies is certainly much more efficient in detecting viruses in pathological sanrples. The ELISA test can be adequately used more properly in serology to test for specific antibodies and in some specific cases where only one virus is searched for.
2
ANIN @ 00oft, 0
3
5
6
P0s
ACKNOWLEDGMENTS The authors wish to acknowledge the skillful technical assistance of Celine Tremblay and the support of the Conseil des Recherches et Services agricoles du Quebec by their grant IAF-78-780.
NEG NON-IMMAUNE
IMMAUNE GUINEA PIG SERA 1:2000
Fig. 1. ELISA diagrnostic results with six bovine samples, a positive and a negative control. All samples were reacted against an immune and a nonimmune guinea pig serum to detect nonspecific reactions. The positive control and stools 2. 5 and 6 are positive. A slight nonapecific reaction is observed with sample 5 and the positive control.
TABLE I. Detection of Viruses in Stool or rissue Samples by Electron Microscopy and the ELISA-Rota Assay
Viruses Observed by EM None 2a/ 85
ELISA-Rota (No. Positive/No
Coronaviruses Rotaviruses Rotaviruses coronaviruses Rotaviruses picomnaviruses
Samplesa)
0 /31 6 /6 2/ 2 1 / 1 11 /125 -These samples upon reexamination were found positive by IEM
Volume 43 - July, 1979
REFERENCES 1. ANDERSON, N. and F. W. DOANE. Specific identification of enteroviruses by immunoelectron microscopy using a sei um-in-agar diffusion method. Can. J. Microbiol. 19: 585-589. 1973. 2. ELLENS D. J. and P. W. de LEEUW. Enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves. J. clin. Microbiol. 6: 530-532. 1977. 3. ENGVALL, E. Quantitative enzyme immunoassay (ELISA) in microbiology. Med. Virol. 55: 193-200. 1977. 4. FAUVEL, M., L. SPENCE, L. A. BABIUK, R. PETRO and S. BLOCH. Hemagglutination and hemagglutination-inhibition studies with a strain of nebraska calf diarrhea virus (bovine rotavirus). Intervirology 9: 95-105. 1978. 5. MARSOLAIS, G., R. ASSAF, C. MONTPETIT and P. MAROIS. Diagnosis of viral agents associated with neonatal calf diar rhea. Can. J. comp. Med. 42: 168171. 1978. 6. 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. 7. WELCH, A. B. Purification, morphology and partial characterization of a reovirus-like agent associated with neonatal calf diarrhea. Can. J. comp. Med. 35: 195202. 1971. 8. YOLKEN, R. H., H. W. KIM, T. CLEM, R. G. WYATT, R. M. CHANOCK, A. R. KALICA and A. Z. KAPIKIAN. Enzyme-linked immunosorbent assay (ELISA) for detection of human reovirus-like agent of infantile gastroenteritis. Lancet 1: 263-267. 1977.
329
