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Pre-Incubation Inoculation of Eggs with Newcastle Disease Virus*†
Pre-Incubation Inoculation of Eggs with Newcastle Disease Virus*1" E. R. DOLL, M. ELIZABETH WALLACE AND WILLIAM H. MCCOLLTJM Department of Animal Pathology, Kentucky Agricultural Experiment Station, Lexington, Kentucky (Received for publication March 6, 1950)
HE possibility of infection in newly hatched chicks from Newcastle disease virus present in the eggs of sick, recovered or vaccinated hens has received the attention of several investigators. Van Roekel (1946), Jungherr (1946, 1947), and Thompson and Osteen (1948) isolated Newcastle disease virus from fresh eggs. Beaudette et al. (1948) isolated the virus from the ovarian yolk of affected hens. De Lay (1947) obtained Newcastle disease virus from incubated infertile eggs and from embryos which died on the 15th and 17th days of incubation. Hofstad (1949) isolated Newcastle disease virus at the 10th day of incubation from infertile eggs and dead embryos. Prier et al. (1950) isolated the virus from eggs laid on the 4th and 5 th days following vaccination with living virus vaccine. De Lay (1947) reported isolation of Newcastle disease virus from the yolk-sac content of four-day-old chicks hatched from eggs collected from infected flocks. This investigation was undertaken to determine the effect of injection of known
* The investigation reported in this paper is in connection with a project of the Kentucky Agri-.v cultural Experiment Station and is published by permission of the Director. t This study was conducted as a segment of Research and Marketing Act Regional Project S6— Newcastle Disease of Poultry. The cooperating agencies under this project are Kentucky, Virginia, and Texas Agricultural Experiment Stations. The research reported in this paper was done solely at the Kentucky Station. 582
quantities of Newcastle disease virus into eggs before incubation. MATERIALS AND METHODS The eggs used in this study were obtained from hens known to have had no previous infection or vaccination with Newcastle disease virus. Injections of virus were made into the yolk or albumen immediately before placing the eggs in the incubator. The eggs were kept in the horizontal position for several hours before the injections were made. For injection of the yolk, the shell surface was disinfected with an iodine-alcohol mixture, the shell pierced with a lancet, and the inoculum injected through a 27-gauge, |-inch needle. Injections into the albumen were made at the small end of the egg or at the airsac margin. The holes in the shells were sealed with flexible collodion immediately after the injections were completed. The volume of inoculum was 0.05 ml. of physiological saline dilutions of amnioallantoic fluid virus. The doses of Newcastle disease virus were 101, 102, 103, and 104 embryo LD50, calculated by the method of Reed and Muench (1938). Incubation was in a forced draft incubator at 99°F. For isolation of virus from the inoculated eggs, entire embryos were collected when death occurred previous to the 8th or 9th day candling. For embryos dying from the 10th to 15th days, amnioallantoic fluids and embryo livers were used. Suspensions of embryo liver were used for those dying from the 16th to 21st days,
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
T
PRE-INCUBATION INOCULATION OF EGGS WITH NEWCASTLE VIRUS
and suspensions of liver and lung were used for chicks that died. EXPERIMENTAL
Two groups of 54 eggs were inoculated into the yolk with 103 and 104 embryo LD50 of Newcastle disease virus, Iowa 23131. When candled on the 8th day, all eggs receiving injection of 104 embryo LD6o of virus were removed as infertile or containing dead embryos. Of the eggs injected with 10* embryo LD50 of virus, 48 were removed as infertile or containing dead embryos on the 8th day. Two embryos were dead on the 9th day, one on the 12th, two on the 14th and one on the 16th day. Newcastle disease virus was recovered by chick embryo inoculation from the embryos dying on the 14th and 16th days. Since positive isolations were obtained from these, it appeared unnecessary to attempt isolation of Newcastle d sease virus from the infertile eggs or those with embryos dying at shorter periods. The Iowa virus 23131 was injected into
the albumen of 54 eggs each for doses 10s and 104 embryo LD 60 of virus. On the 8th day following inoculation, 44 eggs were removed as infertile or containing dead embryos from the group receiving 103 embryo LD50 of virus. Two embryos dying on the 9th day and one dying on the 15th day yielded positive isolations of Newcastle disease virus. From the eggs inoculated with 104 embryo LD50 of virus, 47 were removed as infertile and containing dead embryos on the 8th day of incubation. One embryo died on the 15th day and yielded Newcastle disease virus upon inoculation of 10-day embryonated eggs. On the 19th day, the remaining seven embryos of the eggs receiving 103 embryo LD50 of virus were living, and the six remaining embryos of the group receiving 104 embryo LD5o were living. These eggs were inadvertently discarded with routine testing eggs, and the hatching results were not obtained. Injections of Newcastle disease virus, strain GB, were made into the albumen of 107, 110, and 110 eggs respectively for doses of 102, 103, and 104 embryo LD 60 . On the 9th day of incubation, the number of infertiles and dead embryos were 50 for the 102 LD 60 group, 67 for the 103 LD 60 group and 77 for the 104 LD50 group. Newcastle disease virus was isolated from dead embryos from each group. The virus was isolated from three embryos dying on the 10th day in the 103 LD50 group, but not from one embryo in the 102 LD60 group. Newcastle disease virus was not isolated from embryos dying on the 11th, 15th, and 19th days in the 102 LD 60 group, on the 11th and 19th days in the 103 LD50 group, and on the 13th and 19th days in the 104 LD60 group. Likewise, it was not possible to isolate Newcastle disease virus from liver suspensions prepared from 33 embryos which failed to hatch. The number of chicks hatched were
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
Inoculation of 100 embryo LD50 of Newcastle disease virus, Ky-2, was made into the yolk of 126 eggs. When candled on the 9th day, 48 eggs were removed as infertile or containing dead embryos. On the 18th day eleven eggs contained dead embryos. Virus isolation was not attempted from infertile eggs or those containing dead embryos. From the remaining eggs, 40 chicks were hatched. During an observation period of three weeks there were no symptoms of Newcastle disease. Serum samples were obtained from the chicks at three weeks of age and pooled, using five chicks for each pool. There was no neutralizing activity against Newcastle disease virus in any of the serums. All of the chicks were susceptib'e to exposure with virulent virus at four weeks of age.
583
584
E. R. DOLL, M. ELIZABETH WALLACE AND WILLIAM H. MCCOLLUM
DISCUSSION A total of 818 eggs were injected with living Newcastle disease virus. Inoculations were made into the yolk of 383 fresh eggs before incubation. Of these, 176, 104 and 103 eggs respectively received do?es of 102, 103, and 104 embryo LD50 of virus. Infertiles and dead embryos at the first candling on the 8th or 9th day of incubation accounted for 284 (73.6%) of the
inoculated eggs. Embryo deaths occurred in 17 (4.4%) of the eggs from the 10th to 18th days of incubation, 32 (8.3%) of the eggs contained living embryos on the 18th day but failed to hatch, and 50 (13%) hatched. Newcastle disease virus was injected into the albumen of 107, 164, and 164 fresh eggs respectively for doses of 102, 103, and 104 embryo LD 60 . At the 9th day candling 287 (65.9%) of the eggs were removed as infertile or containing dead embryos. Death of embryos occurred in 19 (4.3%) of the eggs from the 10th to 19th days of incubation. On the 19th day 129 (29.6%) of the eggs contained living embryos. Of these, 33 (7.5%) failed to hatch, 83 (19.0%) hatched, and hatching results were not obtained on 13 (2.9%). The majority of embryos were dead at the first candling on the 8th and 9th days. As estimated by the size of the embryos, most of the deaths occurred between the 3rd and 6th days. Embryo deaths continued from the 10th through the 19th days. Isolations of Newcastle disease virus were successful from embryos dead at the first candling on the 9th day and from embryos dying on 10th, 14th, 15th, and 16th days of incubation. Virus was not isolated from embryos that were dead on the 19th day or embryos which failed to hatch. Six chicks which died during the first week showed no symptoms of infection of the respiratory or nervous systems, and lung and liver suspensions from individual chicks did not yield virus upon inoculation into embryonated eggs. The surviving chicks remained healthy and at three weeks of age showed no serological evidence of experience with Newcastle disease virus. All were susceptible when challenged with virulent virus at three weeks of age. In these experiments, the known presence of Newcastle disease virus in fresh eggs did not provide a source of active
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
38, 23, and 22 for the respectively larger dosage groups. Of these, 5 died during the first six days following hatching, and liver and lung suspensions from individual chicks failed to yield virus in chick embryo cultures. The chicks were observed for a period of three weeks, during which there were no additional deaths and no symptoms of Newcastle disease. At the end of the three week period their serum showed no hemagglutination inhibition activity and all were susceptible to challenge exposure. Inoculations with the GB strain of Newcastle disease virus were made into the yolk of 50, 50, and 49 eggs for the respective doses of 102, 103, and 104 embryo LD 60 . On the 8th day the infertile and dead embryos were 37, 47, and 48 for the respective doses of virus. Since it was anticipated that later embryo deaths would occur, virus isolation was not attempted on the eggs that were removed on the 8th day. One embryo in each of the 102 and 103 groups died on the 10th day and yielded negative results on virus isolation tests. All remaining embryos were living on the 19th day. Virus was not isolated from five embryos which failed to hatch. Ten chicks hatched and survived. During an observation period of three weeks, there were no symptoms of Newcastle disease. Serum of the chicks had no hemagglutination inhibition activity and all were susceptible to challenge exposure.
PRE-INCUBATION INOCULATION OF EGGS WITH NEWCASTLE VIRUS
SUMMARY
Newcastle disease virus was injected into the yolk and albumen of 818 fresh eggs immediately before incubation. Embryos dying from the 9th to 16th days following inoculation of the eggs yielded Newcastle disease virus in chick embryo cultures. Virus was not recovered from embryos dying on the 19th day or those failing to hatch. Virus was not recovered
from chicks dying during the first six days after hatching. Surviving chicks showed neither symptomatic nor serological evidence of infection with Newcastle disease virus and were susceptible to artificial exposure at three weeks of age. REFERENCES Beaudette, F. R., J. A. Bivins, B. R. Miller, C. B. Hudson and J. J. Black, 1948. Studies on diagnosis of Newcastle disease in New Jersey. Am. Jour. Vet. Res., 9: 69-76. De Lay, P. D., 1947. Isolation of avian pneumoencephalitis (Newcastle disease) virus from the yolk-sac of four-day-old chicks, embryos, and infertile eggs. Science 106: 546. Hofstad, M. S., 1949. A study on the epizootiology of Newcastle disease (pneumoencephalitis). Poultry Sci. 28:-530-533. Jungherr, E., 1946. Proceedings of the conference on Newcastle disease. U. S. Dept. Agric. 56. Jungherr, E., 1947. Panel discussion on poultry diseases. Proc. U. S. Livestock Sanitary Assoc. 300. Prier, J. E., T. W. Millen and J. O. Alberts, 1950. Studies on Newcastle disease. IV. The presence of Newcastle disease virus in eggs of hens vaccinated with live vaccine. Jour. Am. Vet. Med. Assoc. 114: 54-55. Reed, L. J. and H. Muench, 1938. A simple method for estimating fifty percent end points. Am. Jour. Hyg. 27:493-497. Thompson, C. H. and O. L. Osteen, 1948. A technique for the isolation of Newcastle disease virus using streptomycin as a bacterial inhibitor. Am. Jour. Vet. Res. 9: 303-305. Van Roekel, H., 1946. Proceedings of the conference on Newcastle disease. U. S. Dept. Agric. 17.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
or latent infection in newly hatched chicks. The virus was not present in chicks living in the shell at the end of the hatching period or in those which died on the 19th day of incubation or later. By this token the weak chicks that broke the shell but failed to hatch were not a source of infection. The survival limit of embryos from which virus was recovered was the 1,6th day of incubation. Embryos dying at this age and at earlier periods could provide a source of incubator and hatchery infection if such eggs were broken in the incubator or after removal from the incubator. By these indices hatching eggs from hens infected with Newcastle disease virus, either naturally or by vaccination with living virus vaccines, are likely to be a source of hatchery infection primarily by exterior contamination or breakage in the hatchery.
585
HE possibility of infection in newly hatched chicks from Newcastle disease virus present in the eggs of sick, recovered or vaccinated hens has received the attention of several investigators. Van Roekel (1946), Jungherr (1946, 1947), and Thompson and Osteen (1948) isolated Newcastle disease virus from fresh eggs. Beaudette et al. (1948) isolated the virus from the ovarian yolk of affected hens. De Lay (1947) obtained Newcastle disease virus from incubated infertile eggs and from embryos which died on the 15th and 17th days of incubation. Hofstad (1949) isolated Newcastle disease virus at the 10th day of incubation from infertile eggs and dead embryos. Prier et al. (1950) isolated the virus from eggs laid on the 4th and 5 th days following vaccination with living virus vaccine. De Lay (1947) reported isolation of Newcastle disease virus from the yolk-sac content of four-day-old chicks hatched from eggs collected from infected flocks. This investigation was undertaken to determine the effect of injection of known
* The investigation reported in this paper is in connection with a project of the Kentucky Agri-.v cultural Experiment Station and is published by permission of the Director. t This study was conducted as a segment of Research and Marketing Act Regional Project S6— Newcastle Disease of Poultry. The cooperating agencies under this project are Kentucky, Virginia, and Texas Agricultural Experiment Stations. The research reported in this paper was done solely at the Kentucky Station. 582
quantities of Newcastle disease virus into eggs before incubation. MATERIALS AND METHODS The eggs used in this study were obtained from hens known to have had no previous infection or vaccination with Newcastle disease virus. Injections of virus were made into the yolk or albumen immediately before placing the eggs in the incubator. The eggs were kept in the horizontal position for several hours before the injections were made. For injection of the yolk, the shell surface was disinfected with an iodine-alcohol mixture, the shell pierced with a lancet, and the inoculum injected through a 27-gauge, |-inch needle. Injections into the albumen were made at the small end of the egg or at the airsac margin. The holes in the shells were sealed with flexible collodion immediately after the injections were completed. The volume of inoculum was 0.05 ml. of physiological saline dilutions of amnioallantoic fluid virus. The doses of Newcastle disease virus were 101, 102, 103, and 104 embryo LD50, calculated by the method of Reed and Muench (1938). Incubation was in a forced draft incubator at 99°F. For isolation of virus from the inoculated eggs, entire embryos were collected when death occurred previous to the 8th or 9th day candling. For embryos dying from the 10th to 15th days, amnioallantoic fluids and embryo livers were used. Suspensions of embryo liver were used for those dying from the 16th to 21st days,
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
T
PRE-INCUBATION INOCULATION OF EGGS WITH NEWCASTLE VIRUS
and suspensions of liver and lung were used for chicks that died. EXPERIMENTAL
Two groups of 54 eggs were inoculated into the yolk with 103 and 104 embryo LD50 of Newcastle disease virus, Iowa 23131. When candled on the 8th day, all eggs receiving injection of 104 embryo LD6o of virus were removed as infertile or containing dead embryos. Of the eggs injected with 10* embryo LD50 of virus, 48 were removed as infertile or containing dead embryos on the 8th day. Two embryos were dead on the 9th day, one on the 12th, two on the 14th and one on the 16th day. Newcastle disease virus was recovered by chick embryo inoculation from the embryos dying on the 14th and 16th days. Since positive isolations were obtained from these, it appeared unnecessary to attempt isolation of Newcastle d sease virus from the infertile eggs or those with embryos dying at shorter periods. The Iowa virus 23131 was injected into
the albumen of 54 eggs each for doses 10s and 104 embryo LD 60 of virus. On the 8th day following inoculation, 44 eggs were removed as infertile or containing dead embryos from the group receiving 103 embryo LD50 of virus. Two embryos dying on the 9th day and one dying on the 15th day yielded positive isolations of Newcastle disease virus. From the eggs inoculated with 104 embryo LD50 of virus, 47 were removed as infertile and containing dead embryos on the 8th day of incubation. One embryo died on the 15th day and yielded Newcastle disease virus upon inoculation of 10-day embryonated eggs. On the 19th day, the remaining seven embryos of the eggs receiving 103 embryo LD50 of virus were living, and the six remaining embryos of the group receiving 104 embryo LD5o were living. These eggs were inadvertently discarded with routine testing eggs, and the hatching results were not obtained. Injections of Newcastle disease virus, strain GB, were made into the albumen of 107, 110, and 110 eggs respectively for doses of 102, 103, and 104 embryo LD 60 . On the 9th day of incubation, the number of infertiles and dead embryos were 50 for the 102 LD 60 group, 67 for the 103 LD 60 group and 77 for the 104 LD50 group. Newcastle disease virus was isolated from dead embryos from each group. The virus was isolated from three embryos dying on the 10th day in the 103 LD50 group, but not from one embryo in the 102 LD60 group. Newcastle disease virus was not isolated from embryos dying on the 11th, 15th, and 19th days in the 102 LD 60 group, on the 11th and 19th days in the 103 LD50 group, and on the 13th and 19th days in the 104 LD60 group. Likewise, it was not possible to isolate Newcastle disease virus from liver suspensions prepared from 33 embryos which failed to hatch. The number of chicks hatched were
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
Inoculation of 100 embryo LD50 of Newcastle disease virus, Ky-2, was made into the yolk of 126 eggs. When candled on the 9th day, 48 eggs were removed as infertile or containing dead embryos. On the 18th day eleven eggs contained dead embryos. Virus isolation was not attempted from infertile eggs or those containing dead embryos. From the remaining eggs, 40 chicks were hatched. During an observation period of three weeks there were no symptoms of Newcastle disease. Serum samples were obtained from the chicks at three weeks of age and pooled, using five chicks for each pool. There was no neutralizing activity against Newcastle disease virus in any of the serums. All of the chicks were susceptib'e to exposure with virulent virus at four weeks of age.
583
584
E. R. DOLL, M. ELIZABETH WALLACE AND WILLIAM H. MCCOLLUM
DISCUSSION A total of 818 eggs were injected with living Newcastle disease virus. Inoculations were made into the yolk of 383 fresh eggs before incubation. Of these, 176, 104 and 103 eggs respectively received do?es of 102, 103, and 104 embryo LD50 of virus. Infertiles and dead embryos at the first candling on the 8th or 9th day of incubation accounted for 284 (73.6%) of the
inoculated eggs. Embryo deaths occurred in 17 (4.4%) of the eggs from the 10th to 18th days of incubation, 32 (8.3%) of the eggs contained living embryos on the 18th day but failed to hatch, and 50 (13%) hatched. Newcastle disease virus was injected into the albumen of 107, 164, and 164 fresh eggs respectively for doses of 102, 103, and 104 embryo LD 60 . At the 9th day candling 287 (65.9%) of the eggs were removed as infertile or containing dead embryos. Death of embryos occurred in 19 (4.3%) of the eggs from the 10th to 19th days of incubation. On the 19th day 129 (29.6%) of the eggs contained living embryos. Of these, 33 (7.5%) failed to hatch, 83 (19.0%) hatched, and hatching results were not obtained on 13 (2.9%). The majority of embryos were dead at the first candling on the 8th and 9th days. As estimated by the size of the embryos, most of the deaths occurred between the 3rd and 6th days. Embryo deaths continued from the 10th through the 19th days. Isolations of Newcastle disease virus were successful from embryos dead at the first candling on the 9th day and from embryos dying on 10th, 14th, 15th, and 16th days of incubation. Virus was not isolated from embryos that were dead on the 19th day or embryos which failed to hatch. Six chicks which died during the first week showed no symptoms of infection of the respiratory or nervous systems, and lung and liver suspensions from individual chicks did not yield virus upon inoculation into embryonated eggs. The surviving chicks remained healthy and at three weeks of age showed no serological evidence of experience with Newcastle disease virus. All were susceptible when challenged with virulent virus at three weeks of age. In these experiments, the known presence of Newcastle disease virus in fresh eggs did not provide a source of active
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
38, 23, and 22 for the respectively larger dosage groups. Of these, 5 died during the first six days following hatching, and liver and lung suspensions from individual chicks failed to yield virus in chick embryo cultures. The chicks were observed for a period of three weeks, during which there were no additional deaths and no symptoms of Newcastle disease. At the end of the three week period their serum showed no hemagglutination inhibition activity and all were susceptible to challenge exposure. Inoculations with the GB strain of Newcastle disease virus were made into the yolk of 50, 50, and 49 eggs for the respective doses of 102, 103, and 104 embryo LD 60 . On the 8th day the infertile and dead embryos were 37, 47, and 48 for the respective doses of virus. Since it was anticipated that later embryo deaths would occur, virus isolation was not attempted on the eggs that were removed on the 8th day. One embryo in each of the 102 and 103 groups died on the 10th day and yielded negative results on virus isolation tests. All remaining embryos were living on the 19th day. Virus was not isolated from five embryos which failed to hatch. Ten chicks hatched and survived. During an observation period of three weeks, there were no symptoms of Newcastle disease. Serum of the chicks had no hemagglutination inhibition activity and all were susceptible to challenge exposure.
PRE-INCUBATION INOCULATION OF EGGS WITH NEWCASTLE VIRUS
SUMMARY
Newcastle disease virus was injected into the yolk and albumen of 818 fresh eggs immediately before incubation. Embryos dying from the 9th to 16th days following inoculation of the eggs yielded Newcastle disease virus in chick embryo cultures. Virus was not recovered from embryos dying on the 19th day or those failing to hatch. Virus was not recovered
from chicks dying during the first six days after hatching. Surviving chicks showed neither symptomatic nor serological evidence of infection with Newcastle disease virus and were susceptible to artificial exposure at three weeks of age. REFERENCES Beaudette, F. R., J. A. Bivins, B. R. Miller, C. B. Hudson and J. J. Black, 1948. Studies on diagnosis of Newcastle disease in New Jersey. Am. Jour. Vet. Res., 9: 69-76. De Lay, P. D., 1947. Isolation of avian pneumoencephalitis (Newcastle disease) virus from the yolk-sac of four-day-old chicks, embryos, and infertile eggs. Science 106: 546. Hofstad, M. S., 1949. A study on the epizootiology of Newcastle disease (pneumoencephalitis). Poultry Sci. 28:-530-533. Jungherr, E., 1946. Proceedings of the conference on Newcastle disease. U. S. Dept. Agric. 56. Jungherr, E., 1947. Panel discussion on poultry diseases. Proc. U. S. Livestock Sanitary Assoc. 300. Prier, J. E., T. W. Millen and J. O. Alberts, 1950. Studies on Newcastle disease. IV. The presence of Newcastle disease virus in eggs of hens vaccinated with live vaccine. Jour. Am. Vet. Med. Assoc. 114: 54-55. Reed, L. J. and H. Muench, 1938. A simple method for estimating fifty percent end points. Am. Jour. Hyg. 27:493-497. Thompson, C. H. and O. L. Osteen, 1948. A technique for the isolation of Newcastle disease virus using streptomycin as a bacterial inhibitor. Am. Jour. Vet. Res. 9: 303-305. Van Roekel, H., 1946. Proceedings of the conference on Newcastle disease. U. S. Dept. Agric. 17.
Downloaded from http://ps.oxfordjournals.org/ at North Dakota State University on June 28, 2015
or latent infection in newly hatched chicks. The virus was not present in chicks living in the shell at the end of the hatching period or in those which died on the 19th day of incubation or later. By this token the weak chicks that broke the shell but failed to hatch were not a source of infection. The survival limit of embryos from which virus was recovered was the 1,6th day of incubation. Embryos dying at this age and at earlier periods could provide a source of incubator and hatchery infection if such eggs were broken in the incubator or after removal from the incubator. By these indices hatching eggs from hens infected with Newcastle disease virus, either naturally or by vaccination with living virus vaccines, are likely to be a source of hatchery infection primarily by exterior contamination or breakage in the hatchery.
585