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A Study on the Epizootiology of Newcastle Disease (Pneumoencephalitis)
A Study on the Epizootiology of Newcastle Disease (Pneumoencephalitis) M . S. HOFSTAD Veterinary Research Institute, Ames, Iowa (Received for publication December 8, 1948)
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of Newcastle disease was made on January 27 by a positive hemagglutinationinhibition (HI) test. The virus was later isolated from eggs. At the time of diagnosis the hatchery was requested to remove eggs from the incubator and to bring them to the laboratory. Two hundred sixteen eggs from the January 19 setting which had been in the incubator for 10 days were received. The 216 eggs were candled to remove dead embryos or infertile eggs. Eleven dead embryos were removed and 32 eggs were classed as infertile. Each of the 11 dead embryos was inoculated into 10-day-old embryos to determine the presence of Newcastle disease virus. Six of the dead embryos yielded the virus. From the 32 infertile eggs, 12 pooled inocula were made and 9 of these inocula yielded Newcastle disease virus on inoculation into 10-day-old embryos. The remaining live embryos were allowed to incubate until the 17th day of incubation when they were killed. From the latter embryos 43 pooled inocula were made from the yolks and yolk sacs—each inocula usually comprising material from four embryos. None of these 43 inocula yielded Newcastle disease* virus. In addition to eggs from the incubator, 10 dozen eggs were received directly from the farm and represented the first eggs the birds had laid on their return to egg production. Of 117 eggs set, 80 infertile and 5
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EWCASTLE disease has spread rapidly in the U. S. since its first recognition. The means by which the disease has spread is not clearly understood. Hatcheries have been incriminated in disseminating Newcastle disease. DeLay (1947) isolated Newcastle disease (pneumonecephalitis) virus from eggs and chicks hatched from eggs laid by an infected flock. Jungherr (1947) has isolated Newcastle virus from yolks of eggs from presumably recovered hens. The present study was made in cooperation with a large hatchery in Iowa to determine what part the hatchery might play in the spread of Newcastle disease. The objectives were to determine the presence or absence of Newcastle disease virus in (1) eggs laid by hens during the decline in production and in (2) first eggs laid by hens during their return to production after recovery from Newcastle disease infection. The study was made on three large breeding flocks where Newcastle disease was diagosed by virus isolation. The experimental procedure and results follow for each of the three flocks. Flock 205. The disease occurred in January 1948 involving 1,300 breeders. There was no death loss but birds began to moult after an almost complete cessation of egg production. The egg deliveries to the hatchery were as follows: January 5— 3,200, January 8—1,600, January 12— 1,600 and January 19—525. A diagnosis
EPIZOOTIC-LOGY OF NEWCASTLE DISEASE
were negative for Newcastle disease virus. Eighteen inocula from infertile eggs failed to yield Newcastle disease virus when inoculated into embryonated eggs. One hundred and twelve chicks were hatched from this group of eggs. They were placed under an electric floor brooder and 50 banded susceptible chicks placed with them. Chicks bled from the susceptible group on the 18th day and at the end of the exposure period of three months were negative for Newcastle antibodies. From the passively immune group, one chick bled on the 18th day was positive for 1000 ND by the SN test. On the 23rd day one pooled sample from two birds was positive for 1000 ND while another chick bled on the 23rd day was positive for 100 ND. On the 30th day, one chick was positive for 10ND while another was negative for 10 ND by the SN test. Five birds bled at the end of the exposure period of three months tested negative to the HI test. These birds were also susceptible to Newcastle disease virus when inoculated intranasally. Flock 228. This group consisted of 600 breeding hens. Newcastle disease virus was isolated from hens submitted for examination. Egg production dropped from 435 to zero in four days. There were no eggs for four days, but the return to production was rapid. Six birds died during the course of the disease and three had nervous symptoms. Three hundred and sixty eggs were received from this flock representing the first eggs layed by the flock after their return to egg production. After seven days of incubation, nine dead embryos and 212 infertile eggs were removed. On the 17th day of incubation, 10 more dead embryos were removed. Nineteen inocula from the dead embryos proved negative for Newcastle virus. Thirty-four pooled inocula from infertile eggs were negative for Newcastle disease virus. Eighty chicks were hatched and
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
dead embryos were removed on the 6th day of incubation. Material from each of the five dead embryos was inoculated into 10-day-old embryos, but none yielded the virus. Nineteen pooled inocula from the 80 infertile eggs failed to yield Newcastle disease virus when inoculated into 10-dayold embryonated eggs. One pooled sample from embryos dead at the time of hatching failed to yield Newcastle disease virus. Twenty six chicks were hatched and placed in a battery. Twenty normal chicks were mixed with the group in the same battery. Paper was used to cover the floor of the battery each day so that the chicks had contact with droppings. From time to time chicks from both groups were bled and their sera tested for Newcastle disease antibodies, either by HI or serum neutralization (SN) tests. Following are results of serum testing. Susceptible chicks were bled on the 5 th, 16th, 25th and 38th days and each sample was negative for 10 neutralizing doses (ND) by the SN test. At .the end of three months exposure, three susceptible chickens were negative to the HI test. In the passively immune group, one pooled sample from two chicks bled on the 5 th day, one bled on the 16th, 25th and 38th day tested positive for 10,000 ND, 1,000 ND, 100 ND and negative for 10 ND, respectively, by the SN test. At the end of three months exposure, three birds tested negative to the HI test. During the exposure period the birds were observed daily while caring for them. The birds remained normal throughout the period. Since most of the first eggs from Flock 205 were infertile, another group of eggs was obtained from the flock at the time they had returned to about half their full production. These chicks were hatched by the hatchery in an isolated incubator. The dead and infertile eggs were removed at 10 days. Nine inocula from dead embryos
531
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M . S. HOFSTAD
From the passively immune group, one bird bled on the 12th day, 13th day, 20th day, 24th day and 29th day after hatching tested 1,000 ND, 1,000 ND, 100 ND and 10 ND by the SN test, respectively. Birds bled from both groups at the end of two and three months tested negative by the HI test. At no time was there any evidence of Newcastle disease among the group. After the exposure period birds from both groups, used for another experiment, proved susceptible to Newcastle disease virus.
DISCUSSION
A frequent question from hatcherymen, following an outbreak of Newcastle disease in a supply flock, is when the eggs from that flock can be used again for hatching. J t has been recommended (Jungherr, 1947) that eggs be withheld for one month after full production has been reached. This question has been given added importance recently following the introduction of a live virus Newcastle disease vaccine. Newcastle virus was isolated from many of the eggs collected from a flock during the stage of the disease when the production was dropping. The virus killed the embryos the first three to six days (judging from the size of the embryos), and embryos that survived the first 10 days of incubation were apparently free from Newcastle disease virus. From eggs collected during the period when birds were beginning to return to egg production, it was not possible to isolate Newcastle disease virus. Since yolk in eggs from recovered birds is high in antibodies, it might be anticipated that the antibodies would combine with the virus and render it incapable of attacking the cells of the yolk sac in the embryonated egg. It was of interest to follow the chicks through until they had lost all the passive antibodies received from the egg yolk. The four groups of chicks hatched and reared with susceptible chicks remained free from Newcastle disease during the observation period of three months. From these observations, it is difficult to believe that true egg transmission can occur in Newcastle disease. It would appear that hatcheries can use eggs from a recovered flock as soon as fertility has come back to make it profitable to do so. There is great danger in handling eggs from a flock which is declining in produc-
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
placed with 50 susceptible chicks under an electric floor brooder. The chicks were cared for daily when observations were made. From the passively immune group one pooled serum sample from two chicks bled the 2nd day inhibited red cell agglutination by Newcastle disease virus at a final dilution of 1:640 of'serum. One bird bled the 39th day was negative for HI antibodies. After an exposure period of three months, four birds from the passively immune group and three birds from the susceptible group were bled and all tested negative for HI antibodies. Flock 239. A typical Newcastle disease outbreak in a large breeding flock was diagnosed by virus isolation. Three hundred and sixty eggs, representing the first eggs laid by the flock after reaching zero production, were received for incubation. The eggs were candled after 10 days of incubation when 238 infertile and 18 dead embryos were removed. At the 15th day two more dead embryos were candled out. Twenty inoculations into chick embryos from the 20 dead embryos failed to yield Newcastle disease virus. Forty one pooled inocula from infertile eggs were negative for Newcastle disease virus. Eighty chicks were hatched and 60 susceptible chicks were placed with them under a floor brooder. The chicks were cared for once daily when observations were made.
EPIZOOTIOLOGY OF NEWCASTLE DISEASE
tion due to Newcastle disease. Breakage of these infected eggs might contaminate the hatchery with Newcastle disease virus. SUMMARY
recovered hens failed to develop Newcastle disease. ACKNOWLEDGMENT
The author wishes to express appreciation for the cooperation given by the Staffs of the Veterinary Research Institute and the Iowa Veterinary Diagnostic Laboratory. REFERENCES
DeLay, 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: 545-546. Jungherr, E., 1947. Panel discussion on poultry diseases. Proceedings U. S. Livestock Sanitary Association, 1947: 299-300.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
Newcastle disease virus was isolated from a high percentage of dead embryos and infertile eggs taken from a group of eggs collected during the decline in egg production following an outbreak of Newcastle disease. Newcastle disease virus, however, could not be demonstrated in the first eggs laid by three flocks during their return to egg production following outbreaks of Newcastle disease. Likewise, chicks hatched from these first eggs of
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of Newcastle disease was made on January 27 by a positive hemagglutinationinhibition (HI) test. The virus was later isolated from eggs. At the time of diagnosis the hatchery was requested to remove eggs from the incubator and to bring them to the laboratory. Two hundred sixteen eggs from the January 19 setting which had been in the incubator for 10 days were received. The 216 eggs were candled to remove dead embryos or infertile eggs. Eleven dead embryos were removed and 32 eggs were classed as infertile. Each of the 11 dead embryos was inoculated into 10-day-old embryos to determine the presence of Newcastle disease virus. Six of the dead embryos yielded the virus. From the 32 infertile eggs, 12 pooled inocula were made and 9 of these inocula yielded Newcastle disease virus on inoculation into 10-day-old embryos. The remaining live embryos were allowed to incubate until the 17th day of incubation when they were killed. From the latter embryos 43 pooled inocula were made from the yolks and yolk sacs—each inocula usually comprising material from four embryos. None of these 43 inocula yielded Newcastle disease* virus. In addition to eggs from the incubator, 10 dozen eggs were received directly from the farm and represented the first eggs the birds had laid on their return to egg production. Of 117 eggs set, 80 infertile and 5
530
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
EWCASTLE disease has spread rapidly in the U. S. since its first recognition. The means by which the disease has spread is not clearly understood. Hatcheries have been incriminated in disseminating Newcastle disease. DeLay (1947) isolated Newcastle disease (pneumonecephalitis) virus from eggs and chicks hatched from eggs laid by an infected flock. Jungherr (1947) has isolated Newcastle virus from yolks of eggs from presumably recovered hens. The present study was made in cooperation with a large hatchery in Iowa to determine what part the hatchery might play in the spread of Newcastle disease. The objectives were to determine the presence or absence of Newcastle disease virus in (1) eggs laid by hens during the decline in production and in (2) first eggs laid by hens during their return to production after recovery from Newcastle disease infection. The study was made on three large breeding flocks where Newcastle disease was diagosed by virus isolation. The experimental procedure and results follow for each of the three flocks. Flock 205. The disease occurred in January 1948 involving 1,300 breeders. There was no death loss but birds began to moult after an almost complete cessation of egg production. The egg deliveries to the hatchery were as follows: January 5— 3,200, January 8—1,600, January 12— 1,600 and January 19—525. A diagnosis
EPIZOOTIC-LOGY OF NEWCASTLE DISEASE
were negative for Newcastle disease virus. Eighteen inocula from infertile eggs failed to yield Newcastle disease virus when inoculated into embryonated eggs. One hundred and twelve chicks were hatched from this group of eggs. They were placed under an electric floor brooder and 50 banded susceptible chicks placed with them. Chicks bled from the susceptible group on the 18th day and at the end of the exposure period of three months were negative for Newcastle antibodies. From the passively immune group, one chick bled on the 18th day was positive for 1000 ND by the SN test. On the 23rd day one pooled sample from two birds was positive for 1000 ND while another chick bled on the 23rd day was positive for 100 ND. On the 30th day, one chick was positive for 10ND while another was negative for 10 ND by the SN test. Five birds bled at the end of the exposure period of three months tested negative to the HI test. These birds were also susceptible to Newcastle disease virus when inoculated intranasally. Flock 228. This group consisted of 600 breeding hens. Newcastle disease virus was isolated from hens submitted for examination. Egg production dropped from 435 to zero in four days. There were no eggs for four days, but the return to production was rapid. Six birds died during the course of the disease and three had nervous symptoms. Three hundred and sixty eggs were received from this flock representing the first eggs layed by the flock after their return to egg production. After seven days of incubation, nine dead embryos and 212 infertile eggs were removed. On the 17th day of incubation, 10 more dead embryos were removed. Nineteen inocula from the dead embryos proved negative for Newcastle virus. Thirty-four pooled inocula from infertile eggs were negative for Newcastle disease virus. Eighty chicks were hatched and
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
dead embryos were removed on the 6th day of incubation. Material from each of the five dead embryos was inoculated into 10-day-old embryos, but none yielded the virus. Nineteen pooled inocula from the 80 infertile eggs failed to yield Newcastle disease virus when inoculated into 10-dayold embryonated eggs. One pooled sample from embryos dead at the time of hatching failed to yield Newcastle disease virus. Twenty six chicks were hatched and placed in a battery. Twenty normal chicks were mixed with the group in the same battery. Paper was used to cover the floor of the battery each day so that the chicks had contact with droppings. From time to time chicks from both groups were bled and their sera tested for Newcastle disease antibodies, either by HI or serum neutralization (SN) tests. Following are results of serum testing. Susceptible chicks were bled on the 5 th, 16th, 25th and 38th days and each sample was negative for 10 neutralizing doses (ND) by the SN test. At .the end of three months exposure, three susceptible chickens were negative to the HI test. In the passively immune group, one pooled sample from two chicks bled on the 5 th day, one bled on the 16th, 25th and 38th day tested positive for 10,000 ND, 1,000 ND, 100 ND and negative for 10 ND, respectively, by the SN test. At the end of three months exposure, three birds tested negative to the HI test. During the exposure period the birds were observed daily while caring for them. The birds remained normal throughout the period. Since most of the first eggs from Flock 205 were infertile, another group of eggs was obtained from the flock at the time they had returned to about half their full production. These chicks were hatched by the hatchery in an isolated incubator. The dead and infertile eggs were removed at 10 days. Nine inocula from dead embryos
531
532
M . S. HOFSTAD
From the passively immune group, one bird bled on the 12th day, 13th day, 20th day, 24th day and 29th day after hatching tested 1,000 ND, 1,000 ND, 100 ND and 10 ND by the SN test, respectively. Birds bled from both groups at the end of two and three months tested negative by the HI test. At no time was there any evidence of Newcastle disease among the group. After the exposure period birds from both groups, used for another experiment, proved susceptible to Newcastle disease virus.
DISCUSSION
A frequent question from hatcherymen, following an outbreak of Newcastle disease in a supply flock, is when the eggs from that flock can be used again for hatching. J t has been recommended (Jungherr, 1947) that eggs be withheld for one month after full production has been reached. This question has been given added importance recently following the introduction of a live virus Newcastle disease vaccine. Newcastle virus was isolated from many of the eggs collected from a flock during the stage of the disease when the production was dropping. The virus killed the embryos the first three to six days (judging from the size of the embryos), and embryos that survived the first 10 days of incubation were apparently free from Newcastle disease virus. From eggs collected during the period when birds were beginning to return to egg production, it was not possible to isolate Newcastle disease virus. Since yolk in eggs from recovered birds is high in antibodies, it might be anticipated that the antibodies would combine with the virus and render it incapable of attacking the cells of the yolk sac in the embryonated egg. It was of interest to follow the chicks through until they had lost all the passive antibodies received from the egg yolk. The four groups of chicks hatched and reared with susceptible chicks remained free from Newcastle disease during the observation period of three months. From these observations, it is difficult to believe that true egg transmission can occur in Newcastle disease. It would appear that hatcheries can use eggs from a recovered flock as soon as fertility has come back to make it profitable to do so. There is great danger in handling eggs from a flock which is declining in produc-
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
placed with 50 susceptible chicks under an electric floor brooder. The chicks were cared for daily when observations were made. From the passively immune group one pooled serum sample from two chicks bled the 2nd day inhibited red cell agglutination by Newcastle disease virus at a final dilution of 1:640 of'serum. One bird bled the 39th day was negative for HI antibodies. After an exposure period of three months, four birds from the passively immune group and three birds from the susceptible group were bled and all tested negative for HI antibodies. Flock 239. A typical Newcastle disease outbreak in a large breeding flock was diagnosed by virus isolation. Three hundred and sixty eggs, representing the first eggs laid by the flock after reaching zero production, were received for incubation. The eggs were candled after 10 days of incubation when 238 infertile and 18 dead embryos were removed. At the 15th day two more dead embryos were candled out. Twenty inoculations into chick embryos from the 20 dead embryos failed to yield Newcastle disease virus. Forty one pooled inocula from infertile eggs were negative for Newcastle disease virus. Eighty chicks were hatched and 60 susceptible chicks were placed with them under a floor brooder. The chicks were cared for once daily when observations were made.
EPIZOOTIOLOGY OF NEWCASTLE DISEASE
tion due to Newcastle disease. Breakage of these infected eggs might contaminate the hatchery with Newcastle disease virus. SUMMARY
recovered hens failed to develop Newcastle disease. ACKNOWLEDGMENT
The author wishes to express appreciation for the cooperation given by the Staffs of the Veterinary Research Institute and the Iowa Veterinary Diagnostic Laboratory. REFERENCES
DeLay, 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: 545-546. Jungherr, E., 1947. Panel discussion on poultry diseases. Proceedings U. S. Livestock Sanitary Association, 1947: 299-300.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 20, 2015
Newcastle disease virus was isolated from a high percentage of dead embryos and infertile eggs taken from a group of eggs collected during the decline in egg production following an outbreak of Newcastle disease. Newcastle disease virus, however, could not be demonstrated in the first eggs laid by three flocks during their return to egg production following outbreaks of Newcastle disease. Likewise, chicks hatched from these first eggs of
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