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Differential Response of Cultured Chicken Glial Cells to Infection with Virulent and Non-Virulent Strains of Newcastle Disease Virus1
BLUECOMB T R E A T M E N T S
glucose solution were not necessarily those which were the least ill and they did not seem to benefit from the glucose solution. ACKNOWLEDGMENTS
REFERENCES Duke, G. E., H. E. Dziuk and L. Hawkins, 1969a. Gastrointestinal transit-times in normal and bluecomb diseased turkeys. Poultry Sci. 48:
835-842. Duke, G. E., H. E. Dziuk and 0. A. Evanson, 1969b. Fluxes of ions, glucose, and water in isolated jejunal segments in normal and bluecomb diseased turkeys. Poultry Sci. 48: 2114-2123. Dziuk, H. E., G. E. Duke, O. A. Evanson, D. E. Nelson and P. N. Schultz, 1969a. Force-feeding turkeys during bluecomb disease. Poultry Sci. 48: 843-846. Dziuk, H. E., O. A. Evanson and C. T. Larsen, 1969b. Physiologic effects of fasting and bluecomb in turkeys. Am. J. Vet. Res. 30: 1045-1056. Schultz, P. N., D. E. Nelson, H. E. Dziuk, G. E. Duke and C. T. Larsen, 1970. Hemic studies in normal and bluecomb diseased turkeys. Poultry Sci. 49: 136-145. Talbot, N. B., J. D. Crawford and A. M. Butler, 1953. Homeostatic limits to safe parenteral fluid therapy. New England J. Med. 248: 1100-1108
Differential Response of Cultured Chicken Glial Cells to Infection with Virulent and Non-Virulent Strains of Newcastle Disease Virus 1 E U G E N E V. ADAMS Department of Animal Science, Rutgers—The State University of New Jersey, New Brunswick, New Jersey 08903 (Received for publication September 10, 1969)
T
H E culture characteristics of chicken brain neuroglial cells has been described by Adams (1965). Trypsin-dispersed chicken brains yield a population of cells which readily grow and multiply to form confluent monolayers of glial cells. Although astrocytes and various transitional astrocytic forms predominate in these cultures, small colonies of oligodendroglia usually are also present. Cultured chicken neuroglial cells readily support the growth of Rous sarcoma virus (Adams, 1966, 1968) and avian encephalomyelitis
1 Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers—The State University of New Jersey, Department of Animal Sciences, New Brunswick, New Jersey 08903.
virus (Mancini and Yates, 1967). Although these cells support the multiplication of these two viruses, they do not undergo any apparent cytopathogenic changes. T h e differentiation of velogenic (highly virulent for chickens), mesogenic (less virulent) and lentogenic (non-virulent) strains of Newcastle disease virus ( N D V ) , according to their plaque-forming ability on monolayers of chicken embryo fibroblasts, has been described by Schloer and Hanson (1968) and b y Daniel and Hanson (1968). This report describes the differentiation of neurotropic and non-neurotropic strains of N D V in cultured chicken brain neuroglial cells.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
The authors are grateful to Mrs. Connie Bissonnette and Dr. C. T. Larsen for help in preparing infective material, to D . E. Nelson for technical help, and to Dr. P . E . Poss, Willmar, Minnesota, for advice based on observations in treating field flocks which had bluecomb.
229
230
E. V. ADAMS
MATERIALS AND METHODS
however, their growth curve patterns were not determined. The Rutgers field strain had all the characteristics of a typical velogenic virus when tested in chickens and chicken enbryos. Although the F strain of NDV is supposed to be lentogenic, it behaved more like a mesogenic type when tested in chickens and chicken embryos in our laboratories at Rutgers University. RESULTS
The activities of the various strains of NDV are summarized in Table 1 and Figure 1. The GB strain and Rutgers field isolate (velogenic types) induced the earliest cytopathogenic effects (giant cell formation) which were consistently evident at 24-36 hours after infection. Giant cell induction by the mesogenic Roakin strain of NDV was delayed and usually was not evident until 48 hours after infection. This was also true with the F strain of NDV. In the case of the lentogenic La Sota strain, cytopathogenicity was never observed. However, all NDV strains tested Stock allantoic fluid preparations of multiplied in cultured glial cells. The GB Gilbert-Boney (GB), Roakin and La Sota strain multiplied at the fastest rate, Roastrains of ND V were maintained at — 70°C. kin was intermediate, and the LaSota until used. The tube cultures and the strain had the slowest rate of multiplicaLeighton tube cultures were infected with tion. At no time was a cytopathogenic approximately 2,000 and 5,000 infectious units of virus, respectively. At various effect observed in control cultures. The time intervals after infection, coverslips supernatant fluids from control cultures were removed from the culture tubes, were also negative when tested in chicken washed in Hanks' salt solution, fixed in embryos. methanol and stained with Jenner-Giemsa. Control cells and infected cells exhibitOne-half ml. samples of the Leighton tube ing giant cell formation are shown in Fig. supernatant fluids were taken at specific TABLE 1.—Activity in NDV strains in time intervals, sealed in ampules, and ctdtured chicken glial cells stored at —70° for future titration in 10day-old chicken embryos to determine virus growth patterns. A recent Rutgers field isolate of NDV and the F strain of NDV obtained from the University of Wisconsin NDV repository were also tested in glial cell cultures;
LaSota Roakin F strain Gilbert-Boney Rutgers-field strain
Lentogenic Mesogenic Mesogenic* Velogenic Velogenic
no yes yes yes yes
— 2 days 2 days 1 day 1 day
yes yes yes yes yes
* Although the F strain is considered to be lentogenic, it behaved more like a mesogenic type when tested in our laboratories.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
The tissue culture growth medium consisted of the following: Scherer's maintenance solution, 68%; tryptose phosphate broth, 20%; heat inactivated calf serum, 10%; penicillin G, 100 units/ml; and streptomycin, 100 /ig./ml. The pH was adjusted to 7.4 with 7.5% sodium bicarbonate solution. Whole brains from 5, two-week-old chickens were minced and washed 5 times in Hanks' salt solution and trypsinized for 1 hour in 0.25% trypsin (Difco 1:250) in Hanks' salt solution, The resultant suspension of material was then washed 3 times and suspended at a 1:100 dilution in growth medium. Rubber-stoppered 16X150 mm. culture tubes containing coverslips and 28X120 mm. Leighton tubes were inoculated with 2 ml. and 4 ml., respectively, of the cell suspension. All cultures were incubated at 38°C. and the growth medium was changed every 2 or 3 days until a confluent monolayer of cells developed, generally within 7 to 14 days.
231
N E W CASTLE D I S E A S E V I R U S
Fusion of cultured cells to form giant cells is apparently preceded by viral induced alteration and dissolution of cell membranes (Buthala and Matthews, 1957; and Kohn, 1965). Concomitant
t/
f
f
FIG. 2. Normal cultured chicken brain glial cells, Jenner-Giemsa stain, X400.
with the start of fusion was the disappearance of the distinctive glial cell processes. Cell fusion was rapid and was accompanied by the formation of large vacuoles. Nucleoli were no longer recognizable and clumping of nuclear chromatin material occurred. Nuclear aggregation and fusion was also observed. DISCUSSION The data indicates that there apparently is a direct correlation between the capacity of a N D V strain to induce an early cytopathogenic effect ( C P E ) , its
FIG. 1. Multiplication rates of GB, Roakin and LaSota strains of NDV in chicken brain glial cell cultures.
FIG. 3. Cultured glial giant cell formation induced by the GB strain of NDV, Jenner-Giemsa stain, X 1,000.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
ures 2 and 3, respectively. The first evidence of cytopathogenic change was manifested by the appearance of small multinucleated masses containing from 3 to 10 or more nuclei scattered throughout. These multinucleated cytoplasmic masses rapidly increased in size with some attaining diameters of 1,200 or more microns and containing more than 200 nuclei. After 2 days, only a few normal appearing glial cells with their typical processes remained on the coverslips. Three to four days after infection, complete degeneration had occurred and onlyscattered areas of cellular debris remained on the coverslips. With the exception of the lentogenic LaSota strain, all the N D V strains tested had the capacity to induce the formation of giant cells.
232
E. V. ADAMS
T h e response of cultured chicken brain cells to infection with N D V strains provides a rather sensitive in vitro means of determing whether a strain is neurotropic or non-neurotropic. Schloer and Hanson (1968) and Daniel and Hanson (1968) found t h a t the plaque forming ability of representative N D V strains was also related to virulence for chickens. Large heterogeneous plaques could be associated with velogenic strains while only small plaques were produced by mesogenic strains of N D V . Plaques were rarely formed by lentogenic strains. T h e formation of multinucleated giant cells or polykaryocytes in vitro is one of a number of cytopathogenic effects induced by animal viruses. This type of cellular response is characteristically produced b y measles virus (Enders and Peebles, 1954; Cascardo and Karzon, 1965; Atherton, et al., 1965), mumps virus (Henle et al., 1954), and other viruses. Buthala and Mathews (1957) described the effect of N D V on cultures of chicken embryo kidney tissue. An early effect was the clumping of groups of cells concomitant with
cell wall dissolution. This activity tended toward the formation of large multinucleated polykaryocytes. Within 48 hours, cellular destruction was usually complete. Recently, Kohn (1965) described the induction of polykaryocytes (containing 3 to 150 nuclei) in a variety of continuous lines of cells following infection with a high input multiplicity of N D V . Polykaryocytosis began within an hour after infection, and the extent of cell fusion was proportional to the multiplicity of the virus. Sequential microscopic observations of virus induced polykaryocytosis has led to the general belief t h a t the formation of giant cells is a result of alteration of cell membranes and subsequent aggregation and fusion of affected cells. This also appears to be the mechanism of giant cell formation in the case of chicken brain glial cells infected with N D V . Atherton et al. (1965) also was able to show t h a t polykaryocytes were formed by cell fusion through the use of radioautographic techniques (tritiated thymidine labeled H E p - 2 and h u m a n amnion cells infected with measles virus).
SUMMARY T h e activity of velogenic, mesogenic and lentogenic strains of Newcastle disease virus (NDV) in cultured chicken brain glial cells is related directly to their virulence for chickens. T h e velogenic GB strain was t h e most virulent in chickens, and induced the earliest cytopathogenic effect (giant cells) and multiplied at the fastest rate in vitro. I n contrast, the lentogenic LaSota strain is not virulent in chickens, and multiplied a t the slowest rate in tissue culture and did not induce a cytopathogenic effect. T h e mesogenic Roakin strain was intermediate between the velogenic and lentogenic strains in regard to its virulence in the chicken and its
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
rate of multiplication, titers attained in cultured brain glial cells and its virulence in chickens. I n other words, the velogenic GB strain is most virulent in chickens, and multiplies at the fastest rate, attains the highest titers and induces the earliest C P E in cultured glial cells. The lentogenic LaSota strain is non-virulent in chickens, and does not induce a C P E in vitro, has the slowest multiplication rate and attains the lowest titers in glial cells. Daniel and Hanson (1968) reported t h a t 6 lentogenic strains of N D V failed to produce plaques in chicken embryo monolayer cultures. T h e mesogenic Roakin strain is intermediate between the velogenic and lentogenic strains in regard to its virulence in the chicken and its activity in cultured glial cells.
N E W CASTLE DISEASE VIRUS a c t i v i t y in c u l t u r e d glial cells. O t h e r N D V s t r a i n s w e r e also t e s t e d . REFERENCES
Daniel, M. D., and R. P. Hanson, 1968. Differentiation of representative Newcastle disease virus strains by their plaque-forming ability on monolayers of chick embryo fibroblasts. Avian Dis. 12: 423-433. Enders, J. F., and T. C. Peebles, 1954. Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exptl. Biol. Med. 86:277-286. Henle, G., F. Deinhardt and A. Giardi, 1954. Cytolytic effects of mumps virus in tissue cultures of epithelial cells. Proc. Soc. Exptl. Biol. Med. 87:386-393. Kohn, A., 1965. Polykaryocytosis induced by Newcastle disease virus (NDV) in monolayers of animal cells. Virology, 26: 228-245. Mancini, L. O., and V. J. Yates, 1967. Cultivation of avian encephalomyelitis virus in vitro. 1. In chick embryo neuroglial cell culture. Avian Dis. 11:672-679. Schloer, G., and R. P. Hanson, 1968. Plaque morphology of Newcastle disease virus as influenced by cell type and environmental factors. Am. Vet. Res. 29: 883-895.
Dietary Pesticides and Contamination of Yolks and Abdominal Fat of Laying Hens SANDRA I. SMITH, C. W. WEBER AND B. L. REID Poultry Science Department, University of Arizona, Tucson, Arizona 85721 (Received for publication September 10, 1969)
have shown that chlorinated SURVEYS hydrocarbon pesticide contaminations are widespread and persist in the environment for several years following application. These pesticides are fat soluble, and tend to accumulate in the fatty tissues of hens and in the egg yolk. Hauver (1965) reported that the U.S.D.A. had tested more than 2,000 poultry fat samples for residues and found low levels in chickens, turkeys, ducks, and geese of all ages and in all locations examined. Stadelman et al. (1965) found that exposure to the equivalent of 10 to 15 p.p.m. Arizona Agricultural Experiment Station Journal Article No. 1546.
DDT resulted in 4.6 p.p.m. residue in the egg yolk. The residue remained for 26 weeks after removal of the pesticide from the diet. Many factors which affect pesticide metabolism have been studied in order to develop methods of hastening pesticide depletion from body tissues of poultry. The effects of force molting and dietary protein levels on pesticide depletion were studied by Wesley et al. (1966). They found that forced molting followed by feed restriction to 20% of normal intake of a high protein diet resulted in higher pesticide depletion rates than the same procedure with a low protein diet or a con-
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
Adams, E. V., 1965. Monolayer growth of chicken neuroglial cells in tissue culture. Poultry Sci. 44: 1558-1561. Adams, E. V., 1966. Response of cultured chicken brain neuroglial cells to infection with Rous sarcoma virus. J. Nat. Cancer Inst. 37: 347-352. Adams, E. V., 1968. Culture characteristics of brain neuroglial cells from chickens infected with Rous sarcoma virus. Poultry Sci. 47 951-956. Atherton, J. G., S. D. Chaparas, M. M. Cremer and I. Gordon, 1965. Mechanism of polykaryocytosis associated with noncytopathic infection by measles virus. J. Bacteriol. 90: 213-219. Buthala, D. A., and J. Mathews, 1957. Use of cellular cultures of chicken embryo kidney tissue in virus studies. Cornell Vet. 47: 143-140. Cascardo, M. R., and D. T. Karzon, 1965. Measles virus giant cell inducing factor (fusion factor). Virology, 26: 311-325.
233
glucose solution were not necessarily those which were the least ill and they did not seem to benefit from the glucose solution. ACKNOWLEDGMENTS
REFERENCES Duke, G. E., H. E. Dziuk and L. Hawkins, 1969a. Gastrointestinal transit-times in normal and bluecomb diseased turkeys. Poultry Sci. 48:
835-842. Duke, G. E., H. E. Dziuk and 0. A. Evanson, 1969b. Fluxes of ions, glucose, and water in isolated jejunal segments in normal and bluecomb diseased turkeys. Poultry Sci. 48: 2114-2123. Dziuk, H. E., G. E. Duke, O. A. Evanson, D. E. Nelson and P. N. Schultz, 1969a. Force-feeding turkeys during bluecomb disease. Poultry Sci. 48: 843-846. Dziuk, H. E., O. A. Evanson and C. T. Larsen, 1969b. Physiologic effects of fasting and bluecomb in turkeys. Am. J. Vet. Res. 30: 1045-1056. Schultz, P. N., D. E. Nelson, H. E. Dziuk, G. E. Duke and C. T. Larsen, 1970. Hemic studies in normal and bluecomb diseased turkeys. Poultry Sci. 49: 136-145. Talbot, N. B., J. D. Crawford and A. M. Butler, 1953. Homeostatic limits to safe parenteral fluid therapy. New England J. Med. 248: 1100-1108
Differential Response of Cultured Chicken Glial Cells to Infection with Virulent and Non-Virulent Strains of Newcastle Disease Virus 1 E U G E N E V. ADAMS Department of Animal Science, Rutgers—The State University of New Jersey, New Brunswick, New Jersey 08903 (Received for publication September 10, 1969)
T
H E culture characteristics of chicken brain neuroglial cells has been described by Adams (1965). Trypsin-dispersed chicken brains yield a population of cells which readily grow and multiply to form confluent monolayers of glial cells. Although astrocytes and various transitional astrocytic forms predominate in these cultures, small colonies of oligodendroglia usually are also present. Cultured chicken neuroglial cells readily support the growth of Rous sarcoma virus (Adams, 1966, 1968) and avian encephalomyelitis
1 Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers—The State University of New Jersey, Department of Animal Sciences, New Brunswick, New Jersey 08903.
virus (Mancini and Yates, 1967). Although these cells support the multiplication of these two viruses, they do not undergo any apparent cytopathogenic changes. T h e differentiation of velogenic (highly virulent for chickens), mesogenic (less virulent) and lentogenic (non-virulent) strains of Newcastle disease virus ( N D V ) , according to their plaque-forming ability on monolayers of chicken embryo fibroblasts, has been described by Schloer and Hanson (1968) and b y Daniel and Hanson (1968). This report describes the differentiation of neurotropic and non-neurotropic strains of N D V in cultured chicken brain neuroglial cells.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
The authors are grateful to Mrs. Connie Bissonnette and Dr. C. T. Larsen for help in preparing infective material, to D . E. Nelson for technical help, and to Dr. P . E . Poss, Willmar, Minnesota, for advice based on observations in treating field flocks which had bluecomb.
229
230
E. V. ADAMS
MATERIALS AND METHODS
however, their growth curve patterns were not determined. The Rutgers field strain had all the characteristics of a typical velogenic virus when tested in chickens and chicken enbryos. Although the F strain of NDV is supposed to be lentogenic, it behaved more like a mesogenic type when tested in chickens and chicken embryos in our laboratories at Rutgers University. RESULTS
The activities of the various strains of NDV are summarized in Table 1 and Figure 1. The GB strain and Rutgers field isolate (velogenic types) induced the earliest cytopathogenic effects (giant cell formation) which were consistently evident at 24-36 hours after infection. Giant cell induction by the mesogenic Roakin strain of NDV was delayed and usually was not evident until 48 hours after infection. This was also true with the F strain of NDV. In the case of the lentogenic La Sota strain, cytopathogenicity was never observed. However, all NDV strains tested Stock allantoic fluid preparations of multiplied in cultured glial cells. The GB Gilbert-Boney (GB), Roakin and La Sota strain multiplied at the fastest rate, Roastrains of ND V were maintained at — 70°C. kin was intermediate, and the LaSota until used. The tube cultures and the strain had the slowest rate of multiplicaLeighton tube cultures were infected with tion. At no time was a cytopathogenic approximately 2,000 and 5,000 infectious units of virus, respectively. At various effect observed in control cultures. The time intervals after infection, coverslips supernatant fluids from control cultures were removed from the culture tubes, were also negative when tested in chicken washed in Hanks' salt solution, fixed in embryos. methanol and stained with Jenner-Giemsa. Control cells and infected cells exhibitOne-half ml. samples of the Leighton tube ing giant cell formation are shown in Fig. supernatant fluids were taken at specific TABLE 1.—Activity in NDV strains in time intervals, sealed in ampules, and ctdtured chicken glial cells stored at —70° for future titration in 10day-old chicken embryos to determine virus growth patterns. A recent Rutgers field isolate of NDV and the F strain of NDV obtained from the University of Wisconsin NDV repository were also tested in glial cell cultures;
LaSota Roakin F strain Gilbert-Boney Rutgers-field strain
Lentogenic Mesogenic Mesogenic* Velogenic Velogenic
no yes yes yes yes
— 2 days 2 days 1 day 1 day
yes yes yes yes yes
* Although the F strain is considered to be lentogenic, it behaved more like a mesogenic type when tested in our laboratories.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
The tissue culture growth medium consisted of the following: Scherer's maintenance solution, 68%; tryptose phosphate broth, 20%; heat inactivated calf serum, 10%; penicillin G, 100 units/ml; and streptomycin, 100 /ig./ml. The pH was adjusted to 7.4 with 7.5% sodium bicarbonate solution. Whole brains from 5, two-week-old chickens were minced and washed 5 times in Hanks' salt solution and trypsinized for 1 hour in 0.25% trypsin (Difco 1:250) in Hanks' salt solution, The resultant suspension of material was then washed 3 times and suspended at a 1:100 dilution in growth medium. Rubber-stoppered 16X150 mm. culture tubes containing coverslips and 28X120 mm. Leighton tubes were inoculated with 2 ml. and 4 ml., respectively, of the cell suspension. All cultures were incubated at 38°C. and the growth medium was changed every 2 or 3 days until a confluent monolayer of cells developed, generally within 7 to 14 days.
231
N E W CASTLE D I S E A S E V I R U S
Fusion of cultured cells to form giant cells is apparently preceded by viral induced alteration and dissolution of cell membranes (Buthala and Matthews, 1957; and Kohn, 1965). Concomitant
t/
f
f
FIG. 2. Normal cultured chicken brain glial cells, Jenner-Giemsa stain, X400.
with the start of fusion was the disappearance of the distinctive glial cell processes. Cell fusion was rapid and was accompanied by the formation of large vacuoles. Nucleoli were no longer recognizable and clumping of nuclear chromatin material occurred. Nuclear aggregation and fusion was also observed. DISCUSSION The data indicates that there apparently is a direct correlation between the capacity of a N D V strain to induce an early cytopathogenic effect ( C P E ) , its
FIG. 1. Multiplication rates of GB, Roakin and LaSota strains of NDV in chicken brain glial cell cultures.
FIG. 3. Cultured glial giant cell formation induced by the GB strain of NDV, Jenner-Giemsa stain, X 1,000.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
ures 2 and 3, respectively. The first evidence of cytopathogenic change was manifested by the appearance of small multinucleated masses containing from 3 to 10 or more nuclei scattered throughout. These multinucleated cytoplasmic masses rapidly increased in size with some attaining diameters of 1,200 or more microns and containing more than 200 nuclei. After 2 days, only a few normal appearing glial cells with their typical processes remained on the coverslips. Three to four days after infection, complete degeneration had occurred and onlyscattered areas of cellular debris remained on the coverslips. With the exception of the lentogenic LaSota strain, all the N D V strains tested had the capacity to induce the formation of giant cells.
232
E. V. ADAMS
T h e response of cultured chicken brain cells to infection with N D V strains provides a rather sensitive in vitro means of determing whether a strain is neurotropic or non-neurotropic. Schloer and Hanson (1968) and Daniel and Hanson (1968) found t h a t the plaque forming ability of representative N D V strains was also related to virulence for chickens. Large heterogeneous plaques could be associated with velogenic strains while only small plaques were produced by mesogenic strains of N D V . Plaques were rarely formed by lentogenic strains. T h e formation of multinucleated giant cells or polykaryocytes in vitro is one of a number of cytopathogenic effects induced by animal viruses. This type of cellular response is characteristically produced b y measles virus (Enders and Peebles, 1954; Cascardo and Karzon, 1965; Atherton, et al., 1965), mumps virus (Henle et al., 1954), and other viruses. Buthala and Mathews (1957) described the effect of N D V on cultures of chicken embryo kidney tissue. An early effect was the clumping of groups of cells concomitant with
cell wall dissolution. This activity tended toward the formation of large multinucleated polykaryocytes. Within 48 hours, cellular destruction was usually complete. Recently, Kohn (1965) described the induction of polykaryocytes (containing 3 to 150 nuclei) in a variety of continuous lines of cells following infection with a high input multiplicity of N D V . Polykaryocytosis began within an hour after infection, and the extent of cell fusion was proportional to the multiplicity of the virus. Sequential microscopic observations of virus induced polykaryocytosis has led to the general belief t h a t the formation of giant cells is a result of alteration of cell membranes and subsequent aggregation and fusion of affected cells. This also appears to be the mechanism of giant cell formation in the case of chicken brain glial cells infected with N D V . Atherton et al. (1965) also was able to show t h a t polykaryocytes were formed by cell fusion through the use of radioautographic techniques (tritiated thymidine labeled H E p - 2 and h u m a n amnion cells infected with measles virus).
SUMMARY T h e activity of velogenic, mesogenic and lentogenic strains of Newcastle disease virus (NDV) in cultured chicken brain glial cells is related directly to their virulence for chickens. T h e velogenic GB strain was t h e most virulent in chickens, and induced the earliest cytopathogenic effect (giant cells) and multiplied at the fastest rate in vitro. I n contrast, the lentogenic LaSota strain is not virulent in chickens, and multiplied a t the slowest rate in tissue culture and did not induce a cytopathogenic effect. T h e mesogenic Roakin strain was intermediate between the velogenic and lentogenic strains in regard to its virulence in the chicken and its
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
rate of multiplication, titers attained in cultured brain glial cells and its virulence in chickens. I n other words, the velogenic GB strain is most virulent in chickens, and multiplies at the fastest rate, attains the highest titers and induces the earliest C P E in cultured glial cells. The lentogenic LaSota strain is non-virulent in chickens, and does not induce a C P E in vitro, has the slowest multiplication rate and attains the lowest titers in glial cells. Daniel and Hanson (1968) reported t h a t 6 lentogenic strains of N D V failed to produce plaques in chicken embryo monolayer cultures. T h e mesogenic Roakin strain is intermediate between the velogenic and lentogenic strains in regard to its virulence in the chicken and its activity in cultured glial cells.
N E W CASTLE DISEASE VIRUS a c t i v i t y in c u l t u r e d glial cells. O t h e r N D V s t r a i n s w e r e also t e s t e d . REFERENCES
Daniel, M. D., and R. P. Hanson, 1968. Differentiation of representative Newcastle disease virus strains by their plaque-forming ability on monolayers of chick embryo fibroblasts. Avian Dis. 12: 423-433. Enders, J. F., and T. C. Peebles, 1954. Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exptl. Biol. Med. 86:277-286. Henle, G., F. Deinhardt and A. Giardi, 1954. Cytolytic effects of mumps virus in tissue cultures of epithelial cells. Proc. Soc. Exptl. Biol. Med. 87:386-393. Kohn, A., 1965. Polykaryocytosis induced by Newcastle disease virus (NDV) in monolayers of animal cells. Virology, 26: 228-245. Mancini, L. O., and V. J. Yates, 1967. Cultivation of avian encephalomyelitis virus in vitro. 1. In chick embryo neuroglial cell culture. Avian Dis. 11:672-679. Schloer, G., and R. P. Hanson, 1968. Plaque morphology of Newcastle disease virus as influenced by cell type and environmental factors. Am. Vet. Res. 29: 883-895.
Dietary Pesticides and Contamination of Yolks and Abdominal Fat of Laying Hens SANDRA I. SMITH, C. W. WEBER AND B. L. REID Poultry Science Department, University of Arizona, Tucson, Arizona 85721 (Received for publication September 10, 1969)
have shown that chlorinated SURVEYS hydrocarbon pesticide contaminations are widespread and persist in the environment for several years following application. These pesticides are fat soluble, and tend to accumulate in the fatty tissues of hens and in the egg yolk. Hauver (1965) reported that the U.S.D.A. had tested more than 2,000 poultry fat samples for residues and found low levels in chickens, turkeys, ducks, and geese of all ages and in all locations examined. Stadelman et al. (1965) found that exposure to the equivalent of 10 to 15 p.p.m. Arizona Agricultural Experiment Station Journal Article No. 1546.
DDT resulted in 4.6 p.p.m. residue in the egg yolk. The residue remained for 26 weeks after removal of the pesticide from the diet. Many factors which affect pesticide metabolism have been studied in order to develop methods of hastening pesticide depletion from body tissues of poultry. The effects of force molting and dietary protein levels on pesticide depletion were studied by Wesley et al. (1966). They found that forced molting followed by feed restriction to 20% of normal intake of a high protein diet resulted in higher pesticide depletion rates than the same procedure with a low protein diet or a con-
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 27, 2015
Adams, E. V., 1965. Monolayer growth of chicken neuroglial cells in tissue culture. Poultry Sci. 44: 1558-1561. Adams, E. V., 1966. Response of cultured chicken brain neuroglial cells to infection with Rous sarcoma virus. J. Nat. Cancer Inst. 37: 347-352. Adams, E. V., 1968. Culture characteristics of brain neuroglial cells from chickens infected with Rous sarcoma virus. Poultry Sci. 47 951-956. Atherton, J. G., S. D. Chaparas, M. M. Cremer and I. Gordon, 1965. Mechanism of polykaryocytosis associated with noncytopathic infection by measles virus. J. Bacteriol. 90: 213-219. Buthala, D. A., and J. Mathews, 1957. Use of cellular cultures of chicken embryo kidney tissue in virus studies. Cornell Vet. 47: 143-140. Cascardo, M. R., and D. T. Karzon, 1965. Measles virus giant cell inducing factor (fusion factor). Virology, 26: 311-325.
233