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Complement-dependent serum neutralization with virulent and a virulent Bucyrus strains of equine arteritis virus
Veterinary Microbiology, 36 ( 1993 ) 379-383 Elsevier Science Publishers B.V., Amsterdam
379
Short Communication Complement-dependent serum neutralization with virulent and avirulent Bucyrus strains of equine arteritis virus Y. Fukunaga, H. Imagawa, T. Kanemaru and M. Kamada Epizootic Research Station, Equine Research Institute, The Japan Racing Association, Shiba, Japan (Accepted 5 February 1993)
ABSTRACT Virulent and avirulent strains of Bucyrus equine arteritis virus (EAV) were used to raise antiserum in horses. Serum neutralization (SN) tests were performed with and without the addition of guinea pig complement. The inclusion of ten percent guinea pig serum in the virus suspension was sufficient for optimal enhancement of SN titres at any immune stages after immunization. Immune serum prepared against avirulent virus reacted only with homologous virus and there was no complement enhancement. Immune sera raised against live or inactivated virulent virus neutralized both virulent and avirulent virus. The reaction with virulent virus demonstrated complement enhancement. There was also moderate potentiation in the presence of complement when serum raised against inactivated virulent virus reacted with avirulent virus.
INTRODUCTION
Equine arteritis virus (EAV) reveals complement requiring serum neutralization (SN). The complement requiring SN antibody against EAV was associated with the IgG fraction of immune sera collected from horses and other experimental animals (Hyllseth and Pettersson, 1970; Radwan and Burger, 1973). Senne et al. ( 1985 ) described a micro-neutralization test detecting immunity to EAV by using a low passage level of the virulent Bueyrus strain of EAV in the presence of fresh quinea pig serum. McCollum (1970) presented a Correspondence to: Y. Fukunaga, Epizootic Research Station, Equine Research Institute, The Japan Racing Association, 1400-4, Shiba, Kokubunji-Machi, Shimotsuga-Gun, Tochigi 329-04, Japan.
0378-1135/93/$06.00 © 1993 Elsevier Science Publishers B.V. All fights reserved.
380
Y. FUKUNAGAETAL.
plaque reduction neutralization test using the avirulent Bucyrus strain of EAV with a high passage history in cell cultures (McCollum, 1969), but in the absence of complement (McCollum and Swerczek, 1978). We describe in this paper the different complement requirements and diverse neutralizing capacities of equine antisera against the virulent and avirulent Bucyrus strains of EAV. MATERIALS AND METHODS
An antiserum against the virulent Bucyrus strain of EAV was prepared by inoculating horses intranassaly (Fukunaga et al., 1981 ) with 5 ml of plural fluid from an experimentally infected horse. This contained the Bucyrus strain of EAV at titers of 105.8 pfu/ml. An antiserum was also prepared by intramuscular injections (Fukunaga et al., 1990) of 2 separate doses, at 4 weekintervals, of a formalin inactivated whole virus preparation, of the virulent Bucyrus strain of EAV which had contained at least 108.5pfu of the virus per dose before inactivation. An antiserum against the avirulent Bucyrus strain of EAV was prepared by injecting horses intramuscularly (Fukunaga et al., 1982) with one ml of a modified live virus of the Bucyrus strain of EAV at titers of 108.3pfu/ml. The modified virus had been passed 131 times in horse kidney, 80 times in rabbit kidney and 16 times in equine dermal (E. Derm) cells (McCollum, 1969). Serum was collected from each horse and stored at - 2 0 ° C until use. Of the viruses used for the SN test, the virulent Bucyrus strain had one passage in E. Derm cells followed by 2 passages in RK- 13 cells. The avirulent Bucyrus strain was of the same passage level described above. The complement was supplied by lyophilized guinea pig serum (Denka Seiken Co. Ltd., Japan). It was rehydrated with distilled water and a pool of 3 batches of the guinea pig serum was added to cold virus diluent. The virus suspension was mixed with immune horse sera which had been previously inactivated at 56°C for 30 min and the mixture was incubated at 37°C for one hour. The SN test was conducted by the 50% plaque reduction method (Fukunaga et al., 1981 ). RESULTS AND DISCUSSION
In order to confirm an optimal complement concentration to enhance SN antibody response, SN tests were conducted in the presence of various concentrations of complement (Table 1 ). When suspensions of the virulent Bucyrus strain of EAV contained more than 5% complement, SN antibody titers were potentiated 16 times higher at all 3 immune stages after infection. Similar potentiations were also observed in the other 2 immune horse sera (data not shown). Therefore, 10% complement would be a sufficient dose for SN testing of serum samples. An excessive amount of complement did not further
COMPLEMENT-DEPENDENTSERUMNEUTRALIZATION
381
TABLE 1 Serum neutralizing antibody titers of three representative sera collected periodically from a horse infected with virulent Bucyrus strain of equine arteritis virus detected by homologous virus in the presence of various concentrations of guinea pig complement Time after inaction
Concentration of complement in virus diluent (%) 40
20
10
5
2.5
9 days l month 6months
320* 1280 1280
320 1280 1280
160 1280 1280
320 1280 1280
160 1280 640
1.25
0.63
0.31
0.0
40 640 -640
20 320 320
20 320 320
20 80 80
*Reciprocal of the highest serum dilution that expressed 50% neutralization against 80-100 plaques of virus. (The test was conducted in duplicate).
enhance SN antibody titers. These observations were in agreement with those of earlier studies. Hyllseth and Pettersson (1970) described similar SN enhancement with immune horse and rabbit sera when 15% unheated guinea pig serum was added to a serum-virus mixture. MoraiUon and Moraillon (1978) and Senne et al. (1985) applied 50/0and 10% guinea pig sera in the virus suspensions, respectively, to detect reactors to EAV in SN tests. Radwan and Burger (1973) reported that complement requiring SN antibody was found only in the later immune sera, whereas we demonstrated enhancement not only one and 6 months after infection but also as early as 9 days after infection. This discrepancy might be due to the different immune sera examined. They used immune sera from horses, rabbits, guinea pigs, hamsters and mice which had received multiple intramuscular injections of EAV antigens. However, what is of interest is the predominant immunoglobulin class of the immune horse serum collected 9 days after infection. As shown in Table 2, immune horse serum was tested with the virulent and avirulent Bucyrus strains of EAV in the presence or absence of 10% complement. Antiserum against the virulent Bucyrus strain of EAV neutralized both virus strains. Antibody titers against the virulent Bucyrus strain were 16 to 32 times higher with complement than without complement. However, the avirulent Bucyrus strain was neutralized constantly, regardless of the presence or absence of complement. This supports findings by McCollum and Swerczek (1978). Their avirulent Bucyrus strain of EAV, used for routine serology, did not require the presence of complement for neutralization. Immune sera induced by inactivated virus of the virulent Bucyrus strain of EAV revealed similar neutralization patterns to those of live virus. However, antibody titers against the virulent Bucyrus strain, even in the presence of complement were 16 times lower than against the avirulent Bucyrus strain without complement. Furthermore, when the avirulent Bucyrus strain was
382
Y. FUKUNAGAETAL.
TABLE 2 Cross serum neutralization of virulent and avirulent Bucyrus strains of equine arteritis virus by their specific immune horse sera in the presence or absence of guinea pig complement Immune horse serum
Days after inoculation
Virus suspension Virulent Bucyrus W/O C 'a
Live virulent Bucyrus Inactivated virulent Bucyrus Live avirulent Bucyrus
11 10 10
40¢ 80 80
Avirulent Bucyrus With C 'b
W/O C'
With C'
640 1280 2560
1280 1280 2560
1280 2560 2560
7 7 7
<5 <5 5
10 40 160
160 640 2560
640 5120 10240
11 10 11
<5 <5 <5
<5 <5 <5
160 320 640
320 320 640
aWithout guinea pig complement: Eagle's MEM was added in virus diluent instead of complement. bWith guinea pig complement: 10% complement was added. CReciprocal of the highest serum dilution that expressed 50% neutralization against 80-100 plaques of virus. (The test was conducted in duplicate).
tested in the presence of complement, SN antibody titers were moderately enhanced (4 to 8 times). Antiserum against the avirulent Bucyrus strain of EAV neutralized only homologous virus and complement-requiringSN potentiation was not present. The one way cross-reaction, as well as the lack of complement enhancement, may be attributable to the modification of this virus strain during repeated passages (227) in cell cultures. In contrast, the avirulent Bucyrus strain of EAV showed good responses to all the immune horse sera tested. The results were similar to findings in the previous study of complement fixation reactions (Fukunaga and McCollum, 1977 ). Variability of SN antibody response was notably demonstrated in the immune sera against the inactivated Bucyrus strain of EAV. This is possibly due to different immunizations, in that the antisera were induced by injection with 2 separate doses of inactivated virus, while the other antisera were raised by infection with live virus. REFERENCES Fukunaga, Y., Imagawa, H. Tabuchi, E. and Akiyama, Y., 1981. Clinical and virological findings on experimental equine viral arteritis in horses. Bull. Equine Res. Inst., 18:110-118. Fukunaga, Y. and McCollum, W.H., 1977. Complement fixation reactions in equine viral arteritis. Am. J. Vet. Res., 38: 2043-2046. Fukunaga, Y., Wada, R., Hirasawa, K., Kamada, M., Kumanomido, T. and Akiyama, Y., 1982.
COMPLEMENT-DEPENDENTSERUMNEUTRALIZATION
383
Effect of the modified Bucyrus strain of equine arteritis virus experimentally inoculated into horses. Bull. Equine Res. Inst., 19: 97-101. Fukunaga, Y., Wada, R., Matsumura, T., Sugiura, T. and Imagawa, H., 1990: Induction of immune response and protection from equine viral arteritis (EVA) by formalin inactivatedvirus vaccine for EVA in horses. J. Vet. Med., B., 37:135-141. Hyllseth, B. and Pettersson, U., 1970. Neutralization of equine arteritis virus: Enhancing effect of guinea pig serum. Arch. ges. Virusforsch., 32: 337-247. McCollum, W.H., 1969. Development of a modified virus strain and vaccine for equine viral arteritis. J. Am. Vet. Med. Ass., 155: 318-322. McCollum, W.H., 1970. Vaccination for equine viral arteritis. In: J.T. Bryans and H. Gerber (Editors), Equine Infectious Disease II. S. Karger, Basel, pp. 143-151. McCollum, W.H. and Swerczek, T.W., 1978. Studies of an epizootic of equine viral arteritis in racehorses. J. Equine Med. Surg., 2: 293-299. Moraillon, A. and Moraillon, R., 1978. Results of a serological survey of viral arteritis in France and in several European and African countries. In: J.T. Bryans and H. Gerber (Editors), Equine Infectious Disease IV. Veterinary Publications, Inc., Princeton, N.J., pp. 467-473. Radwan, A.I. and Burger, D., 1973. The complement-requiring neutralization of equine arteritis virus by late antisera. Virology, 51: 71-77. Senne, D.A., Pearson, J.E. and Carbrey, E.A., 1985. Equine viral arteritis: A standard procedure for the virus neutralization test and comparison of results of a proficiency test performed at five laboratories. Proc. 89th Annu. Meet. US Anim. Health Ass. Milwaukee, Wis., 89: 2934.
379
Short Communication Complement-dependent serum neutralization with virulent and avirulent Bucyrus strains of equine arteritis virus Y. Fukunaga, H. Imagawa, T. Kanemaru and M. Kamada Epizootic Research Station, Equine Research Institute, The Japan Racing Association, Shiba, Japan (Accepted 5 February 1993)
ABSTRACT Virulent and avirulent strains of Bucyrus equine arteritis virus (EAV) were used to raise antiserum in horses. Serum neutralization (SN) tests were performed with and without the addition of guinea pig complement. The inclusion of ten percent guinea pig serum in the virus suspension was sufficient for optimal enhancement of SN titres at any immune stages after immunization. Immune serum prepared against avirulent virus reacted only with homologous virus and there was no complement enhancement. Immune sera raised against live or inactivated virulent virus neutralized both virulent and avirulent virus. The reaction with virulent virus demonstrated complement enhancement. There was also moderate potentiation in the presence of complement when serum raised against inactivated virulent virus reacted with avirulent virus.
INTRODUCTION
Equine arteritis virus (EAV) reveals complement requiring serum neutralization (SN). The complement requiring SN antibody against EAV was associated with the IgG fraction of immune sera collected from horses and other experimental animals (Hyllseth and Pettersson, 1970; Radwan and Burger, 1973). Senne et al. ( 1985 ) described a micro-neutralization test detecting immunity to EAV by using a low passage level of the virulent Bueyrus strain of EAV in the presence of fresh quinea pig serum. McCollum (1970) presented a Correspondence to: Y. Fukunaga, Epizootic Research Station, Equine Research Institute, The Japan Racing Association, 1400-4, Shiba, Kokubunji-Machi, Shimotsuga-Gun, Tochigi 329-04, Japan.
0378-1135/93/$06.00 © 1993 Elsevier Science Publishers B.V. All fights reserved.
380
Y. FUKUNAGAETAL.
plaque reduction neutralization test using the avirulent Bucyrus strain of EAV with a high passage history in cell cultures (McCollum, 1969), but in the absence of complement (McCollum and Swerczek, 1978). We describe in this paper the different complement requirements and diverse neutralizing capacities of equine antisera against the virulent and avirulent Bucyrus strains of EAV. MATERIALS AND METHODS
An antiserum against the virulent Bucyrus strain of EAV was prepared by inoculating horses intranassaly (Fukunaga et al., 1981 ) with 5 ml of plural fluid from an experimentally infected horse. This contained the Bucyrus strain of EAV at titers of 105.8 pfu/ml. An antiserum was also prepared by intramuscular injections (Fukunaga et al., 1990) of 2 separate doses, at 4 weekintervals, of a formalin inactivated whole virus preparation, of the virulent Bucyrus strain of EAV which had contained at least 108.5pfu of the virus per dose before inactivation. An antiserum against the avirulent Bucyrus strain of EAV was prepared by injecting horses intramuscularly (Fukunaga et al., 1982) with one ml of a modified live virus of the Bucyrus strain of EAV at titers of 108.3pfu/ml. The modified virus had been passed 131 times in horse kidney, 80 times in rabbit kidney and 16 times in equine dermal (E. Derm) cells (McCollum, 1969). Serum was collected from each horse and stored at - 2 0 ° C until use. Of the viruses used for the SN test, the virulent Bucyrus strain had one passage in E. Derm cells followed by 2 passages in RK- 13 cells. The avirulent Bucyrus strain was of the same passage level described above. The complement was supplied by lyophilized guinea pig serum (Denka Seiken Co. Ltd., Japan). It was rehydrated with distilled water and a pool of 3 batches of the guinea pig serum was added to cold virus diluent. The virus suspension was mixed with immune horse sera which had been previously inactivated at 56°C for 30 min and the mixture was incubated at 37°C for one hour. The SN test was conducted by the 50% plaque reduction method (Fukunaga et al., 1981 ). RESULTS AND DISCUSSION
In order to confirm an optimal complement concentration to enhance SN antibody response, SN tests were conducted in the presence of various concentrations of complement (Table 1 ). When suspensions of the virulent Bucyrus strain of EAV contained more than 5% complement, SN antibody titers were potentiated 16 times higher at all 3 immune stages after infection. Similar potentiations were also observed in the other 2 immune horse sera (data not shown). Therefore, 10% complement would be a sufficient dose for SN testing of serum samples. An excessive amount of complement did not further
COMPLEMENT-DEPENDENTSERUMNEUTRALIZATION
381
TABLE 1 Serum neutralizing antibody titers of three representative sera collected periodically from a horse infected with virulent Bucyrus strain of equine arteritis virus detected by homologous virus in the presence of various concentrations of guinea pig complement Time after inaction
Concentration of complement in virus diluent (%) 40
20
10
5
2.5
9 days l month 6months
320* 1280 1280
320 1280 1280
160 1280 1280
320 1280 1280
160 1280 640
1.25
0.63
0.31
0.0
40 640 -640
20 320 320
20 320 320
20 80 80
*Reciprocal of the highest serum dilution that expressed 50% neutralization against 80-100 plaques of virus. (The test was conducted in duplicate).
enhance SN antibody titers. These observations were in agreement with those of earlier studies. Hyllseth and Pettersson (1970) described similar SN enhancement with immune horse and rabbit sera when 15% unheated guinea pig serum was added to a serum-virus mixture. MoraiUon and Moraillon (1978) and Senne et al. (1985) applied 50/0and 10% guinea pig sera in the virus suspensions, respectively, to detect reactors to EAV in SN tests. Radwan and Burger (1973) reported that complement requiring SN antibody was found only in the later immune sera, whereas we demonstrated enhancement not only one and 6 months after infection but also as early as 9 days after infection. This discrepancy might be due to the different immune sera examined. They used immune sera from horses, rabbits, guinea pigs, hamsters and mice which had received multiple intramuscular injections of EAV antigens. However, what is of interest is the predominant immunoglobulin class of the immune horse serum collected 9 days after infection. As shown in Table 2, immune horse serum was tested with the virulent and avirulent Bucyrus strains of EAV in the presence or absence of 10% complement. Antiserum against the virulent Bucyrus strain of EAV neutralized both virus strains. Antibody titers against the virulent Bucyrus strain were 16 to 32 times higher with complement than without complement. However, the avirulent Bucyrus strain was neutralized constantly, regardless of the presence or absence of complement. This supports findings by McCollum and Swerczek (1978). Their avirulent Bucyrus strain of EAV, used for routine serology, did not require the presence of complement for neutralization. Immune sera induced by inactivated virus of the virulent Bucyrus strain of EAV revealed similar neutralization patterns to those of live virus. However, antibody titers against the virulent Bucyrus strain, even in the presence of complement were 16 times lower than against the avirulent Bucyrus strain without complement. Furthermore, when the avirulent Bucyrus strain was
382
Y. FUKUNAGAETAL.
TABLE 2 Cross serum neutralization of virulent and avirulent Bucyrus strains of equine arteritis virus by their specific immune horse sera in the presence or absence of guinea pig complement Immune horse serum
Days after inoculation
Virus suspension Virulent Bucyrus W/O C 'a
Live virulent Bucyrus Inactivated virulent Bucyrus Live avirulent Bucyrus
11 10 10
40¢ 80 80
Avirulent Bucyrus With C 'b
W/O C'
With C'
640 1280 2560
1280 1280 2560
1280 2560 2560
7 7 7
<5 <5 5
10 40 160
160 640 2560
640 5120 10240
11 10 11
<5 <5 <5
<5 <5 <5
160 320 640
320 320 640
aWithout guinea pig complement: Eagle's MEM was added in virus diluent instead of complement. bWith guinea pig complement: 10% complement was added. CReciprocal of the highest serum dilution that expressed 50% neutralization against 80-100 plaques of virus. (The test was conducted in duplicate).
tested in the presence of complement, SN antibody titers were moderately enhanced (4 to 8 times). Antiserum against the avirulent Bucyrus strain of EAV neutralized only homologous virus and complement-requiringSN potentiation was not present. The one way cross-reaction, as well as the lack of complement enhancement, may be attributable to the modification of this virus strain during repeated passages (227) in cell cultures. In contrast, the avirulent Bucyrus strain of EAV showed good responses to all the immune horse sera tested. The results were similar to findings in the previous study of complement fixation reactions (Fukunaga and McCollum, 1977 ). Variability of SN antibody response was notably demonstrated in the immune sera against the inactivated Bucyrus strain of EAV. This is possibly due to different immunizations, in that the antisera were induced by injection with 2 separate doses of inactivated virus, while the other antisera were raised by infection with live virus. REFERENCES Fukunaga, Y., Imagawa, H. Tabuchi, E. and Akiyama, Y., 1981. Clinical and virological findings on experimental equine viral arteritis in horses. Bull. Equine Res. Inst., 18:110-118. Fukunaga, Y. and McCollum, W.H., 1977. Complement fixation reactions in equine viral arteritis. Am. J. Vet. Res., 38: 2043-2046. Fukunaga, Y., Wada, R., Hirasawa, K., Kamada, M., Kumanomido, T. and Akiyama, Y., 1982.
COMPLEMENT-DEPENDENTSERUMNEUTRALIZATION
383
Effect of the modified Bucyrus strain of equine arteritis virus experimentally inoculated into horses. Bull. Equine Res. Inst., 19: 97-101. Fukunaga, Y., Wada, R., Matsumura, T., Sugiura, T. and Imagawa, H., 1990: Induction of immune response and protection from equine viral arteritis (EVA) by formalin inactivatedvirus vaccine for EVA in horses. J. Vet. Med., B., 37:135-141. Hyllseth, B. and Pettersson, U., 1970. Neutralization of equine arteritis virus: Enhancing effect of guinea pig serum. Arch. ges. Virusforsch., 32: 337-247. McCollum, W.H., 1969. Development of a modified virus strain and vaccine for equine viral arteritis. J. Am. Vet. Med. Ass., 155: 318-322. McCollum, W.H., 1970. Vaccination for equine viral arteritis. In: J.T. Bryans and H. Gerber (Editors), Equine Infectious Disease II. S. Karger, Basel, pp. 143-151. McCollum, W.H. and Swerczek, T.W., 1978. Studies of an epizootic of equine viral arteritis in racehorses. J. Equine Med. Surg., 2: 293-299. Moraillon, A. and Moraillon, R., 1978. Results of a serological survey of viral arteritis in France and in several European and African countries. In: J.T. Bryans and H. Gerber (Editors), Equine Infectious Disease IV. Veterinary Publications, Inc., Princeton, N.J., pp. 467-473. Radwan, A.I. and Burger, D., 1973. The complement-requiring neutralization of equine arteritis virus by late antisera. Virology, 51: 71-77. Senne, D.A., Pearson, J.E. and Carbrey, E.A., 1985. Equine viral arteritis: A standard procedure for the virus neutralization test and comparison of results of a proficiency test performed at five laboratories. Proc. 89th Annu. Meet. US Anim. Health Ass. Milwaukee, Wis., 89: 2934.