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The Effects of Sonication, Freezing and Lyophilization on JMV Leukosis Strain*
The Effects of Sonication, Freezing and Lyophilization on JMV Leukosis Strain* KEITH HAFFER and MARTIN SEVOIAN Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01002 (Received for publication July 18, 1977) ABSTRACT JMV lymphoblastic leukemic cells were subjected to sonication, followed by freezing and lyophilization in an attempt to learn the tolerance of JMV cells to these treatments. Sonication experiments indicated that a high percentage of cell breakage (>89%) is necessary for any decrease in lethality to be observed. Freezing experiments involving a wide range of cryoprotectors demonstrated 2M glycerol to be the best for JMV preservation. Subsequent freeze-drying of sonicated, frozen JMV preparations, of high titer, consistently resulted in all loss of lethality.
MATERIALS AND METHODS Chicks. Day-old DeKalb Warren (commercial flock) or Tillson farm (University of Massachusetts flock) N.H. Red chicks were used in the experiments. Cryoprotective agents. The following cryoprotective agents were used in the freezing and freeze drying trials: Phosphate buffered saline (PBS), pH 7.2, 5% polyvinylpyrrolidone (PVP); 20% dimethyl sulfoxide (DMSO) in 15% calf serum (Spencer and Calnek, 1967); SPGA (0.218M sucrose, 0.0038M monopotassium phosphate, 0.0072M dipotassium phosphate, 0.0049M monosodium glutamate, and 1% bovine albumin powder according to Bovarnick et al, 1950); SPGA + .2% EDTA; 2M glycerol; skim milk + 2% NZ amine (Calnek et al, 1970) and Eagle's medium with 15 glycerol and 20% calf serum (Rapp and Benyesh-Melnick, 1963). JMV leukosis strain. JMV was obtained from blood, livers and spleens of JMV moribund chicks, 4 days post-inoculation. Livers and spleens were diluted 1:5 (w./v.) with PBS or SPGA and ground in a Ten-Broek tissue grinder. This homogenate and whole citrated blood were the sources of JMV used in the experiments. Experiment 1. Sonication. JMV blood samples were kept in an ice bath and sonicated (Bronwill Biosonik IIA, 20 kc./sec. at 100% intensity) at 60% intensity for 30 sec. or 100% intensity for 10 sec, 1 min. or 2 min. Cell counts were made prior to and following sonic disruptions with a Neubauer Hemacytometer. Control (unsonicated JMV blood samples) and
•Contribution #2150 of the Massachusetts Agricultural Experiment Station. 95
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
INTRODUCTION By rapid passage of JM strain of Marek's disease (Sevoian et al, 1962) in chicks, a highly antigenic and virulent strain (JMV) was obtained that could produce a lethal lymphoblastic leukemia in 3—8 days (Sevoian, 1967). Infectivity can be experimentally transferred by inoculation of tumor suspensions or blood obtained from JMV moribund chicks. It has been demonstrated by Ficoll-Paque separations and fluorescent antibody studies, that infectivity is transferred with the white elements of the blood, specifically lymphoblasts (unpublished data). The true nature of JMV has yet to be conclusively established, as both evidence for a viral nature (Hamdy and Sevoian, 1973; Kenyon et al, 1969; and Sevoian and Weston, 1972), and a cellular nature (Olmstead and Kenyon, 1971; Stephens et al, 1976; Witter et al, 1975; Munch and Sevoian, 1977) have been indicated. This study describes: 1. the degree of cell breakage and significant changes in lethality (LDso/ml.) that various degrees and lengths of sonication have on JMV strain; 2. the protection of various cryoprotective agents on the viability and infectivity of JMV strain during the freezing process; 3. the effects of freeze drying on sonicated JMV preparations.
96
HAFFER AND SEVOIAN
RESULTS Experiment 1. Sonication (Table 1 and Fig. 1). JMV blood sonicated for 10 sec. at 100% intensity caused a 60% breakage of the white cell population, but was not reflected as a significant change in lethality (LD s 0 /ml.). Similarly, no significant changes in lethality were observed when 76% of the white cell population was destroyed, when sonicated at 60% intensity for 30 sec. Sonication of JMV strain at 100% intensity for 1 min. resulted in an 89% breakage of the white cell population and was reflected as a 3 log 1 0 loss in lethality. Sonication at 100% intensity, total time of 2 min., resulted in 100% cell breakage and a total loss of lethality (LD 5 0 /ml. = 0.00).
TABLE 1.-Changes in JMVLD50/ml. induced by sonicated cell breakage Sonication intensity 60% 100%
Duration of sonication (seconds)
% white cell breakage
Change LD5 0 /ml. (logi 0 )
30 10 60 120
76 60 89 100
0.375 0.00 3.0741 6.1981
'Statistically significant change in LDS0/ml., determined by Spaerman-Karber method.
Experiment 2. Freezing (Table 2). The freezing of sonicated JMV suspensions without the addition of a cryoprotector, or diluted 1:1 with PBS, resulted in a total loss of lethality. The various cryoprotectors (penetrating, colloidal and polymeric) varied in their degree of protectivity during the freezing process. Significant protection was only observed with 2 M glycerol as a diluent. Experiment 3. Freeze-Drying (Table 3). Freeze-drying of sonicated, frozen JMV suspensions (SPGA, SPGA + .2% EDTA, 5% PVP, or skim milk + 2% NZ Amine) resulted in a total loss of lethality in all trials. Experiment 4. Studies of cell-free components of JMV cells. The supernatant obtained from centrifugation of sonicated JMV preparations, 2120g. for 15 min. (a force that sedi-
Percent Remaining Whole _ Cells *
0
L0
20
30
'»U
50
60
70
80
90
LOU
LL0
120
Duration of Sonionlion at 100% Intensity (sec)
FIG. 1. Percentage of whole white cells remaining after various durations of sonication at 100% intensity. 2100% whole cells corresponds to unsonicated materials. 3 Corresponds to 76% cell breakage due to sonication at 60% intensity for 30 seconds.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
sonicated samples were serially diluted ten fold and inoculated intraperitoneally into day-old chicks (0.2 ml./chick) for titrations. Uninfected controls were also kept in each experiment. Experiment 2. Freezing. Sonicated (60% for 30 sec.) JMV suspensions (previously titrated) were diluted 1:1 with the various cryoprotectors and slow frozen on the shelf of the Virtis Automatic Freeze Dryer, 10—800. The frozen samples were allowed to thaw at room temperature, were then serially diluted ten-fold, and inoculated intraperitoneally into chicks (0.2 ml, chick). Experiment 3. Freeze-Drying. Frozen JMV suspensions (previously titrated) were freeze dried for 48 hr. on the shelf of the Virtis Automatic Freeze Dryer, 10—800. The samples were sealed under vacuum and allowed to come to room temperature. At this time they were rehydrated, serially diluted, ten-fold and inoculated intraperitoneally into day-old chicks (0.2 ml. chick). Experiment 4. Studies on cell-free components of JMV cells. Sonicated (60% for 30 sec. or 100% for 1 or 2 min.) JMV suspensions were studied in order to isolate a cell-free component of the JMV cells. Centrifugation at 2120g for 15 min. and filtration studies through 0.45 |U. Millipore filters were used to define if a cell-free infectious component did exist. Statistical Analysis. Lethality fifty percent end points (LD 5 0 /ml.) were made using the method described by Reed and Muench (Reed and Muench, 1938). The Spaerman-Karber method (Finney, 1952) was used to obtain the 95% confidence limits of the results.
JMV LEUKOSIS STRAIN
97
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
TABLE 2.—Effects of various cryoprotective agents vaccinated birds. Although Marek's disease tuon the preservation of JMV cells, mors do not often manifest themselves in these expressed as losses in LD5 0/ml birds, a dual infection is present (Purchase et al., 1972; Sevoian 89%) have to
98
HAFFER AND SEVOIAN
be disrupted before any significant change in L D 5 0 / m l . is n o t e d ; 2. J M V can b e successfully frozen w i t h o u t significant losses in lethality; 3. sonicated J M V preparations c a n n o t be lyophilized w i t h o u t total loss in lethality; and 4. t h e infectious agent in J M V l y m p h o b l a s t i c leukemia appears t o be an intact cell.
REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
Bovarnick, Marianna R., Judith C. Miller and J. C. Snyder. 1950. The influence of certain salts, amino acid, sugars and proteins on the stability of rickettsiae. J. Bacter. 59:509-522. Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herepesvirus and a herpesvirus of turkeys. Appl. Micro., 20:723-726. Finney, D. J., 1952. Statistical Methods in Biological Assay. Charles Griffin and Co., Ltd., London, England. Hamdy, F. M., and M. Sevoian, 1973. Immunologic relationship between JM and JMV leukosis strains and h e r p e s v i r u s of turkeys. Avian Dis., 17:476-485. Hong, C. C , and M. Sevoian, 1974. The comparative influence of passive (maternal) antibodies in early esposure to JM virus of progeny chicks. Avian Dis., 18:305-317. Kenyon, A. J.; M. Sevoian, M. Horwitz, N. D. Jones and C. F. Helmboldt. Lymphoproliferative disease of fowl — immunologic factors associated with passage of a lymphoblastic leukemia (JMV), 1969. Avian Dis., 13:585-595. Munch, David, and M. Sevoian, 1977. The in vitro establishment of avian lymphoblastic leukemia cells (JMV). Infection and Immunity. (In press). Olmsted, J., and A. J. Kenyon, 1971. Lymphoproliferative disease of fowl — radiologic evidence of a transplantable nature of lymphoblastic leukemia (JMV). Avian Dis., 15:672-679.
Purchase, H. G., W. Okazaki and B. R. Burmester, 1972. Long term field trials with the herpesvirus of turkeys vaccine against Marek's disease. Avian Dis., 16:57-71. Rapp, F., and M. Benyesh-Melnick, 1963. Plaque assay for measurement of cells infected with zoster virus. Science, 141:433-434. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Am. J. Hyg., 27:493-497. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. I. Experimental reproduction of neural and visceral forms. Vet. Med., 57:500-501. Sevoian, M. Immunity studies in chickens inoculated with a virulent Type II avian leukosis strain (JMV), 1967. Proc. 39th N.E. Conference on Avian Dis., State Univ. of N.Y., Stony Brook, Long Island, New York, June 1 9 - 2 1 . Sevoian, M., and C. R. Weston, 1972. The effects of JM and JMV leukosis strain on chicks vaccinated with herpesvirus of turkeys. Poultry Sci., 51:513— 516. Sevoian, M., C. C. Hong and H. Shieh, 1973. Immunologic studies of Type II (Marek's leukosis). World Vet. Poultry Congress, Munich, p. 178—180. Spencer, J. L., and B. W. Calnek, 1967. Storage of cells infected with Rous sarcoma virus or JM strain avian l y m p h o m a t o s i s agent. Avian Dis., 11:274-287. Stephens, E. A., R. L. Witter, L. F. Lee, J. M. Sharma, K. Nazerian and B. M. Longenecker. Characteristics of JMV Marek's disease tumor: A non-productively infected transplantable cell lacking in rescuable virus. J. Natl. Cancer Inst., 1976, in press. 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. Witter, R. L., E. A. Stephens, J. M. Sharma and K. Nazerian, 1975. Demonstration of a tumor-associated surface antigen in Marek's disease. J. Imm., 115:177-183.
MATERIALS AND METHODS Chicks. Day-old DeKalb Warren (commercial flock) or Tillson farm (University of Massachusetts flock) N.H. Red chicks were used in the experiments. Cryoprotective agents. The following cryoprotective agents were used in the freezing and freeze drying trials: Phosphate buffered saline (PBS), pH 7.2, 5% polyvinylpyrrolidone (PVP); 20% dimethyl sulfoxide (DMSO) in 15% calf serum (Spencer and Calnek, 1967); SPGA (0.218M sucrose, 0.0038M monopotassium phosphate, 0.0072M dipotassium phosphate, 0.0049M monosodium glutamate, and 1% bovine albumin powder according to Bovarnick et al, 1950); SPGA + .2% EDTA; 2M glycerol; skim milk + 2% NZ amine (Calnek et al, 1970) and Eagle's medium with 15 glycerol and 20% calf serum (Rapp and Benyesh-Melnick, 1963). JMV leukosis strain. JMV was obtained from blood, livers and spleens of JMV moribund chicks, 4 days post-inoculation. Livers and spleens were diluted 1:5 (w./v.) with PBS or SPGA and ground in a Ten-Broek tissue grinder. This homogenate and whole citrated blood were the sources of JMV used in the experiments. Experiment 1. Sonication. JMV blood samples were kept in an ice bath and sonicated (Bronwill Biosonik IIA, 20 kc./sec. at 100% intensity) at 60% intensity for 30 sec. or 100% intensity for 10 sec, 1 min. or 2 min. Cell counts were made prior to and following sonic disruptions with a Neubauer Hemacytometer. Control (unsonicated JMV blood samples) and
•Contribution #2150 of the Massachusetts Agricultural Experiment Station. 95
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
INTRODUCTION By rapid passage of JM strain of Marek's disease (Sevoian et al, 1962) in chicks, a highly antigenic and virulent strain (JMV) was obtained that could produce a lethal lymphoblastic leukemia in 3—8 days (Sevoian, 1967). Infectivity can be experimentally transferred by inoculation of tumor suspensions or blood obtained from JMV moribund chicks. It has been demonstrated by Ficoll-Paque separations and fluorescent antibody studies, that infectivity is transferred with the white elements of the blood, specifically lymphoblasts (unpublished data). The true nature of JMV has yet to be conclusively established, as both evidence for a viral nature (Hamdy and Sevoian, 1973; Kenyon et al, 1969; and Sevoian and Weston, 1972), and a cellular nature (Olmstead and Kenyon, 1971; Stephens et al, 1976; Witter et al, 1975; Munch and Sevoian, 1977) have been indicated. This study describes: 1. the degree of cell breakage and significant changes in lethality (LDso/ml.) that various degrees and lengths of sonication have on JMV strain; 2. the protection of various cryoprotective agents on the viability and infectivity of JMV strain during the freezing process; 3. the effects of freeze drying on sonicated JMV preparations.
96
HAFFER AND SEVOIAN
RESULTS Experiment 1. Sonication (Table 1 and Fig. 1). JMV blood sonicated for 10 sec. at 100% intensity caused a 60% breakage of the white cell population, but was not reflected as a significant change in lethality (LD s 0 /ml.). Similarly, no significant changes in lethality were observed when 76% of the white cell population was destroyed, when sonicated at 60% intensity for 30 sec. Sonication of JMV strain at 100% intensity for 1 min. resulted in an 89% breakage of the white cell population and was reflected as a 3 log 1 0 loss in lethality. Sonication at 100% intensity, total time of 2 min., resulted in 100% cell breakage and a total loss of lethality (LD 5 0 /ml. = 0.00).
TABLE 1.-Changes in JMVLD50/ml. induced by sonicated cell breakage Sonication intensity 60% 100%
Duration of sonication (seconds)
% white cell breakage
Change LD5 0 /ml. (logi 0 )
30 10 60 120
76 60 89 100
0.375 0.00 3.0741 6.1981
'Statistically significant change in LDS0/ml., determined by Spaerman-Karber method.
Experiment 2. Freezing (Table 2). The freezing of sonicated JMV suspensions without the addition of a cryoprotector, or diluted 1:1 with PBS, resulted in a total loss of lethality. The various cryoprotectors (penetrating, colloidal and polymeric) varied in their degree of protectivity during the freezing process. Significant protection was only observed with 2 M glycerol as a diluent. Experiment 3. Freeze-Drying (Table 3). Freeze-drying of sonicated, frozen JMV suspensions (SPGA, SPGA + .2% EDTA, 5% PVP, or skim milk + 2% NZ Amine) resulted in a total loss of lethality in all trials. Experiment 4. Studies of cell-free components of JMV cells. The supernatant obtained from centrifugation of sonicated JMV preparations, 2120g. for 15 min. (a force that sedi-
Percent Remaining Whole _ Cells *
0
L0
20
30
'»U
50
60
70
80
90
LOU
LL0
120
Duration of Sonionlion at 100% Intensity (sec)
FIG. 1. Percentage of whole white cells remaining after various durations of sonication at 100% intensity. 2100% whole cells corresponds to unsonicated materials. 3 Corresponds to 76% cell breakage due to sonication at 60% intensity for 30 seconds.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
sonicated samples were serially diluted ten fold and inoculated intraperitoneally into day-old chicks (0.2 ml./chick) for titrations. Uninfected controls were also kept in each experiment. Experiment 2. Freezing. Sonicated (60% for 30 sec.) JMV suspensions (previously titrated) were diluted 1:1 with the various cryoprotectors and slow frozen on the shelf of the Virtis Automatic Freeze Dryer, 10—800. The frozen samples were allowed to thaw at room temperature, were then serially diluted ten-fold, and inoculated intraperitoneally into chicks (0.2 ml, chick). Experiment 3. Freeze-Drying. Frozen JMV suspensions (previously titrated) were freeze dried for 48 hr. on the shelf of the Virtis Automatic Freeze Dryer, 10—800. The samples were sealed under vacuum and allowed to come to room temperature. At this time they were rehydrated, serially diluted, ten-fold and inoculated intraperitoneally into day-old chicks (0.2 ml. chick). Experiment 4. Studies on cell-free components of JMV cells. Sonicated (60% for 30 sec. or 100% for 1 or 2 min.) JMV suspensions were studied in order to isolate a cell-free component of the JMV cells. Centrifugation at 2120g for 15 min. and filtration studies through 0.45 |U. Millipore filters were used to define if a cell-free infectious component did exist. Statistical Analysis. Lethality fifty percent end points (LD 5 0 /ml.) were made using the method described by Reed and Muench (Reed and Muench, 1938). The Spaerman-Karber method (Finney, 1952) was used to obtain the 95% confidence limits of the results.
JMV LEUKOSIS STRAIN
97
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
TABLE 2.—Effects of various cryoprotective agents vaccinated birds. Although Marek's disease tuon the preservation of JMV cells, mors do not often manifest themselves in these expressed as losses in LD5 0/ml birds, a dual infection is present (Purchase et al., 1972; Sevoian 89%) have to
98
HAFFER AND SEVOIAN
be disrupted before any significant change in L D 5 0 / m l . is n o t e d ; 2. J M V can b e successfully frozen w i t h o u t significant losses in lethality; 3. sonicated J M V preparations c a n n o t be lyophilized w i t h o u t total loss in lethality; and 4. t h e infectious agent in J M V l y m p h o b l a s t i c leukemia appears t o be an intact cell.
REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 23, 2015
Bovarnick, Marianna R., Judith C. Miller and J. C. Snyder. 1950. The influence of certain salts, amino acid, sugars and proteins on the stability of rickettsiae. J. Bacter. 59:509-522. Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herepesvirus and a herpesvirus of turkeys. Appl. Micro., 20:723-726. Finney, D. J., 1952. Statistical Methods in Biological Assay. Charles Griffin and Co., Ltd., London, England. Hamdy, F. M., and M. Sevoian, 1973. Immunologic relationship between JM and JMV leukosis strains and h e r p e s v i r u s of turkeys. Avian Dis., 17:476-485. Hong, C. C , and M. Sevoian, 1974. The comparative influence of passive (maternal) antibodies in early esposure to JM virus of progeny chicks. Avian Dis., 18:305-317. Kenyon, A. J.; M. Sevoian, M. Horwitz, N. D. Jones and C. F. Helmboldt. Lymphoproliferative disease of fowl — immunologic factors associated with passage of a lymphoblastic leukemia (JMV), 1969. Avian Dis., 13:585-595. Munch, David, and M. Sevoian, 1977. The in vitro establishment of avian lymphoblastic leukemia cells (JMV). Infection and Immunity. (In press). Olmsted, J., and A. J. Kenyon, 1971. Lymphoproliferative disease of fowl — radiologic evidence of a transplantable nature of lymphoblastic leukemia (JMV). Avian Dis., 15:672-679.
Purchase, H. G., W. Okazaki and B. R. Burmester, 1972. Long term field trials with the herpesvirus of turkeys vaccine against Marek's disease. Avian Dis., 16:57-71. Rapp, F., and M. Benyesh-Melnick, 1963. Plaque assay for measurement of cells infected with zoster virus. Science, 141:433-434. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Am. J. Hyg., 27:493-497. Sevoian, M., D. M. Chamberlain and F. T. Counter, 1962. Avian lymphomatosis. I. Experimental reproduction of neural and visceral forms. Vet. Med., 57:500-501. Sevoian, M. Immunity studies in chickens inoculated with a virulent Type II avian leukosis strain (JMV), 1967. Proc. 39th N.E. Conference on Avian Dis., State Univ. of N.Y., Stony Brook, Long Island, New York, June 1 9 - 2 1 . Sevoian, M., and C. R. Weston, 1972. The effects of JM and JMV leukosis strain on chicks vaccinated with herpesvirus of turkeys. Poultry Sci., 51:513— 516. Sevoian, M., C. C. Hong and H. Shieh, 1973. Immunologic studies of Type II (Marek's leukosis). World Vet. Poultry Congress, Munich, p. 178—180. Spencer, J. L., and B. W. Calnek, 1967. Storage of cells infected with Rous sarcoma virus or JM strain avian l y m p h o m a t o s i s agent. Avian Dis., 11:274-287. Stephens, E. A., R. L. Witter, L. F. Lee, J. M. Sharma, K. Nazerian and B. M. Longenecker. Characteristics of JMV Marek's disease tumor: A non-productively infected transplantable cell lacking in rescuable virus. J. Natl. Cancer Inst., 1976, in press. 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. Witter, R. L., E. A. Stephens, J. M. Sharma and K. Nazerian, 1975. Demonstration of a tumor-associated surface antigen in Marek's disease. J. Imm., 115:177-183.