INFECTION
AND
IMMUNITY, May 1976,
p.
1321-1324
Copyright C 1976 American Society for Microbiology
Vol. 13, No. 5 Printed in USA.
Modified Hemolytic Plaque Technique for the Detection of Bluetongue Virus Antibody-Forming Cells R. A. OELLERMANN,* P. CARTER, AND M. J. MARX
Department of Biochemistry, Veterinary Research Institute, Onderstepoort, 0110, Republic of South Africa Received for publication 14 October 1975
A hemolytic plaque assay was developed for the detection of antibody-forming cells to bluetongue virus (BTV). Sheep erythrocytes (SRBC), onto which BTV had been adsorbed, served as the indicator of lysis due to the presence of BTV antibody-forming cells. The ratio of BTV to SRBC was found to be critical for optimum hemolytic plaque formation. For routine use, 50 Al of 12% BTV SRBC, 0.1 ml of a spleen cell suspension, and 0.5 ml of 0.5% agarose in a balanced salt solution were mixed and plated on a microscope slide precoated with 0.1% aqueous agarose. Slides were incubated for 1 h at 37 C in a humidified incubator and subsequently flooded with 0.4 ml of a 1:15 dilution of complement. Incubation was continued for a further 2 h before the hemolytic plaques were scored. It was not possible to establish BTV serotype specificity by this technique. Antiserum production. At 10-day intervals, three 5-month-old rabbits from the local colony were given three intramuscular injections of approximately 1011 infectious plaque-forming units of purified BTV type 10. Blood was collected by cardiac puncture 2 weeks after the last BTV injection and pooled, and antiserum was separated by centrifugation 2 h after clot formation. Antiserum was inactivated at 56 C for 30 min and stored at -20 C. Similarly antiserum to SRBC was prepared by 4 weekly injections of 2 x 109 SRBC per rabbit, followed by bleeding 10 days after the final injection. ECBO virus antiserum was prepared as described previously (11). Preparation of erythrocyte suspensions. For the duration of this study, blood was collected from one sheep. A 7% suspension of SRBC was prepared in a balanced salt solution (BSS), as described (6), and used within 2 days. Different amounts of BTV were allowed to adsorb onto aliquots of 7% SRBC at room temperature for 30 min. To remove unbound BTV, the erythrocytes were washed three times with BSS and resuspended to a final concentration of 12% SRBC in BSS. In the passive hemolysis test, a 50% erythrocyte suspension was prepared after adsorption with 40 yig of BTV per ml of 7% SRBC. BTV SRBC were used within 1 h after preparation. In comparative studies, chicken erythrocyte (CRBC) suspensions were prepared by the same procedure. Complement. Blood was collected from five mature guinea pigs from the local colony by cardiac MATERIALS AND METHODS puncture and pooled, and the serum was separated BTV antigen preparation. The production and by centrifugation 2 h after clot formation. Suitable purification of BTV has been described in detail by fractions were stored at -20 C for future use as a Verwoerd et al. (10). The virulent strains of BTV source of complement. types 4 and 10 were used in this investigation. The Passive hemolysis of erythrocyte suspensions. A protein concentration of purified BTV suspensions 0.6-ml antiserum dilution was added to a mixture of was determined spectrophotometrically by the 0.3 ml of BTV-treated or untreated erythrocyte susmethod of differential absorbance (7). pensions and 0.3 ml of complement diluted 1:15. 1321
Sixteen serological types of bluetongue virus (BTV) are known (2), and significant differences between their capsid polypeptides have been demonstrated (1). BTV has been shown to consist of seven polypeptides, two of which are present as a diffuse protein layer surrounding the capsid (10). Very little is known, however, of the basic immunological response to these serotypes, either in vivo or in vitro. A sensitive assay for the detection of BTV antibody-forming cells (PFC) is therefore desirable. It would be advantageous if the BTV serotype specificity could be determined by such an assay. A technique for the detection of cells producing hemolytic antibody to sheep erythrocytes (SRBC) has been described by Jerne et al. (4). This technique has been adapted to soluble antigens (5, 8) and foot-and-mouth disease virus (12) by chemically coupling the antigen to SRBC. The use of a similar technique was therefore contemplated for BTV. The relative instability of BTV (3) made chemical coupling less attractive, and a different approach had to be resorted to. For this study, the characteristic of BTV to adsorb onto cellular membranes (9) was successfully exploited in the development of a hemolytic BTV PFC assay.
1322
INFECT. IMMUN.
OELLERMANN, CARTER, AND MARX
Thereupon 0.5 ml was dispensed into two adjacent wells of a hemagglutination plate. Incubation was carried out for 3 h at 37 C in a humidified incubator, and the individual wells were examined for hemolysis. Erythrocytes, mixed with saline, complement, or antiserum alone, were included as controls. Spleen cell suspensions. For each experiment, three 10-week-old male albino mice from the local colony were immunized with approximately 20 ug of BTV, expressed as protein, per mouse. After 4 days mice were killed by cervical dislocation, the spleen was removed aseptically, and pooled cell suspensions were prepared as described (6). After determination of the spleen cell concentration with an electronic particle counter (Coulter Electronics Inc., Hialeah, Fla.), the cell suspension was diluted to contain 3 x 106, 10, and 3 x 105 cells per 0.1 ml. Hemolytic plaque assay. The method for the detection of BTV PFC was similar to the hemolytic plaque assay described by Oellermann (6). A solution of 0.5% molten agarose (Miles-Seravac, Cape Town) in BSS was prepared, and 0.5 ml was pipetted into tubes maintained at 43 C. To each tube was added 30 to 70 Al of 12% BTV SRBC kept at 30 C, followed immediately by 0.1 ml of a prewarmed spleen cell suspension. The contents of each tube were rapidly mixed and poured onto labeled microscope slides, precoated with 0.1% aqueous agarose. For the determination of optimum temperature, the slides were incubated for 1 h at 31 to 39 C in a humidified incubator. The slides were then flooded with 0.4 ml of a 1:15 dilution of complement, and incubation continued for a further 2 h. For direct
plaques were scored either directly or under indirect illumination at an eightfold magnification. In all experiments, suitable plaque counts were obtained by using 106 cells per slide. Based on these counts, the numbers of PFC per slide were calculated as the mean + standard error from three experiments, with duplicate determinations per experiment.
RESULTS
Passive hemolysis of erythrocytes by different antisera. BTV-treated and untreated SRBC and CRBC were incubated with dilutions of SRBC-, BTV-, and ECBO virus-specific antisera in the presence of complement (Fig. 1). SRBC-specific antiserum resulted in lysis of SRBC and BTV SRBC, but not of any CRBC. BTV-specific antiserum, however, resulted in lysis of BTV SRBC and BTV CRBC, and the reaction was complement dependent. In no case did ECBO virus-specific antiserum produce lysis of erythrocytes. The results clearly demonstrate the specificity of lysis of BTV-treated erythrocytes by BTV-specific antiserum, thereby supporting the feasibility of a hemolytic plaque assay for the detection of BTV PFC. 0
0
*
comparison of the hemolytic plaques formed, slides
were prepared from the same spleen cell suspensions and BTV SRBC, but incubated in different incubators set at the desired temperatures. Hemolytic
*
*
v
9
i
.*
0
0
O
0
*
0
0
*
,
*
0
I
t:CI e
j
, IN
&.
0
,
, 0
*
00
*
0
*
0
*
.
,
0
C,-
-_,,,;
C1
0
-r
c,,
:,
C,,
dL.
CP
I.I.4
f
-1
ANT I SE RJ M FIG. 1. Passive hemolysis of BTV-treated and untreated sheep and chicken erythrocytes b; different antisera. The number after each antiserum is the log4 of the dilution used in the test.
VOL. 13, 1976
BLUETONGUE VIRUS HEMOLYTIC PFC DETECTION
Effect of BTV-to-SRBC ratio on PFC detection. The following experiment was conducted partly to determine the practicability of the BTV PFC assay. Different concentrations of BTV types 4 and 10 were adsorbed onto SRBC before plating in agarose with spleen cell suspensions from mice immunized with BTV types 4 and 10, respectively. The reciprocal titration was carried out to determine whether serotype specificity could be demonstrated by using the BTV PFC assay. The volume of washed BTV SRBC was kept constant at 50 ul per slide. The results are presented in Table 1. Low concentrations of BTV resulted in diffuse and poorly defined plaques. Increasing concentrations of BTV resulted in more clearly defined plaques, but decreasing in size. With excessive concentrations of BTV, hemolytic plaques were hardly discernible and aggregation of SRBC occurred, resulting in a heterogeneous appearance of the agarose gel. It would also appear as if slightly higher concentrations of BTV type 4 than of type 10 are necessary for binding to SRBC to ensure efficient PFC detection. The ratio of BTV to SRBC is therefore critical for optimum hemolytic plaque formation. No definite serotype specificity could be demonstrated under the existing experimental conditions, and marked cross-reactivity occurred between the two serotypes tested. Effect of SRBC concentration on PFC detection. For these experiments, 20 ug of BTV type 10 per ml of 7% SRBC was used for adsorpTABLE 1. Effect of BTV serotype and concentration on hemolytic plaque formation by spleen cells from mice immunized with BTV serotypes 4 and 10 BTV serotype lAg of BTV- PFC/slide m of (ean -Plaque descripImmu- Adsorp- 7%per tion SRBC SE)a nization
tion
4
4 4 4
0 20 40
3±3 66 + 11 210 + 15
4 4
80 120
200 + 15 164 + 12
10
20
133 + 10
10 10 10
0 10 20
2±2 205 ± 17 233 ± 15
10
10
40
190 + 13
10
80
160 + 11
4
40
110 ± 11
Faint, diffuse Faint, sharper outline Clear, small Microscopic, SRBC aggregates Mostly clear, small
Diffuse, larger Mostly clear, small Clear, microscopic Poor, SRBC aggregates
Faint, sharper
tion. A description of the hemolytic plaques obtained upon the addition of different concentrations of washed BTV SRBC is presented in Table 2. With lower concentrations of erythrocytes, plaques with a relatively sharp outline were observed, but they were faint because of insufficient erythrocytes being present. Increased erythrocyte concentrations resulted in hemolytic plaques that were reduced in size but more clearly defined. Improved contrast was also observed at the higher erythrocyte concentrations, facilitating PFC detection. Fewer PFC were found, however, when 70 Al of 12% SRBC was used per slide. The SRBC concentration is therefore of considerable importance in the detection of BTV PFC; 50 ,lI per slide was found to be the optimum and was routinely used. Effect of temperature on PFC detection. BTV is relatively unstable at 37 C (3), and the influence of a lower incubation temperature on hemolytic plaque formation was therefore investigated. For these experiments, 25 Mg of BTV type 10 was used per ml of 7% SRBC. After washing of erythrocytes, 50 ,l of 12% SRBC was applied per slide (Table 3). No real differences were observed between numbers of BTV PFC obtained at incubation temperatures of 34 and 37 C; furthermore, hemolytic plaques obtained at 37 C were clearer, in general, than those obtained at 34 C. At 31 C, hemolytic plaques were fainter and had a more diffuse outline than those observed at the higher temperatures. Out of interest, an incubation temperature of 39 C was included in the experiments. Although clearly defined plaques were observed, they were microscopic in size, with the number of plaques observed per slide tending to decrease. For routine purposes, therefore, an incubation temperature of 37 C was used.
DISCUSSION Basic study of the immunological response to BTV is hampered by the lack of a sensitive TABLE 2. Effect of SRBC concentration on BTV PFC detection ,ul of 12% PFC/slide SRBC/slide (mean ± SE)
SE, Standard error.
Plaque description
30 40
227 + 16 211 ± 14
Sharp outline, faint Sharp outline, fairly
50 60
209 + 15 193 ± 12
clear Sharp outline, clear Sharp outline, clear,
70
162 ± 13
small
outline a
1323
SE, Standard error.
Sharp outline, microscopic
1324
OELLERMANN, CARTER, AND MARX
TABLE 3. Effect of temperature of incubation on BTV hemolytic plaque formation Temp (C) PFC/slide (mean
Plaque description
31 201 ± 17 34 195 ± 14 210 ± 16 37 171 ± 11 39 " SE, Standard error.
Diffuse, small Less clear, small Clear, small Clear, microscopic
for BTV PFC. The Jerne hemolytic plaque technique (4) has been adapted to studies of the immunological response to various antigens (5, 8, 12). A further modification of this technique using BTV as antigen was therefore investigated. The property of BTV to adsorb onto cellular membranes was used to advantage in this study, eliminating the need for chemical coupling of BTV, which is relatively unstable (3), to a suitable erythrocyte. The specificity of lysis of BTV-treated erythrocytes by BTV antiserum opened the way to the successful development of an assay specific for BTV PFC. From the results shown in Fig. 1, it is evident that lysis of BTV SRBC by BTV antiserum is more sensitive than lysis of BTV CRBC. Throughout this investigation, SRBC were therefore used as an indicator of lysis for the detection of BTV PFC. For their optimal detection, it is essential that the correct ratio of BTV to SRBC for each serotype of BTV studied be determined, as well as the most suitable concentration of BTV SRBC, to achieve good contrast between the lysed plaque area and the background. The difference in optimal concentration between BTV serotypes adsorbed onto SRBC for PFC detection could possibly be due to inherent differences between the outer proteins of the different serotypes. Although differences in the sizes of the outer polypeptide components between different serotypes have been indicated (1), their further biochemical characterization has not yet been possible. Preliminary experiments on the BTV serotype specificity of the hemolytic plaques were conducted. A certain degree of specificity was observed using the hemolytic BTV PFC assay, assay
INFECT. IMMUN.
but considerable cross-reactivity was apparent. According to Howell (personal communication), there is no cross-reaction between the two serotypes used in this investigation. Work is now in progress to clarify this apparent discrepancy. ACKNOWLEDGMENTS We are most grateful to Dr. K. E. Weiss, Director of the Veterinary Research Institute, Onderstepoort, for his continued interest in and support of our work. LITERATURE CITED 1. De Villiers, E. M. 1974. Comparison of the capsid polypeptides of various bluetongue virus serotypes. Intervirology 3:47-53. 2. Howell, P. G., N. A. KUimm, and M. J. Botha. 1970. The application of improved techniques to the identification of strains of bluetongue virus. Onderstepoort J. Vet. Res. 37:59-66. 3. Howell, P. G., D. W. Verwoerd, and R. A. Oellermann. 1967. Plaque formation by bluetongue virus. Onderstepoort J. Vet. Res. 34:317-332. 4. Jerne, N. K., A. A. Nordin, and C. Henry. 1963. The agar plaque technique for recognising antibody-producing cells, p. 109-122. In B. Amos and H. Koprowski (ed.), Cell-bound antibodies. Wistar Press Institute, Philadelphia. 5. Lemieux, S., S. Avrameas, and A. E. Bussard. 1974. Local hemolysis plaque assay using a new method of coupling antigens on sheep erythrocytes by glutaraldehyde. Immunochemistry 11:261-269. 6. Oellermann, R. A. 1974. Stimulation of the immune response in vivo by different nucleic acids. Onderstepoort J. Vet. Res. 41:217-220. 7. Oellermann, R. A. 1974. The elimination of ribonucleic acid interference in the spectrophotometric determination of protein concentration. Onderstepoort J. Vet. Res. 41:221-224. 8. Schwenk, H-U., and F. Lehmann-Grube. 1970. The localised hemolysis-in-gel method adapted to the detection of spleen cells releasing virus-specific antibodies. J. Immunol. 197:1184-1186. 9. Verwoerd, D. W. 1969. Purification and characterization of bluetongue virus. Virology 38:203-212. 10. Verwoerd, D. W., H. J. Els, E. M. de Villiers, and H. Huismans. 1972. Structure of the bluetongue virus capsid. J. Virol. 10:783-794. 11. Verwoerd, D. W., R. A. Oellermann, J. Broekman, and K. E. Weiss. 1967. The serological relationship of South African bovine enterovirus strains (ECBO SAI and -IH) and the growth characteristics in cell culture of the prototype strain (ECBO SA-I). Onderstepoort J. Vet. Res. 34:41-52. 12. Wittmann, G., and I. Reda. 1974. Der Nachweis antikorperbildender Zellen mit Hilfe der lokalen Hamolyse im Gel (LHG) bei Mausen und Schweinen nach Immunisierung mit Maul- und KlauenseucheVirus (MKSV) und MKS-Vakzine. Zentralbl. Bakteriol. Hyg. Parasitenkd. Infektionskr. I Abt. Orig. A 227:420-425.
AND
IMMUNITY, May 1976,
p.
1321-1324
Copyright C 1976 American Society for Microbiology
Vol. 13, No. 5 Printed in USA.
Modified Hemolytic Plaque Technique for the Detection of Bluetongue Virus Antibody-Forming Cells R. A. OELLERMANN,* P. CARTER, AND M. J. MARX
Department of Biochemistry, Veterinary Research Institute, Onderstepoort, 0110, Republic of South Africa Received for publication 14 October 1975
A hemolytic plaque assay was developed for the detection of antibody-forming cells to bluetongue virus (BTV). Sheep erythrocytes (SRBC), onto which BTV had been adsorbed, served as the indicator of lysis due to the presence of BTV antibody-forming cells. The ratio of BTV to SRBC was found to be critical for optimum hemolytic plaque formation. For routine use, 50 Al of 12% BTV SRBC, 0.1 ml of a spleen cell suspension, and 0.5 ml of 0.5% agarose in a balanced salt solution were mixed and plated on a microscope slide precoated with 0.1% aqueous agarose. Slides were incubated for 1 h at 37 C in a humidified incubator and subsequently flooded with 0.4 ml of a 1:15 dilution of complement. Incubation was continued for a further 2 h before the hemolytic plaques were scored. It was not possible to establish BTV serotype specificity by this technique. Antiserum production. At 10-day intervals, three 5-month-old rabbits from the local colony were given three intramuscular injections of approximately 1011 infectious plaque-forming units of purified BTV type 10. Blood was collected by cardiac puncture 2 weeks after the last BTV injection and pooled, and antiserum was separated by centrifugation 2 h after clot formation. Antiserum was inactivated at 56 C for 30 min and stored at -20 C. Similarly antiserum to SRBC was prepared by 4 weekly injections of 2 x 109 SRBC per rabbit, followed by bleeding 10 days after the final injection. ECBO virus antiserum was prepared as described previously (11). Preparation of erythrocyte suspensions. For the duration of this study, blood was collected from one sheep. A 7% suspension of SRBC was prepared in a balanced salt solution (BSS), as described (6), and used within 2 days. Different amounts of BTV were allowed to adsorb onto aliquots of 7% SRBC at room temperature for 30 min. To remove unbound BTV, the erythrocytes were washed three times with BSS and resuspended to a final concentration of 12% SRBC in BSS. In the passive hemolysis test, a 50% erythrocyte suspension was prepared after adsorption with 40 yig of BTV per ml of 7% SRBC. BTV SRBC were used within 1 h after preparation. In comparative studies, chicken erythrocyte (CRBC) suspensions were prepared by the same procedure. Complement. Blood was collected from five mature guinea pigs from the local colony by cardiac MATERIALS AND METHODS puncture and pooled, and the serum was separated BTV antigen preparation. The production and by centrifugation 2 h after clot formation. Suitable purification of BTV has been described in detail by fractions were stored at -20 C for future use as a Verwoerd et al. (10). The virulent strains of BTV source of complement. types 4 and 10 were used in this investigation. The Passive hemolysis of erythrocyte suspensions. A protein concentration of purified BTV suspensions 0.6-ml antiserum dilution was added to a mixture of was determined spectrophotometrically by the 0.3 ml of BTV-treated or untreated erythrocyte susmethod of differential absorbance (7). pensions and 0.3 ml of complement diluted 1:15. 1321
Sixteen serological types of bluetongue virus (BTV) are known (2), and significant differences between their capsid polypeptides have been demonstrated (1). BTV has been shown to consist of seven polypeptides, two of which are present as a diffuse protein layer surrounding the capsid (10). Very little is known, however, of the basic immunological response to these serotypes, either in vivo or in vitro. A sensitive assay for the detection of BTV antibody-forming cells (PFC) is therefore desirable. It would be advantageous if the BTV serotype specificity could be determined by such an assay. A technique for the detection of cells producing hemolytic antibody to sheep erythrocytes (SRBC) has been described by Jerne et al. (4). This technique has been adapted to soluble antigens (5, 8) and foot-and-mouth disease virus (12) by chemically coupling the antigen to SRBC. The use of a similar technique was therefore contemplated for BTV. The relative instability of BTV (3) made chemical coupling less attractive, and a different approach had to be resorted to. For this study, the characteristic of BTV to adsorb onto cellular membranes (9) was successfully exploited in the development of a hemolytic BTV PFC assay.
1322
INFECT. IMMUN.
OELLERMANN, CARTER, AND MARX
Thereupon 0.5 ml was dispensed into two adjacent wells of a hemagglutination plate. Incubation was carried out for 3 h at 37 C in a humidified incubator, and the individual wells were examined for hemolysis. Erythrocytes, mixed with saline, complement, or antiserum alone, were included as controls. Spleen cell suspensions. For each experiment, three 10-week-old male albino mice from the local colony were immunized with approximately 20 ug of BTV, expressed as protein, per mouse. After 4 days mice were killed by cervical dislocation, the spleen was removed aseptically, and pooled cell suspensions were prepared as described (6). After determination of the spleen cell concentration with an electronic particle counter (Coulter Electronics Inc., Hialeah, Fla.), the cell suspension was diluted to contain 3 x 106, 10, and 3 x 105 cells per 0.1 ml. Hemolytic plaque assay. The method for the detection of BTV PFC was similar to the hemolytic plaque assay described by Oellermann (6). A solution of 0.5% molten agarose (Miles-Seravac, Cape Town) in BSS was prepared, and 0.5 ml was pipetted into tubes maintained at 43 C. To each tube was added 30 to 70 Al of 12% BTV SRBC kept at 30 C, followed immediately by 0.1 ml of a prewarmed spleen cell suspension. The contents of each tube were rapidly mixed and poured onto labeled microscope slides, precoated with 0.1% aqueous agarose. For the determination of optimum temperature, the slides were incubated for 1 h at 31 to 39 C in a humidified incubator. The slides were then flooded with 0.4 ml of a 1:15 dilution of complement, and incubation continued for a further 2 h. For direct
plaques were scored either directly or under indirect illumination at an eightfold magnification. In all experiments, suitable plaque counts were obtained by using 106 cells per slide. Based on these counts, the numbers of PFC per slide were calculated as the mean + standard error from three experiments, with duplicate determinations per experiment.
RESULTS
Passive hemolysis of erythrocytes by different antisera. BTV-treated and untreated SRBC and CRBC were incubated with dilutions of SRBC-, BTV-, and ECBO virus-specific antisera in the presence of complement (Fig. 1). SRBC-specific antiserum resulted in lysis of SRBC and BTV SRBC, but not of any CRBC. BTV-specific antiserum, however, resulted in lysis of BTV SRBC and BTV CRBC, and the reaction was complement dependent. In no case did ECBO virus-specific antiserum produce lysis of erythrocytes. The results clearly demonstrate the specificity of lysis of BTV-treated erythrocytes by BTV-specific antiserum, thereby supporting the feasibility of a hemolytic plaque assay for the detection of BTV PFC. 0
0
*
comparison of the hemolytic plaques formed, slides
were prepared from the same spleen cell suspensions and BTV SRBC, but incubated in different incubators set at the desired temperatures. Hemolytic
*
*
v
9
i
.*
0
0
O
0
*
0
0
*
,
*
0
I
t:CI e
j
, IN
&.
0
,
, 0
*
00
*
0
*
0
*
.
,
0
C,-
-_,,,;
C1
0
-r
c,,
:,
C,,
dL.
CP
I.I.4
f
-1
ANT I SE RJ M FIG. 1. Passive hemolysis of BTV-treated and untreated sheep and chicken erythrocytes b; different antisera. The number after each antiserum is the log4 of the dilution used in the test.
VOL. 13, 1976
BLUETONGUE VIRUS HEMOLYTIC PFC DETECTION
Effect of BTV-to-SRBC ratio on PFC detection. The following experiment was conducted partly to determine the practicability of the BTV PFC assay. Different concentrations of BTV types 4 and 10 were adsorbed onto SRBC before plating in agarose with spleen cell suspensions from mice immunized with BTV types 4 and 10, respectively. The reciprocal titration was carried out to determine whether serotype specificity could be demonstrated by using the BTV PFC assay. The volume of washed BTV SRBC was kept constant at 50 ul per slide. The results are presented in Table 1. Low concentrations of BTV resulted in diffuse and poorly defined plaques. Increasing concentrations of BTV resulted in more clearly defined plaques, but decreasing in size. With excessive concentrations of BTV, hemolytic plaques were hardly discernible and aggregation of SRBC occurred, resulting in a heterogeneous appearance of the agarose gel. It would also appear as if slightly higher concentrations of BTV type 4 than of type 10 are necessary for binding to SRBC to ensure efficient PFC detection. The ratio of BTV to SRBC is therefore critical for optimum hemolytic plaque formation. No definite serotype specificity could be demonstrated under the existing experimental conditions, and marked cross-reactivity occurred between the two serotypes tested. Effect of SRBC concentration on PFC detection. For these experiments, 20 ug of BTV type 10 per ml of 7% SRBC was used for adsorpTABLE 1. Effect of BTV serotype and concentration on hemolytic plaque formation by spleen cells from mice immunized with BTV serotypes 4 and 10 BTV serotype lAg of BTV- PFC/slide m of (ean -Plaque descripImmu- Adsorp- 7%per tion SRBC SE)a nization
tion
4
4 4 4
0 20 40
3±3 66 + 11 210 + 15
4 4
80 120
200 + 15 164 + 12
10
20
133 + 10
10 10 10
0 10 20
2±2 205 ± 17 233 ± 15
10
10
40
190 + 13
10
80
160 + 11
4
40
110 ± 11
Faint, diffuse Faint, sharper outline Clear, small Microscopic, SRBC aggregates Mostly clear, small
Diffuse, larger Mostly clear, small Clear, microscopic Poor, SRBC aggregates
Faint, sharper
tion. A description of the hemolytic plaques obtained upon the addition of different concentrations of washed BTV SRBC is presented in Table 2. With lower concentrations of erythrocytes, plaques with a relatively sharp outline were observed, but they were faint because of insufficient erythrocytes being present. Increased erythrocyte concentrations resulted in hemolytic plaques that were reduced in size but more clearly defined. Improved contrast was also observed at the higher erythrocyte concentrations, facilitating PFC detection. Fewer PFC were found, however, when 70 Al of 12% SRBC was used per slide. The SRBC concentration is therefore of considerable importance in the detection of BTV PFC; 50 ,lI per slide was found to be the optimum and was routinely used. Effect of temperature on PFC detection. BTV is relatively unstable at 37 C (3), and the influence of a lower incubation temperature on hemolytic plaque formation was therefore investigated. For these experiments, 25 Mg of BTV type 10 was used per ml of 7% SRBC. After washing of erythrocytes, 50 ,l of 12% SRBC was applied per slide (Table 3). No real differences were observed between numbers of BTV PFC obtained at incubation temperatures of 34 and 37 C; furthermore, hemolytic plaques obtained at 37 C were clearer, in general, than those obtained at 34 C. At 31 C, hemolytic plaques were fainter and had a more diffuse outline than those observed at the higher temperatures. Out of interest, an incubation temperature of 39 C was included in the experiments. Although clearly defined plaques were observed, they were microscopic in size, with the number of plaques observed per slide tending to decrease. For routine purposes, therefore, an incubation temperature of 37 C was used.
DISCUSSION Basic study of the immunological response to BTV is hampered by the lack of a sensitive TABLE 2. Effect of SRBC concentration on BTV PFC detection ,ul of 12% PFC/slide SRBC/slide (mean ± SE)
SE, Standard error.
Plaque description
30 40
227 + 16 211 ± 14
Sharp outline, faint Sharp outline, fairly
50 60
209 + 15 193 ± 12
clear Sharp outline, clear Sharp outline, clear,
70
162 ± 13
small
outline a
1323
SE, Standard error.
Sharp outline, microscopic
1324
OELLERMANN, CARTER, AND MARX
TABLE 3. Effect of temperature of incubation on BTV hemolytic plaque formation Temp (C) PFC/slide (mean
Plaque description
31 201 ± 17 34 195 ± 14 210 ± 16 37 171 ± 11 39 " SE, Standard error.
Diffuse, small Less clear, small Clear, small Clear, microscopic
for BTV PFC. The Jerne hemolytic plaque technique (4) has been adapted to studies of the immunological response to various antigens (5, 8, 12). A further modification of this technique using BTV as antigen was therefore investigated. The property of BTV to adsorb onto cellular membranes was used to advantage in this study, eliminating the need for chemical coupling of BTV, which is relatively unstable (3), to a suitable erythrocyte. The specificity of lysis of BTV-treated erythrocytes by BTV antiserum opened the way to the successful development of an assay specific for BTV PFC. From the results shown in Fig. 1, it is evident that lysis of BTV SRBC by BTV antiserum is more sensitive than lysis of BTV CRBC. Throughout this investigation, SRBC were therefore used as an indicator of lysis for the detection of BTV PFC. For their optimal detection, it is essential that the correct ratio of BTV to SRBC for each serotype of BTV studied be determined, as well as the most suitable concentration of BTV SRBC, to achieve good contrast between the lysed plaque area and the background. The difference in optimal concentration between BTV serotypes adsorbed onto SRBC for PFC detection could possibly be due to inherent differences between the outer proteins of the different serotypes. Although differences in the sizes of the outer polypeptide components between different serotypes have been indicated (1), their further biochemical characterization has not yet been possible. Preliminary experiments on the BTV serotype specificity of the hemolytic plaques were conducted. A certain degree of specificity was observed using the hemolytic BTV PFC assay, assay
INFECT. IMMUN.
but considerable cross-reactivity was apparent. According to Howell (personal communication), there is no cross-reaction between the two serotypes used in this investigation. Work is now in progress to clarify this apparent discrepancy. ACKNOWLEDGMENTS We are most grateful to Dr. K. E. Weiss, Director of the Veterinary Research Institute, Onderstepoort, for his continued interest in and support of our work. LITERATURE CITED 1. De Villiers, E. M. 1974. Comparison of the capsid polypeptides of various bluetongue virus serotypes. Intervirology 3:47-53. 2. Howell, P. G., N. A. KUimm, and M. J. Botha. 1970. The application of improved techniques to the identification of strains of bluetongue virus. Onderstepoort J. Vet. Res. 37:59-66. 3. Howell, P. G., D. W. Verwoerd, and R. A. Oellermann. 1967. Plaque formation by bluetongue virus. Onderstepoort J. Vet. Res. 34:317-332. 4. Jerne, N. K., A. A. Nordin, and C. Henry. 1963. The agar plaque technique for recognising antibody-producing cells, p. 109-122. In B. Amos and H. Koprowski (ed.), Cell-bound antibodies. Wistar Press Institute, Philadelphia. 5. Lemieux, S., S. Avrameas, and A. E. Bussard. 1974. Local hemolysis plaque assay using a new method of coupling antigens on sheep erythrocytes by glutaraldehyde. Immunochemistry 11:261-269. 6. Oellermann, R. A. 1974. Stimulation of the immune response in vivo by different nucleic acids. Onderstepoort J. Vet. Res. 41:217-220. 7. Oellermann, R. A. 1974. The elimination of ribonucleic acid interference in the spectrophotometric determination of protein concentration. Onderstepoort J. Vet. Res. 41:221-224. 8. Schwenk, H-U., and F. Lehmann-Grube. 1970. The localised hemolysis-in-gel method adapted to the detection of spleen cells releasing virus-specific antibodies. J. Immunol. 197:1184-1186. 9. Verwoerd, D. W. 1969. Purification and characterization of bluetongue virus. Virology 38:203-212. 10. Verwoerd, D. W., H. J. Els, E. M. de Villiers, and H. Huismans. 1972. Structure of the bluetongue virus capsid. J. Virol. 10:783-794. 11. Verwoerd, D. W., R. A. Oellermann, J. Broekman, and K. E. Weiss. 1967. The serological relationship of South African bovine enterovirus strains (ECBO SAI and -IH) and the growth characteristics in cell culture of the prototype strain (ECBO SA-I). Onderstepoort J. Vet. Res. 34:41-52. 12. Wittmann, G., and I. Reda. 1974. Der Nachweis antikorperbildender Zellen mit Hilfe der lokalen Hamolyse im Gel (LHG) bei Mausen und Schweinen nach Immunisierung mit Maul- und KlauenseucheVirus (MKSV) und MKS-Vakzine. Zentralbl. Bakteriol. Hyg. Parasitenkd. Infektionskr. I Abt. Orig. A 227:420-425.
