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Vaccination Against Marek's Disease and Infectious Bursal Disease III. Growth Rate of Both Turkey Herpesvirus and Infectious Bursal Disease Virus in Coinfected Cell Cultures1,2
Vaccination Against Marek's Disease and Infectious Bursal Disease III. Growth Rate of Both Turkey Herpesvirus and Infectious Bursal Disease Virus in Coinfected Cell Cultures1,2 J.-D. T. CHANG, 3 C. S. EIDSON, and S. H. KLEVEN 4 Poultry Disease Research Center, Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30605 (Received for publication November 16, 1983) ABSTRACT Titers of the turkey herpesvirus (HVT) and infectious bursal disease virus (IBDV) in coinfected cell cultures, used to produce a live bivalent HVT/IBDV vaccine, were determined daily. Based on the daily titers, cells harvested either 2 or 3 days after inoculating the second virus (IBDV) into HVT infected cell cultures yielded the maximum titers for both viruses. Harvesting coinfected cell cultures on either day provided the most suitable source of the live HVT/IBDV vaccine against Marek's disease and infectious bursal disease. (Key words.- turkey herpesvirus/infectious bursal disease virus vaccine, live bivalent vaccine, Marek's disease, infectious bursal disease) 1985 Poultry Science 64:841-843 INTRODUCTION
Recently we reported the development of a bivalent live turkey herpesvirus/infectious bursal disease virus (HVT/IBDV) vaccine by coinfection of HVT and IBDV in chicken embryo fibroblast (CEF) cell cultures (Chang et al, 1983). We also developed a method for the titration of this bivalent live vaccine (Change et al, 1984). For the present report, the daily titers of the two component viruses of HVT/ IBDV in cell cultures were studied in an attempt to determine the optimal time to harvest the co-infected cultures to provide the maximal yield of both viruses. MATERIALS AND METHODS
Viruses. The FC-126 strain of HVT (Witter et al 1970) (Vineland Laboratories, Vineland, New Jersey) and the BursaVac-M strain of IBDV (Sterwin Laboratories, Inc., Millsboro, Delaware) were used. One vial of each virus was
'Paper No. 2154 of The University of Georgia Veterinary Medical Experiment Station. 2 Support was provided in part by a grant from the Georgia Department of Agriculture and in part from the University of Georgia Veterinary Medical Experiment Station. 'Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. "To whom correspondence should be addressed.
841
reconstituted in 200 ml of medium 199 (GIBCO) and served as the source of primary inoculum for virus propagation. Preparation of Vaccines. The live bivalent HVT/IBDV arid the monovalent HVT or IBDV vaccines were prepared as previously described (Chang et al, 1983). Briefly, 18-hr chicken embryo fibroblast cell cultures were either infected with HVT 24 hr prior to the superinfection of infectious bursal disease vaccine virus or singly infected with HVT or IBDV. Growth Rate of Turkey Herpesvirus and Infectious Bursal Disease Virus in Coinfected Cell Cultures. Six 100-mm plastic petri dishes (Falcon 3003, Div. Becton Dickinson and Co., Oxnard, CA) of the HVT/IBDV coinfected CEF cultures were harvested daily by trypsinization and centrifugation from 24 hr after IBDV superinfection until the infected CEF monolayers were entirely altered and no complete cell sheets were available (4th day after superinfection). The HVT or IBDV singly infected CEF monolayers, six 100-mm cell culture plates each, also were harvested on the same daily schedule as that of the coinfected cultures. After resuspending in 200 ml of medium 199 (GIBCO), the daily harvested HVT/IBDV, HVT, and IBDV-infected CEF suspensions were each divided into two equal aliquots. The first aliquot was used to prepare cell-associated (CA) virus, and the second aliquot was used to prepare cell-free (CF) virus. The aliquots for CF preparations were sonicated with a cell disruptor (Model W185,
842
CHANG ET AL.
Heat Systems-Ultrasonic, Inc., Long Island, NY) at an intensity setting of 7 for 1 min. The harvested CA and CF preparations of HVT/ IBDV were further divided into three equal subaliquots each. The three subaliquots were treated separately as follows: 1) anti-IBDV serum treatment, 2) chloroform (CHCI3) treatment, and 3) untreated control. For titration, the growth rate (daily titer) of each component virus in the daily harvested coinfected cell culture monolayers was determined with the aid of CHC13 or anti-IBDV serum treatment (Chang et al, 1984). Daily titers of HVT and IBDV in daily harvested monovalent HVT or IBDV and bivalent HVT/IBDV were titrated as described (Chang et al, 1984). The HVT plaque counts were recorded as an average count of triplicate CEF cultures. The IBDV titers were obtained from a microtiter system in which quadriplicate wells were used for each virus dilution.
preparations provided no available data regarding the HVT titer. Turkey herpesvirus plaques were not observed in the monolayers inoculated with CHCI3-treated HVT/IBDV preparations for 4 successive days after IBDV superinfection. Daily Titers of Infectious Bursal Disease Virus in Coinfected Preparations. The daily IBDV titers are shown in Table 2. Both CA and CF HVT/IBDV, as well as IBDV, produced a high IBDV titer ranging from 10 6 to more than 10 7 50% tissue culture infective dose (TCID 50 ) throughout the 4-day period. The CHCL3treated CA and CF HVT/IBDV titers remained above 10 7 TCID 50 for the first 3 days postinfection, then a decline followed on Day 4 postinfection to 10 4 to 1 0 s . The IBDV titers of both CA and CF/HVT/IBDV declined immediately to an extremely low level (less than 10 TCID50) after treatment with anti-IBDV serum.
RESULTS
DISCUSSION The infectivity titers of HVT and IBDV indicated that the period between 2 and 3 days postinfection with IBDV was the most suitable time for harvest of the coinfected cell cultures as a live bivalent HVT/IBDV vaccine. Although there was an increasing HVT titer in the singly and serum-treated coinfected preparations at the 4th day post-IBDV infection, the nontreated coinfected preparation was not assayed because of the extensive destruction of the CEF
Daily Titers of Turkey Herpesvirus in Coinfected Preparations. During the propagation of vaccine viruses in CEF cultures, HVT replicated with increasing titers through the observation period of 4 days after IBDV superinfection (Table 1). A similar pattern was found in the monovalent HVT-infected preparation and in coinfected cultures treated with anti-IBDV serum. Four days after superinfection, the untreated CA and CF HVT/IBDV
TABLE 1. Daily turkey herpesvirus (HVT) titers from cell cultures infected with HVT alone or superinfected with infectious bursal disease virus (IBDV) D a y s after IBDV superinfection
Vaccine Type
Component
Cell-associated
Cell-free
1
HVT HVT/IBDV1 HVT/IBDV1 HVT/IBDV1 HVT HVT/IBDV1 HVT/IBDV1 HVT/IBDV'
Treatment
1
2
3
4
Anti-IBDV serum CHCI35
852'3 72 36 0
305 252 119 0
550 117 283 0
603 NA4 450 0
Anti-IBDV serum CHC1 3
80 40 40 0
120 80 200 0
200 160 280 0
320 NA 440 0
From a coinfected chicken embryo fibroblast cell culture source.
2
Plaque forming units per dose of inoculum (.2 ml) counted on the 3rd to 5th day postinoculation.
3
An average count of triplicate cell culture plates.
4
Not assayed.
5
Chloroform.
BIVALENT LIVE VIRUS VACCINE
843
TABLE 2. Daily infectious bursal disease virus (IBDV) titers, 50% tissue culture infective dose (TCIDse), cell cultures infected with IBDValone or superinfected with turkey herpes virus (HVT) Vaccine Type
Cell-associated
Cell-free
1 2 3
from
Days after IBDV superinfection
Component
Treatment
1
2
3
4
IBDV HVT/IBDV 1 HVT/IBDV 1 HVT/IBDV 1
... ... Anti-IBDV serum CHC1 3 2
>1072 >107 <10 >107
>107 10 7 <10 >107
>10' 10 6 <10 >107
>107 >107 <10 10"
IBDV HVT/IBDV1 HVT/IBDV 1 HVT/IBDV 1
... ... Anti-IBDV serum CHC13
10 7 >107 <10 >107
>107 >107 <10 >107
10 6 . 3 10 6 ' 7 <10 >107
>107 >107 <10 10 s
From a coinfected chicken embryo fibroblast cell culture source. TCID 50 per dose (.05 ml). Chloroform.
monolayers by IBDV. Four days after IBDV superinfection, the coinfected cell culture monolayers were entirely affected by cytopathic effects (CPE) of IBDV (Cho et al, 1979), and the foci of HVT plaques were not as clearly identifiable as those obtained earlier. When these coinfected cell cultures were harvested and inoculated directly onto 18hr-old fresh cell cultures for virus titration, the IBDV-induced CPE overwhelmed the HVT plaque formation. This was due to the rapid replication and cytolytic effect of IBDV (Petek, et al, 1973; Lukert and Davis, 1974; Nick et al, 1976; Cho et al, 1979), as well as the less cytolytic effect of HVT (Calnek and Witter, 1978). Therefore, the titer of HVT in the untreated HVT/IBDV preparations obtained 4 days after IBDV superinfection could not be assayed. After 4 days post-IBDV superinfection, the majority of cells in the coinfected cell cultures had become lysed and the integrity of monolayer no longer existed. Thus, the titration was not practical afterward. It was interesting that, at 4 days after superinfection there was a large drop in IBDV titer after CHCI3 treatment. Such a decrease could not be the result of CHCI3 treatment because IBDV is resistant to CHCI3 (Benton et al, 1967). The loss of IBDV might be attributed to variation in sampling or other unknown factors. ACKNOWLEDGMENTS The authors are indebted to P. D. Lukert for helpful review and suggestions.
REFERENCES Benton, W. J., M. S. Cover, J. K. Rosenberger, and R. S. Lake, 1967. Physicochemical properties of the infectious bursal agent (IBA) of chicken. Avian Dis. 11:438-445. Calnek, B. W., and R. L. Witter, 1978. Marek's disease. Pages 385—418 in Diseases of Poultry. 7th ed. M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reed, and H. W. Yoder, Jr., ed. Iowa State Univ. Press, Ames, IA. Chang, J.-D. T., C. S. Eidson, M. J. Dykstra, S. H. Kleven, and O. J. Fletcher, 1983. Vaccination against Marek's disease and infectious bursal disease. I. Development of a bivalent live vaccine by co-cultivating turkey herpesvirus and infectious bursal disease vaccine viruses in chicken embryo fibroblast monolayers. Poultry Sci. 62:2326-2335. Chang, J.-D. T., C. S. Eidson, S. H. Kleven, and O. J. Fletcher, 1984. Vaccination against Marek's disease and infectious bursal disease. II. Titration and in vivo efficacy of the co-infection-derived bivalent live HVT/IBDV vaccine. Poultry Sci. 63:1752-1758. Cho, B. R., R. G. Raymond, and R. W. Hill, 1979. Growth of infectious bursal disease virus with plaque formation in chicken embryo fibroblast cell culture. Avian Dis. 23:209-218. Lukert, P. D., and R. B. Davis, 1974. Infectious bursal disease virus: growth and characterization in cell cultures. Avian Dis. 18:243—250. Nick, H., D. Cursiefen, and H. Becht, 1976. Structure and growth characteristics of infectious bursal disease virus. J. Virol. 18:227-234. Petek, M., P. N. D'Aprile, and F. Cancellotti, 197" Biological and physico-chemical properties of the infectious bursal disease virus (IBDV). Avian Pathol. 2:135-152. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.
Recently we reported the development of a bivalent live turkey herpesvirus/infectious bursal disease virus (HVT/IBDV) vaccine by coinfection of HVT and IBDV in chicken embryo fibroblast (CEF) cell cultures (Chang et al, 1983). We also developed a method for the titration of this bivalent live vaccine (Change et al, 1984). For the present report, the daily titers of the two component viruses of HVT/ IBDV in cell cultures were studied in an attempt to determine the optimal time to harvest the co-infected cultures to provide the maximal yield of both viruses. MATERIALS AND METHODS
Viruses. The FC-126 strain of HVT (Witter et al 1970) (Vineland Laboratories, Vineland, New Jersey) and the BursaVac-M strain of IBDV (Sterwin Laboratories, Inc., Millsboro, Delaware) were used. One vial of each virus was
'Paper No. 2154 of The University of Georgia Veterinary Medical Experiment Station. 2 Support was provided in part by a grant from the Georgia Department of Agriculture and in part from the University of Georgia Veterinary Medical Experiment Station. 'Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. "To whom correspondence should be addressed.
841
reconstituted in 200 ml of medium 199 (GIBCO) and served as the source of primary inoculum for virus propagation. Preparation of Vaccines. The live bivalent HVT/IBDV arid the monovalent HVT or IBDV vaccines were prepared as previously described (Chang et al, 1983). Briefly, 18-hr chicken embryo fibroblast cell cultures were either infected with HVT 24 hr prior to the superinfection of infectious bursal disease vaccine virus or singly infected with HVT or IBDV. Growth Rate of Turkey Herpesvirus and Infectious Bursal Disease Virus in Coinfected Cell Cultures. Six 100-mm plastic petri dishes (Falcon 3003, Div. Becton Dickinson and Co., Oxnard, CA) of the HVT/IBDV coinfected CEF cultures were harvested daily by trypsinization and centrifugation from 24 hr after IBDV superinfection until the infected CEF monolayers were entirely altered and no complete cell sheets were available (4th day after superinfection). The HVT or IBDV singly infected CEF monolayers, six 100-mm cell culture plates each, also were harvested on the same daily schedule as that of the coinfected cultures. After resuspending in 200 ml of medium 199 (GIBCO), the daily harvested HVT/IBDV, HVT, and IBDV-infected CEF suspensions were each divided into two equal aliquots. The first aliquot was used to prepare cell-associated (CA) virus, and the second aliquot was used to prepare cell-free (CF) virus. The aliquots for CF preparations were sonicated with a cell disruptor (Model W185,
842
CHANG ET AL.
Heat Systems-Ultrasonic, Inc., Long Island, NY) at an intensity setting of 7 for 1 min. The harvested CA and CF preparations of HVT/ IBDV were further divided into three equal subaliquots each. The three subaliquots were treated separately as follows: 1) anti-IBDV serum treatment, 2) chloroform (CHCI3) treatment, and 3) untreated control. For titration, the growth rate (daily titer) of each component virus in the daily harvested coinfected cell culture monolayers was determined with the aid of CHC13 or anti-IBDV serum treatment (Chang et al, 1984). Daily titers of HVT and IBDV in daily harvested monovalent HVT or IBDV and bivalent HVT/IBDV were titrated as described (Chang et al, 1984). The HVT plaque counts were recorded as an average count of triplicate CEF cultures. The IBDV titers were obtained from a microtiter system in which quadriplicate wells were used for each virus dilution.
preparations provided no available data regarding the HVT titer. Turkey herpesvirus plaques were not observed in the monolayers inoculated with CHCI3-treated HVT/IBDV preparations for 4 successive days after IBDV superinfection. Daily Titers of Infectious Bursal Disease Virus in Coinfected Preparations. The daily IBDV titers are shown in Table 2. Both CA and CF HVT/IBDV, as well as IBDV, produced a high IBDV titer ranging from 10 6 to more than 10 7 50% tissue culture infective dose (TCID 50 ) throughout the 4-day period. The CHCL3treated CA and CF HVT/IBDV titers remained above 10 7 TCID 50 for the first 3 days postinfection, then a decline followed on Day 4 postinfection to 10 4 to 1 0 s . The IBDV titers of both CA and CF/HVT/IBDV declined immediately to an extremely low level (less than 10 TCID50) after treatment with anti-IBDV serum.
RESULTS
DISCUSSION The infectivity titers of HVT and IBDV indicated that the period between 2 and 3 days postinfection with IBDV was the most suitable time for harvest of the coinfected cell cultures as a live bivalent HVT/IBDV vaccine. Although there was an increasing HVT titer in the singly and serum-treated coinfected preparations at the 4th day post-IBDV infection, the nontreated coinfected preparation was not assayed because of the extensive destruction of the CEF
Daily Titers of Turkey Herpesvirus in Coinfected Preparations. During the propagation of vaccine viruses in CEF cultures, HVT replicated with increasing titers through the observation period of 4 days after IBDV superinfection (Table 1). A similar pattern was found in the monovalent HVT-infected preparation and in coinfected cultures treated with anti-IBDV serum. Four days after superinfection, the untreated CA and CF HVT/IBDV
TABLE 1. Daily turkey herpesvirus (HVT) titers from cell cultures infected with HVT alone or superinfected with infectious bursal disease virus (IBDV) D a y s after IBDV superinfection
Vaccine Type
Component
Cell-associated
Cell-free
1
HVT HVT/IBDV1 HVT/IBDV1 HVT/IBDV1 HVT HVT/IBDV1 HVT/IBDV1 HVT/IBDV'
Treatment
1
2
3
4
Anti-IBDV serum CHCI35
852'3 72 36 0
305 252 119 0
550 117 283 0
603 NA4 450 0
Anti-IBDV serum CHC1 3
80 40 40 0
120 80 200 0
200 160 280 0
320 NA 440 0
From a coinfected chicken embryo fibroblast cell culture source.
2
Plaque forming units per dose of inoculum (.2 ml) counted on the 3rd to 5th day postinoculation.
3
An average count of triplicate cell culture plates.
4
Not assayed.
5
Chloroform.
BIVALENT LIVE VIRUS VACCINE
843
TABLE 2. Daily infectious bursal disease virus (IBDV) titers, 50% tissue culture infective dose (TCIDse), cell cultures infected with IBDValone or superinfected with turkey herpes virus (HVT) Vaccine Type
Cell-associated
Cell-free
1 2 3
from
Days after IBDV superinfection
Component
Treatment
1
2
3
4
IBDV HVT/IBDV 1 HVT/IBDV 1 HVT/IBDV 1
... ... Anti-IBDV serum CHC1 3 2
>1072 >107 <10 >107
>107 10 7 <10 >107
>10' 10 6 <10 >107
>107 >107 <10 10"
IBDV HVT/IBDV1 HVT/IBDV 1 HVT/IBDV 1
... ... Anti-IBDV serum CHC13
10 7 >107 <10 >107
>107 >107 <10 >107
10 6 . 3 10 6 ' 7 <10 >107
>107 >107 <10 10 s
From a coinfected chicken embryo fibroblast cell culture source. TCID 50 per dose (.05 ml). Chloroform.
monolayers by IBDV. Four days after IBDV superinfection, the coinfected cell culture monolayers were entirely affected by cytopathic effects (CPE) of IBDV (Cho et al, 1979), and the foci of HVT plaques were not as clearly identifiable as those obtained earlier. When these coinfected cell cultures were harvested and inoculated directly onto 18hr-old fresh cell cultures for virus titration, the IBDV-induced CPE overwhelmed the HVT plaque formation. This was due to the rapid replication and cytolytic effect of IBDV (Petek, et al, 1973; Lukert and Davis, 1974; Nick et al, 1976; Cho et al, 1979), as well as the less cytolytic effect of HVT (Calnek and Witter, 1978). Therefore, the titer of HVT in the untreated HVT/IBDV preparations obtained 4 days after IBDV superinfection could not be assayed. After 4 days post-IBDV superinfection, the majority of cells in the coinfected cell cultures had become lysed and the integrity of monolayer no longer existed. Thus, the titration was not practical afterward. It was interesting that, at 4 days after superinfection there was a large drop in IBDV titer after CHCI3 treatment. Such a decrease could not be the result of CHCI3 treatment because IBDV is resistant to CHCI3 (Benton et al, 1967). The loss of IBDV might be attributed to variation in sampling or other unknown factors. ACKNOWLEDGMENTS The authors are indebted to P. D. Lukert for helpful review and suggestions.
REFERENCES Benton, W. J., M. S. Cover, J. K. Rosenberger, and R. S. Lake, 1967. Physicochemical properties of the infectious bursal agent (IBA) of chicken. Avian Dis. 11:438-445. Calnek, B. W., and R. L. Witter, 1978. Marek's disease. Pages 385—418 in Diseases of Poultry. 7th ed. M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reed, and H. W. Yoder, Jr., ed. Iowa State Univ. Press, Ames, IA. Chang, J.-D. T., C. S. Eidson, M. J. Dykstra, S. H. Kleven, and O. J. Fletcher, 1983. Vaccination against Marek's disease and infectious bursal disease. I. Development of a bivalent live vaccine by co-cultivating turkey herpesvirus and infectious bursal disease vaccine viruses in chicken embryo fibroblast monolayers. Poultry Sci. 62:2326-2335. Chang, J.-D. T., C. S. Eidson, S. H. Kleven, and O. J. Fletcher, 1984. Vaccination against Marek's disease and infectious bursal disease. II. Titration and in vivo efficacy of the co-infection-derived bivalent live HVT/IBDV vaccine. Poultry Sci. 63:1752-1758. Cho, B. R., R. G. Raymond, and R. W. Hill, 1979. Growth of infectious bursal disease virus with plaque formation in chicken embryo fibroblast cell culture. Avian Dis. 23:209-218. Lukert, P. D., and R. B. Davis, 1974. Infectious bursal disease virus: growth and characterization in cell cultures. Avian Dis. 18:243—250. Nick, H., D. Cursiefen, and H. Becht, 1976. Structure and growth characteristics of infectious bursal disease virus. J. Virol. 18:227-234. Petek, M., P. N. D'Aprile, and F. Cancellotti, 197" Biological and physico-chemical properties of the infectious bursal disease virus (IBDV). Avian Pathol. 2:135-152. Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.