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Development of a syncytia inhibition assay for the detection of antibodies to bovine leukemia virus in naturally infected cattle; comparison with Western blot and agar gel immunodiffusion
Journal of Virological Methods 70 (1998) 177 – 182
Development of a syncytia inhibition assay for the detection of antibodies to bovine leukemia virus in naturally infected cattle; comparison with Western blot and agar gel immunodiffusion Michael Johnson a, Fred Rommel b, Jay Mone´ a,* b
a Department of Biology, Roddy Science Building, Millers6ille Uni6ersity, Millers6ille PA 17551, USA Commonwealth of Pennsyl6ania Department of Agriculture, Bureau of Veterinary Laboratories, 2305 N. Cameron St., Harrisburg PA 17110, USA
Received 17 June 1997; received in revised form 7 October 1997; accepted 10 October 1997
Abstract A syncytia inhibition assay (SIA) for the detection of antibodies to bovine leukemia virus is described. This test involves specific antibody-mediated inhibition of BLV-induced cytopathic effects in an indicator cell line. A total of 300 sera were screened commercially by agar gel immunodiffusion (AGID) and were then screened by Western blot and SIA. The new assay system provided results which were comparable to Western blot and AGID. The results obtained suggest that SIA may be more sensitive than either of the other two assay systems examined for the determination of the infection status of cattle. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Bovine leukemia virus; Syncytia inhibition assay; Agar gel immunodiffusion; Western blot
1. Introduction Bovine leukemia virus (BLV) is an oncogenic retrovirus which is the causative agent of bovine leukemia/lymphoma in a significant minority of infected cows (Schwartz and Levy, 1994). Cattle
* Corresponding author. Fax: +1 717 8723985.
in the US are regularly screened for antibodies to BLV and many foreign countries require imported cattle to be free of BLV infection, making determination of infection status of considerable economic importance. A recent study by the USDA estimated that 89% of dairy operations in the US contain BLV-positive animals; in the majority of affected herds, seroprevalence was \ 25%, with some herds approaching 100% (NAHMS, 1996).
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 1 8 6 - 9
178
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
The most common commercial assay system for BLV is agar gel immunodiffusion (AGID); however, many alternative assay systems are currently being examined for possible advantages over AGID (Naif et al., 1992; Kelly et al., 1993; Nguyen and Maes, 1993). It has been known for some time that BLV is capable of causing syncytia formation in many indicator cell lines (Benton et al., 1978; Ferrer et al., 1981; Bobakova et al., 1985), but no attempt had been made to examine the usefulness of syncytia inhibition to determine infection status of cows. In this study, it was found that fetal lamb kidney (FLK) cells, which are persistently infected with BLV, are capable of inducing syncytia formation in the human B-lymphoblastoid cell line, Raji, following co-cultivation. Furthermore, it was found that pre-incubation of FLK cells with sera obtained from cows infected with BLV, but not from BLV-negative cows, would effectively inhibit syncytia formation, providing the basis for a new way to screen cows for BLV infection. This syncytia inhibition assay (SIA) was used to screen 300 bovine serum samples. The sera were also screened by Western blot and agar gel immunodiffusion and the results from these three assays were compared. The results obtained by syncytia inhibition assay were comparable to those obtained by AGID and Western blot. Syncytia inhibition assay was found to be a reliable, simple and efficient means by which to screen livestock for infection with BLV.
2.2. Agar gel immunodiffusion assay Commercial AGID kits (Symbiotics, San Diego, CA) were used to screen sera for anti-BLV antibodies according to the manufacturers instructions. All AGID assays were performed at the veterinary laboratory of the Commonwealth of Pennsylvania Department of Agriculture.
2.3. Cells All media and supplements, with the exception of fetal bovine serum, were obtained from GibcoBRL, Gaithersburg, MD. Fetal bovine serum was obtained from Sigma, St. Louis, MO. Fetal lamb kidney (FLK) cells were a generous gift from Janice Miller, United States Department of Agriculture, Ames, IA and were cultured in MEM supplemented with penicillin–streptomycin (100 units penicillin, 0.1 mg streptomycin per ml), 1 mM sodium pyruvate, 10 mM HEPES, 0.25% w/v lactalbumin hydrolysate, 0.22% w/v sodium bicarbonate and 5% v/v heat-inactivated fetal bovine serum. Raji cells were a generous gift from Dr Jonathan S. Allan, Southwest Foundation for Biomedical Research, San Antonio, TX and were maintained in RPMI supplemented with penicillin–streptomycin (100 units penicillin, 0.1 mg streptomycin per ml), 1 mM sodium pyruvate, 10 mM HEPES, 0.22% w/v sodium bicarbonate and 10% v/v heat-inactivated fetal bovine serum.
2.4. Western blot assay 2. Materials and methods
2.1. Sera A total of 300 sera were obtained from the Commonwealth of Pennsylvania Department of Agriculture, Harrisburg, PA. The sera were stored frozen at − 80°C until assayed. These serum samples had been collected to be screened for antiBLV antibodies by AGID. After commercial screening, the sera were coded and assayed by Western blot and SIA as described below.
FLK cells were grown to confluency in 75 cm2 tissue culture flasks and lysed by freeze–thawing three times; the contents of the flask were then diluted with an equal volume of 2× reducing sample buffer (0.125 M Tris–HCl, 4% SDS, 20% glycerol, 10% b-mercaptoethanol). The proteins were boiled for 1 min and subjected to discontinuous SDS polyacrylamide gel electrophoresis. Following electrophoresis, the proteins were passively transferred to 0.45 mm nitrocellulose membranes (Micron Separations, Westborough,
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
MA) in a transfer buffer consisting of 10 mM Tris base, 10 mM EDTA and 50 mM NaCl, pH 7.0 at 25°C. Following transfer, the membranes were blocked with 5% non-fat dry milk in phosphate buffered saline (PBS) for 1 h at 37°C, rinsed twice in PBS Tween and stored at − 20oC until used. The membranes were cut into strips and probed with 2 ml of a 1:40 dilution of sera in PBS with 5% non-fat dry milk, 5% FBS and 0.2% Tween 20 for 2 h at 37°C. The strips were washed extensively with PBS+0.2% Tween 20, followed by the addition of biotinylated antibovine IgG (Sigma) diluted in PBS Tween. After 1 h of incubation at 37°C, the strips were washed and reacted with streptavidin-HRP (Sigma) for 45 min at room temperature. After extensive washing, the bands were visualized using diaminobenzidine and H2O2. All reactivity was compared to known positive and negative controls kindly provided by Janice Miller, USDA, Ames, IA.
179
3. Results
3.1. Agar gel immunodiffusion compared with Western blotting Western blot analysis has been demonstrated to be a sensitive method for the detection of antibodies against BLV and has been shown to correlate well with AGID and ELISA assays for diagnosis of BLV in cattle (Kittelberger et al., 1996). For this reason, serum samples were first analyzed by Western blot to determine the presence or absence of anti-BLV antibodies for correlation with the commercial AGID assay. Western blot analysis detected antibodies consistently against two viral proteins designated p15 and p24 (Fig. 1). Previous reports identified p15 and p24 as products of the viral gag gene
2.5. Syncytia inhibition assay Syncytia inhibition assays were carried out in sterile 96-well tissue culture dishes. The following were added to each well of a tissue culture dish: 100 ml of MEM containing 5×104 FLK cells and 50 ml of the sera to be tested. The cells were incubated at room temperature for 30 min before the addition of 100 ml of media containing 5 × 104 Raji cells. The plates were then incubated for 12–16 h, until syncytia were apparent in wells containing FLK and Raji cells with negative control serum. The assay was scored as positive for anti-BLV antibodies if no syncytia were evident and negative if any syncytia were observed. The plates were then incubated for an additional 24 h and re-evaluated.
2.6. Data analysis Calculations to determine test sensitivity and specificity were carried out as previously described (Monke et al., 1992).
Fig. 1. Western blot assay for detection of anti-BLV antibodies in bovine sera. FLK cells were used as the source of BLV antigens. Lane 1, negative control; lane 2, positive control; lanes 3 – 6, bovine sera displaying reactivity against p15, p24 and gp51.
180
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
(Coffin, 1996). Other investigators reported that p24 reactivity was the most reliable marker for determining infection status when compared to commercial assay systems (Kittelberger et al., 1996); we also found that p24 was a reliable indicator of BLV infection, although p15 invariably stained with greater intensity. In the present study, because antibodies to p15 and p24 were consistently detected together, sera displaying reactivity against these two proteins were scored positive. Most sera containing reactivity against p15 and p24 also reacted with the env gene product gp51; however, the intensity of staining of gp51 was inconsistent and was thereby deemed unreliable as a marker for BLV infection. A total of 300 sera were screened by Western blot and agar gel immunodiffusion. The Western blot results were analyzed using the data obtained by AGID as the standard. The agar gel immunodiffusion assay scored 98 animals as being positive for infection with BLV, of these, 93 were positive by Western blot. Conversely, of the 202 animals diagnosed as BLV negative by AGID, 195 were also negative by Western blot. The Western blot technique described therefore, returned five false negative results and seven false positive results as compared to AGID; based on these data, the sensitivity and specificity of this test were calculated at 95 and 96.5%, respectively (Table 1).
3.2. Syncytia inhibition assay compared with Western blotting Co-cultivation of BLV-producing FLK cells with Raji cells induced syncytia, usually within 12 h, Fig. 2a. Syncytia induction was blocked by antibodies against BLV, Fig. 2b. It is also worthy to note the finding that EDTA effectively blocked syncytia formation regardless of the presence or absence of anti-BLV antibodies. This was discovered through side-by-side analysis of duplicate serum samples taken from several cows in both EDTA and heparin tubes. The sera with heparin as the anticoagulant yielded results comparable to AGID, whereas all of the sera containing EDTA were able to block syncytia (data not shown). It has been previously reported that the cytopathic effects of many viruses, including HIV, are calcium
Table 1 Comparison of results from syncytia inhibition assay, agar gel immunodiffusion and Western blot True animal disease status Infected Western blot result a Positive Negative Total animals
Total animals
Not infected
93 5 98
7 195 202
100 200 300
SIA result b Positive Negative Total animals
95 5 100
12 188 200
107 193 300
SIA result c Positive Negative Total animals
98 0 98
9 193 202
107 193 300
a Analysis of Western blot using AGID as the standard data (true infection status). b Analysis of SIA results using Western blot as the standard data. c Analysis of SIA results using AGID as the standard data.
dependent (Tyler and Fields, 1996), and the effect of EDTA on syncytia formation in this assay suggests that BLV may share a similar dependence for divalent cations. The bovine serum samples were screened by SIA and the results were interpreted after 12–16 h and again after 40 h of incubation. The data obtained from the first examination were identical to the second, for all 300 samples. These results were then compared with Western blot analysis. A total of 100 animals tested positive by Western blot and of these, 95 were positive by SIA. Of the 200 sera testing negative by Western blot, 188 were negative by SIA. When using Western blot as the standard assay, SIA was determined to have a sensitivity of 95% and a specificity of 94% (Table 1).
3.3. Syncytia inhibition assay compared with agar gel immunodiffusion The results obtained by the syncytia inhibition assay were also compared to those obtained by
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
181
Fig. 2. Appearance of syncytia inhibition assay after 16 h of incubation. (a) FLK and Raji cells co-cultivated with BLV-negative serum. Syncytia appear as large irregular cells. (b) FLK and Raji cells co-cultivated with BLV-positive serum. Normal Rajis appear as small grape-like clusters.
AGID. Agar gel immunodiffusion scored 98 animals as BLV positive; all 98 of these animals were correctly diagnosed as BLV positive by SIA. The syncytia inhibition assay did, however, score nine animals as BLV positive, which were included in the 202 animals which AGID determined to be BLV negative. Assuming that the results obtained by AGID were reliable, SIA was calculated to have a sensitivity of 100% and a specificity of 95.5%.
4. Discussion Syncytia inhibition has been shown to be a sensitive and reliable method for determining BLV infection in cattle. The results obtained using SIA suggest that this assay may be more sensitive than AGID for determination of the presence of anti-BLV antibodies. Every sample which tested positive by the commercial AGID assay was de-
tected as positive by SIA. A previous study describing the sensitivity and specificity of AGID reported this assay to return 0.2% false-positive and 1.5% false-negative results (Monke et al., 1992). Based on these numbers and the distribution of data obtained in this study, it would be expected that : 1–2 false negatives would be scored by AGID. Two of the samples which were negative by AGID and positive by SIA, were also positive by Western blot (data not shown) and it is reasonable to assume, based on the agreement between Western blot and SIA, and taking into account what is known about the sensitivity of AGID, that one or two of these discrepant samples were indeed BLV positive. This would indicate that SIA is a more sensitive assay than AGID. SIA has additional advantages over commercial AGID assays. Syncytia development occurs reliably within 12–16 h, compared to 48–72 h for the development of precipitin lines with AGID. Eval-
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
182
uation of results based on the presence or absence of syncytia is very straightforward, requiring little interpretation, as opposed to AGID which requires more complicated analysis. Furthermore, due largely to the fact that most of the materials required for performing SIA are inexpensive or self-replicating, the cost of materials for SIA is approximately one tenth that of commercial AGID. The results obtained by Western blot were very comparable to those obtained by AGID. These two assays appeared to have approximately equal sensitivities for detecting anti-BLV antibodies. Although AGID is typically not considered to be as sensitive an assay as Western blot, in this system AGID is detecting the serodominant antibody species to gp51 (Kittelberger et al., 1996), whereas Western blot consistently detects antibodies to p15 and p24. New strategies for the control of BLV infection are currently being planned to prevent the spread of infection in cattle in the US (NAHMS, 1996). Also, of critical importance to control efforts will be early and sensitive detection of infected animals. New assay systems will clearly be needed which show high sensitivity and are capable of rapidly returning data regarding the infection status of cattle. SIA appears to demonstrate both of these qualities and may be a viable alternative to current techniques for large-scale commercial screening.
Acknowledgements We gratefully acknowledge the assistance of Joann Wampler in collecting and coding the sera used in this study. We would like to thank Janice Miller for the gift of the FLK cell line as well as for the control BLV sera, and Jonathan Allan for the gift of the Raji cell line. We also would like to acknowledge Dr Guy Steucek for his assistance .
with data analysis. This work was funded through the Neimeyer–Hodgson Research Grant, Millersville University, Millersville, PA.
References Benton, C.V., Soria, A.E., Gilden, R.V., 1978. Direct syncytial assay for the quantitation of bovine leukemia virus. Infect. Immun. 20, 307 – 309. Bobakova, M., Lesnik, F., Vrtiak, O.J., 1985. Testing the susceptibility of cultured cells to infection with bovine leukemia virus. Vet. Med. (Praha) 30, 267 – 274. Coffin, J.M., 1996. Retroviridae: The viruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol. 3rd. Lippencott – Raven, New York. Ferrer, J.F., Cabradilla, C., Gupta, P., 1981. Use of a feline cell line in the syncytia infectivity assay for the detection of bovine leukemia virus infection in cattle. Am. J. Vet. Res. 42, 9 – 14. Kelly, E.J., Jackson, M.K., Marsolais, G., Morrey, J.D., Callan, R.J., 1993. Early detection of bovine leukemia virus in cattle by use of the polymerase chain reaction. Am. J. Vet. Res. 54, 205 – 209. Kittelberger, R., Laybourn, B.J., Diack, D.S., Penrose, M.E., Reichel, M.P., Motha, J., Molloy, J.B., Merza, M., 1996. Evaluation of electrophoretic immunoblotting for the detection of antibodies against the bovine leukosis virus in cattle. J. Virol. Methods 61, 7 – 22. Monke, D.R., Rohde, R.F., Hueston, W.D., Milburn, R.J., 1992. Estimation of the sensitivity and specificity of the agar gel immunodiffusion test for bovine leukemia virus: 1,296 Cases (1982 – 1989). J. Am. Vet. Med. Assoc. 200, 2001 – 2004. Naif, H.M., Daniel, R.C., Cougle, W.G., Lavin, M.F., 1992. Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle. J. Clin. Microbiol. 30, 675 – 679. NAHMS, National Animal Health Monitoring System, 1996. High Prevalence of BLV in US Dairy Herds. Available on line at www.aphis.usda.gov/vs/ceah/cahm/d96blv.htm Nguyen, V.K., Maes, R.F., 1993. Evaluation of an enzymelinked immunosorbent assay for detection of antibodies to bovine leukemia virus in serum and milk. J. Clin. Microbiol. 31, 979 – 981. Schwartz, I., Levy, D., 1994. Pathobiology of bovine leukemia virus. Vet. Res. 25, 521 – 536. Tyler, K.L., Fields, B.N., 1996. Pathogenesis of viral infections. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol. 3rd. Lippencott – Raven, New York.
Development of a syncytia inhibition assay for the detection of antibodies to bovine leukemia virus in naturally infected cattle; comparison with Western blot and agar gel immunodiffusion Michael Johnson a, Fred Rommel b, Jay Mone´ a,* b
a Department of Biology, Roddy Science Building, Millers6ille Uni6ersity, Millers6ille PA 17551, USA Commonwealth of Pennsyl6ania Department of Agriculture, Bureau of Veterinary Laboratories, 2305 N. Cameron St., Harrisburg PA 17110, USA
Received 17 June 1997; received in revised form 7 October 1997; accepted 10 October 1997
Abstract A syncytia inhibition assay (SIA) for the detection of antibodies to bovine leukemia virus is described. This test involves specific antibody-mediated inhibition of BLV-induced cytopathic effects in an indicator cell line. A total of 300 sera were screened commercially by agar gel immunodiffusion (AGID) and were then screened by Western blot and SIA. The new assay system provided results which were comparable to Western blot and AGID. The results obtained suggest that SIA may be more sensitive than either of the other two assay systems examined for the determination of the infection status of cattle. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Bovine leukemia virus; Syncytia inhibition assay; Agar gel immunodiffusion; Western blot
1. Introduction Bovine leukemia virus (BLV) is an oncogenic retrovirus which is the causative agent of bovine leukemia/lymphoma in a significant minority of infected cows (Schwartz and Levy, 1994). Cattle
* Corresponding author. Fax: +1 717 8723985.
in the US are regularly screened for antibodies to BLV and many foreign countries require imported cattle to be free of BLV infection, making determination of infection status of considerable economic importance. A recent study by the USDA estimated that 89% of dairy operations in the US contain BLV-positive animals; in the majority of affected herds, seroprevalence was \ 25%, with some herds approaching 100% (NAHMS, 1996).
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 1 8 6 - 9
178
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
The most common commercial assay system for BLV is agar gel immunodiffusion (AGID); however, many alternative assay systems are currently being examined for possible advantages over AGID (Naif et al., 1992; Kelly et al., 1993; Nguyen and Maes, 1993). It has been known for some time that BLV is capable of causing syncytia formation in many indicator cell lines (Benton et al., 1978; Ferrer et al., 1981; Bobakova et al., 1985), but no attempt had been made to examine the usefulness of syncytia inhibition to determine infection status of cows. In this study, it was found that fetal lamb kidney (FLK) cells, which are persistently infected with BLV, are capable of inducing syncytia formation in the human B-lymphoblastoid cell line, Raji, following co-cultivation. Furthermore, it was found that pre-incubation of FLK cells with sera obtained from cows infected with BLV, but not from BLV-negative cows, would effectively inhibit syncytia formation, providing the basis for a new way to screen cows for BLV infection. This syncytia inhibition assay (SIA) was used to screen 300 bovine serum samples. The sera were also screened by Western blot and agar gel immunodiffusion and the results from these three assays were compared. The results obtained by syncytia inhibition assay were comparable to those obtained by AGID and Western blot. Syncytia inhibition assay was found to be a reliable, simple and efficient means by which to screen livestock for infection with BLV.
2.2. Agar gel immunodiffusion assay Commercial AGID kits (Symbiotics, San Diego, CA) were used to screen sera for anti-BLV antibodies according to the manufacturers instructions. All AGID assays were performed at the veterinary laboratory of the Commonwealth of Pennsylvania Department of Agriculture.
2.3. Cells All media and supplements, with the exception of fetal bovine serum, were obtained from GibcoBRL, Gaithersburg, MD. Fetal bovine serum was obtained from Sigma, St. Louis, MO. Fetal lamb kidney (FLK) cells were a generous gift from Janice Miller, United States Department of Agriculture, Ames, IA and were cultured in MEM supplemented with penicillin–streptomycin (100 units penicillin, 0.1 mg streptomycin per ml), 1 mM sodium pyruvate, 10 mM HEPES, 0.25% w/v lactalbumin hydrolysate, 0.22% w/v sodium bicarbonate and 5% v/v heat-inactivated fetal bovine serum. Raji cells were a generous gift from Dr Jonathan S. Allan, Southwest Foundation for Biomedical Research, San Antonio, TX and were maintained in RPMI supplemented with penicillin–streptomycin (100 units penicillin, 0.1 mg streptomycin per ml), 1 mM sodium pyruvate, 10 mM HEPES, 0.22% w/v sodium bicarbonate and 10% v/v heat-inactivated fetal bovine serum.
2.4. Western blot assay 2. Materials and methods
2.1. Sera A total of 300 sera were obtained from the Commonwealth of Pennsylvania Department of Agriculture, Harrisburg, PA. The sera were stored frozen at − 80°C until assayed. These serum samples had been collected to be screened for antiBLV antibodies by AGID. After commercial screening, the sera were coded and assayed by Western blot and SIA as described below.
FLK cells were grown to confluency in 75 cm2 tissue culture flasks and lysed by freeze–thawing three times; the contents of the flask were then diluted with an equal volume of 2× reducing sample buffer (0.125 M Tris–HCl, 4% SDS, 20% glycerol, 10% b-mercaptoethanol). The proteins were boiled for 1 min and subjected to discontinuous SDS polyacrylamide gel electrophoresis. Following electrophoresis, the proteins were passively transferred to 0.45 mm nitrocellulose membranes (Micron Separations, Westborough,
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
MA) in a transfer buffer consisting of 10 mM Tris base, 10 mM EDTA and 50 mM NaCl, pH 7.0 at 25°C. Following transfer, the membranes were blocked with 5% non-fat dry milk in phosphate buffered saline (PBS) for 1 h at 37°C, rinsed twice in PBS Tween and stored at − 20oC until used. The membranes were cut into strips and probed with 2 ml of a 1:40 dilution of sera in PBS with 5% non-fat dry milk, 5% FBS and 0.2% Tween 20 for 2 h at 37°C. The strips were washed extensively with PBS+0.2% Tween 20, followed by the addition of biotinylated antibovine IgG (Sigma) diluted in PBS Tween. After 1 h of incubation at 37°C, the strips were washed and reacted with streptavidin-HRP (Sigma) for 45 min at room temperature. After extensive washing, the bands were visualized using diaminobenzidine and H2O2. All reactivity was compared to known positive and negative controls kindly provided by Janice Miller, USDA, Ames, IA.
179
3. Results
3.1. Agar gel immunodiffusion compared with Western blotting Western blot analysis has been demonstrated to be a sensitive method for the detection of antibodies against BLV and has been shown to correlate well with AGID and ELISA assays for diagnosis of BLV in cattle (Kittelberger et al., 1996). For this reason, serum samples were first analyzed by Western blot to determine the presence or absence of anti-BLV antibodies for correlation with the commercial AGID assay. Western blot analysis detected antibodies consistently against two viral proteins designated p15 and p24 (Fig. 1). Previous reports identified p15 and p24 as products of the viral gag gene
2.5. Syncytia inhibition assay Syncytia inhibition assays were carried out in sterile 96-well tissue culture dishes. The following were added to each well of a tissue culture dish: 100 ml of MEM containing 5×104 FLK cells and 50 ml of the sera to be tested. The cells were incubated at room temperature for 30 min before the addition of 100 ml of media containing 5 × 104 Raji cells. The plates were then incubated for 12–16 h, until syncytia were apparent in wells containing FLK and Raji cells with negative control serum. The assay was scored as positive for anti-BLV antibodies if no syncytia were evident and negative if any syncytia were observed. The plates were then incubated for an additional 24 h and re-evaluated.
2.6. Data analysis Calculations to determine test sensitivity and specificity were carried out as previously described (Monke et al., 1992).
Fig. 1. Western blot assay for detection of anti-BLV antibodies in bovine sera. FLK cells were used as the source of BLV antigens. Lane 1, negative control; lane 2, positive control; lanes 3 – 6, bovine sera displaying reactivity against p15, p24 and gp51.
180
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
(Coffin, 1996). Other investigators reported that p24 reactivity was the most reliable marker for determining infection status when compared to commercial assay systems (Kittelberger et al., 1996); we also found that p24 was a reliable indicator of BLV infection, although p15 invariably stained with greater intensity. In the present study, because antibodies to p15 and p24 were consistently detected together, sera displaying reactivity against these two proteins were scored positive. Most sera containing reactivity against p15 and p24 also reacted with the env gene product gp51; however, the intensity of staining of gp51 was inconsistent and was thereby deemed unreliable as a marker for BLV infection. A total of 300 sera were screened by Western blot and agar gel immunodiffusion. The Western blot results were analyzed using the data obtained by AGID as the standard. The agar gel immunodiffusion assay scored 98 animals as being positive for infection with BLV, of these, 93 were positive by Western blot. Conversely, of the 202 animals diagnosed as BLV negative by AGID, 195 were also negative by Western blot. The Western blot technique described therefore, returned five false negative results and seven false positive results as compared to AGID; based on these data, the sensitivity and specificity of this test were calculated at 95 and 96.5%, respectively (Table 1).
3.2. Syncytia inhibition assay compared with Western blotting Co-cultivation of BLV-producing FLK cells with Raji cells induced syncytia, usually within 12 h, Fig. 2a. Syncytia induction was blocked by antibodies against BLV, Fig. 2b. It is also worthy to note the finding that EDTA effectively blocked syncytia formation regardless of the presence or absence of anti-BLV antibodies. This was discovered through side-by-side analysis of duplicate serum samples taken from several cows in both EDTA and heparin tubes. The sera with heparin as the anticoagulant yielded results comparable to AGID, whereas all of the sera containing EDTA were able to block syncytia (data not shown). It has been previously reported that the cytopathic effects of many viruses, including HIV, are calcium
Table 1 Comparison of results from syncytia inhibition assay, agar gel immunodiffusion and Western blot True animal disease status Infected Western blot result a Positive Negative Total animals
Total animals
Not infected
93 5 98
7 195 202
100 200 300
SIA result b Positive Negative Total animals
95 5 100
12 188 200
107 193 300
SIA result c Positive Negative Total animals
98 0 98
9 193 202
107 193 300
a Analysis of Western blot using AGID as the standard data (true infection status). b Analysis of SIA results using Western blot as the standard data. c Analysis of SIA results using AGID as the standard data.
dependent (Tyler and Fields, 1996), and the effect of EDTA on syncytia formation in this assay suggests that BLV may share a similar dependence for divalent cations. The bovine serum samples were screened by SIA and the results were interpreted after 12–16 h and again after 40 h of incubation. The data obtained from the first examination were identical to the second, for all 300 samples. These results were then compared with Western blot analysis. A total of 100 animals tested positive by Western blot and of these, 95 were positive by SIA. Of the 200 sera testing negative by Western blot, 188 were negative by SIA. When using Western blot as the standard assay, SIA was determined to have a sensitivity of 95% and a specificity of 94% (Table 1).
3.3. Syncytia inhibition assay compared with agar gel immunodiffusion The results obtained by the syncytia inhibition assay were also compared to those obtained by
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
181
Fig. 2. Appearance of syncytia inhibition assay after 16 h of incubation. (a) FLK and Raji cells co-cultivated with BLV-negative serum. Syncytia appear as large irregular cells. (b) FLK and Raji cells co-cultivated with BLV-positive serum. Normal Rajis appear as small grape-like clusters.
AGID. Agar gel immunodiffusion scored 98 animals as BLV positive; all 98 of these animals were correctly diagnosed as BLV positive by SIA. The syncytia inhibition assay did, however, score nine animals as BLV positive, which were included in the 202 animals which AGID determined to be BLV negative. Assuming that the results obtained by AGID were reliable, SIA was calculated to have a sensitivity of 100% and a specificity of 95.5%.
4. Discussion Syncytia inhibition has been shown to be a sensitive and reliable method for determining BLV infection in cattle. The results obtained using SIA suggest that this assay may be more sensitive than AGID for determination of the presence of anti-BLV antibodies. Every sample which tested positive by the commercial AGID assay was de-
tected as positive by SIA. A previous study describing the sensitivity and specificity of AGID reported this assay to return 0.2% false-positive and 1.5% false-negative results (Monke et al., 1992). Based on these numbers and the distribution of data obtained in this study, it would be expected that : 1–2 false negatives would be scored by AGID. Two of the samples which were negative by AGID and positive by SIA, were also positive by Western blot (data not shown) and it is reasonable to assume, based on the agreement between Western blot and SIA, and taking into account what is known about the sensitivity of AGID, that one or two of these discrepant samples were indeed BLV positive. This would indicate that SIA is a more sensitive assay than AGID. SIA has additional advantages over commercial AGID assays. Syncytia development occurs reliably within 12–16 h, compared to 48–72 h for the development of precipitin lines with AGID. Eval-
M. Johnson et al. / Journal of Virological Methods 70 (1998) 177–182
182
uation of results based on the presence or absence of syncytia is very straightforward, requiring little interpretation, as opposed to AGID which requires more complicated analysis. Furthermore, due largely to the fact that most of the materials required for performing SIA are inexpensive or self-replicating, the cost of materials for SIA is approximately one tenth that of commercial AGID. The results obtained by Western blot were very comparable to those obtained by AGID. These two assays appeared to have approximately equal sensitivities for detecting anti-BLV antibodies. Although AGID is typically not considered to be as sensitive an assay as Western blot, in this system AGID is detecting the serodominant antibody species to gp51 (Kittelberger et al., 1996), whereas Western blot consistently detects antibodies to p15 and p24. New strategies for the control of BLV infection are currently being planned to prevent the spread of infection in cattle in the US (NAHMS, 1996). Also, of critical importance to control efforts will be early and sensitive detection of infected animals. New assay systems will clearly be needed which show high sensitivity and are capable of rapidly returning data regarding the infection status of cattle. SIA appears to demonstrate both of these qualities and may be a viable alternative to current techniques for large-scale commercial screening.
Acknowledgements We gratefully acknowledge the assistance of Joann Wampler in collecting and coding the sera used in this study. We would like to thank Janice Miller for the gift of the FLK cell line as well as for the control BLV sera, and Jonathan Allan for the gift of the Raji cell line. We also would like to acknowledge Dr Guy Steucek for his assistance .
with data analysis. This work was funded through the Neimeyer–Hodgson Research Grant, Millersville University, Millersville, PA.
References Benton, C.V., Soria, A.E., Gilden, R.V., 1978. Direct syncytial assay for the quantitation of bovine leukemia virus. Infect. Immun. 20, 307 – 309. Bobakova, M., Lesnik, F., Vrtiak, O.J., 1985. Testing the susceptibility of cultured cells to infection with bovine leukemia virus. Vet. Med. (Praha) 30, 267 – 274. Coffin, J.M., 1996. Retroviridae: The viruses and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol. 3rd. Lippencott – Raven, New York. Ferrer, J.F., Cabradilla, C., Gupta, P., 1981. Use of a feline cell line in the syncytia infectivity assay for the detection of bovine leukemia virus infection in cattle. Am. J. Vet. Res. 42, 9 – 14. Kelly, E.J., Jackson, M.K., Marsolais, G., Morrey, J.D., Callan, R.J., 1993. Early detection of bovine leukemia virus in cattle by use of the polymerase chain reaction. Am. J. Vet. Res. 54, 205 – 209. Kittelberger, R., Laybourn, B.J., Diack, D.S., Penrose, M.E., Reichel, M.P., Motha, J., Molloy, J.B., Merza, M., 1996. Evaluation of electrophoretic immunoblotting for the detection of antibodies against the bovine leukosis virus in cattle. J. Virol. Methods 61, 7 – 22. Monke, D.R., Rohde, R.F., Hueston, W.D., Milburn, R.J., 1992. Estimation of the sensitivity and specificity of the agar gel immunodiffusion test for bovine leukemia virus: 1,296 Cases (1982 – 1989). J. Am. Vet. Med. Assoc. 200, 2001 – 2004. Naif, H.M., Daniel, R.C., Cougle, W.G., Lavin, M.F., 1992. Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle. J. Clin. Microbiol. 30, 675 – 679. NAHMS, National Animal Health Monitoring System, 1996. High Prevalence of BLV in US Dairy Herds. Available on line at www.aphis.usda.gov/vs/ceah/cahm/d96blv.htm Nguyen, V.K., Maes, R.F., 1993. Evaluation of an enzymelinked immunosorbent assay for detection of antibodies to bovine leukemia virus in serum and milk. J. Clin. Microbiol. 31, 979 – 981. Schwartz, I., Levy, D., 1994. Pathobiology of bovine leukemia virus. Vet. Res. 25, 521 – 536. Tyler, K.L., Fields, B.N., 1996. Pathogenesis of viral infections. In: Fields, B.N., Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, vol. 3rd. Lippencott – Raven, New York.