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Immunological Value of Various Egg Components from Eggs Infected with Hamster-Adapted Newcastle Disease Virus
Immunological Value of Various Egg Components from Eggs Infected with Hamster-Adapted Newcastle Disease Virus REGINALD L. REAGAN, DOROTHY M. SCHENCK, H. ODETTE WERNER AND A. L. BRUECKNER Maryland Slate Board of Agriculture, Live Stock Sanitary Service, University of Maryland, College Park, Maryland (Received for publication November 9, 1950)
A
CALIFORNIA strain of Newcastle disease virus (No. 11,914) adapted to the Syrian hamster (Reagan et al., 1947) has proved effective, in the form of both hamster brain suspension (Lillie et al., 1948) and allanto-amniotic fluid from embryonated chicken eggs infected with hamster-adapted virus (Reagan et al., 1950), in immunizing chickens against the virulent non-modified virus of this California strain. These successful immunizations prompted further experiments to determine the value, as vaccines, of various components of embryonated chicken eggs infected with hamster-adapted Newcastle disease virus. Twenty 5-week-old Syrian hamsters (average weight 40 grams) were injected intracerebrally with 0.05 ml. of a 10% brain suspension from infected hamsters of the 78th passage. Hamsters were sacrificed in forty-eight to seventy-two hours when typical symptoms of central nervous system involvement appeared. The brains of these hamsters were removed aseptically, ground with alundum, and diluted to a 10% suspension with pysiological saline. Fifteen-hundredths ml. of this suspension was injected into the allantoic sac of each of twenty 8-day 575
embryonated White Leghorn eggs. All eggs were incubated at 37.5°C. None of the embryos died within the first twentyfour hours post inoculation, but all were dead by ninety-six hours. The allantoic and amnionic fluids were harvested and pooled from 10 of the dead eggs for vaccine No. 1; the embryos were removed and pooled for vaccine No. 2; and the yolk sacs were removed and pooled for vaccine No. 3. Yolk sacs and embryos were ground aseptically in large mortars using alundum as an abrasive. Vaccine No. 4 was prepared from the other 10 eggs by emulsifying the entire eggs without the shells in a Waring blender. All vaccine preparations were diluted to 10% suspensions using physiological saline. After storage for one week in a dry ice locker at — 40°C, all four vaccines were titrated in 9-day embryonated eggs. The l.d.so's, as calculated by the Reed and Muench (1938) method, were as follows: vaccine No. 1, 10~2-5; vaccine No. 2, 10~2-5; vaccine No. 3, 10-3-6; vaccine No. 4, 10-3-6. Healthy 8-week-old New Hampshire chickens, identified by wing bands, were used in the experiment. Hemagglutination-inhibition tests performed with serum
576
R. REAGAN, D. SCHENCK, H. WERNER AND A. BRUECKNER TABLE 1.—Results of vaccination and challenge Vaccination
Grou
P
No. of birds
Vaccine
Date
Challenge Symptoms and deaths Typical
Atypical
No. of' birds*
Date
Died
Survived
1
50
Allantoamniotic fluid
6-5-50
6
0
44
7-5-50
1
43
1 RC
6
—
—
0
0
6
7-5-50
5
1
6-5-50
2
0
51*
7-5-50
0
51
—
0
0
6
7-5-50
4
2
6-5-50
10
0
38*
7-5-50
0
38
—
0
0
6
7-5-50
4
2
6-5-50
3
0
46*
7-5-50
3
43
2
54 . Embryo
—
2 RC
6
3
51
3 RC
6
4
52
4 RC
6
—
—
2
0
4
7-5-50
4
0
Control
—
—
—
—
—
54
7-5-50
40
14
Yolk sac
— Whole egg
* In some groups the number of birds challenged does not correlate with the number surviving the vaccination period. This is due to the fact that these birds lost their bands and could not be definitely identified and were discarded before challenge. RC = Room control.
collected in pooled lots before vaccination were negative. The chickens were divided into four groups, and each group was vaccinated with one of the above vaccines. In all instances the dose consisted of 0.25 ml. injected into the base of the left wattle. Six non-vaccinated chickens were placed with each vaccinated group as contact controls. Careful observations of all birds were made twice daily. Results of the 30-day observation period before challenging are shown in Table 1. After thirty days birds of all four vaccinated groups were challenged in the base of the right wattle with 0.5 ml. of a 10_1 dilution of virulent chicken embryopropagated Newcastle disease virus, California strain, No. 11,914 (24th embryo passage). Fifty-four normal control birds of corresponding age were challenged at the same time with the same inoculum. All birds were examined twice daily for fourteen days. The response of the chick-
ens to the challenge is given in Table. 1 The challenge material titrated 10~6-2 in 9-day embryonated chicken eggs and 10~3-2 in 12-week-old chickens. DISCUSSION
The pathogenicity of vaccines 1, 2, 3, and 4 was 12.0%, 3.7%, 19.6% and 5.8%, respectively. Although vaccines No. 2 and No. 4 were less pathogenic than vaccines No. 1 and No. 3, their immunizing power was of slightly lower order as indicated in Table 1. Of the normal room controls added to the vaccinated chickens, 2 chickens out of 6 from vaccine contact in the whole egg vaccine group (No. 4) developed Newcastle disease before challenge. Although no contact controls from the, other 3 vaccine groups showed evidence of Newcastle disease, 16.6 to 33.3% were resistant to challenge. It is possible that these birds had developed some
577
N E W S AND N O T E S
immunity through contact with the vaccinated chickens. SUMMARY Various components;—allanto-amnionic fluid, embryo, yolk sac, and whole egg—• of embryonated eggs infected with hamster-adapted N D V were studied to determine their pathogenicity and immunological value as vaccines for chickens. T h e embryo and whole egg virus proved to be the least pathogenic. T h e immunizing power of the allanto-amnionic fluid and the whole egg was of slightly lower order t h a n t h a t of the other components.
REFERENCES Lillie, M. G., R. L. Reagan, J. E. Hauser and N. J. Kincaid, 1948. Early immunological response of young chickens to modified Newcastle virus. Poultry Sci. 27: 798-801, Reagan, R. L., M. G. Lillie, L. J. Poelma and A. L. Brueckner, 1947. Transmission of the virus of Newcastle disease to the Syrian hamster. Am. J. Vet. Res. 8: 136-138. Reagan, R. L., J. W. Hartley, H. O. Werner and A. L. Brueckner, 19S0. Immunological studies of embryo-propagated hamster-adapted Newcastle virus in chickens. Proc.'Soc. Exptl. Biol. Med. 73:241-243. Reed, L. J., and H. Muench, 1938. A simple method of estimating the 50% end points. Am. J. Hyg. 27: 493-497.
News and Notes (Continued from page 574)
Irving I. L u t s k y received his Master of Science degree from P u r d u e University in J a n u a r y , with a major in poultry physiology. He received his bachelor's degree from Rutgers University and is now continuing in graduate work a t Purdue University in the area of endocrine physiology and the physiology of disease resistance. C. J. Echterling (B.S.A., M.S., P u r d u e University), who has served as part-time graduate research assistant in the P u r d u e Poultry D e p a r t m e n t for the past three a n d a half years, was shifted to junior assistant in agricultural economics (with rank of instructor) in the Agricultural Extension Service a t P u r d u e University, effective F e b r u a r y 1, 1951. M r . Echterling is being employed halftime b y the P u r d u e D e p a r t m e n t of Agricultural Economics and half-time through P.M.A. to develop a regional poultry extension marketing project. H e will be officed a t Lafayette, Indiana, b u t 'willcover the twelve north central states a n d
K e n t u c k y in coordinating the poultry marketing extension program. T h e spring m o n t h s of 1951 will be spent in laying the ground work for t h e program which is of regional n a t u r e . T h e program will be built around the marketing and merchandising of poultry and eggs in the principal metropolitan markets within the area, since a number of these states contribute to such i m p o r t a n t markets as St. Louis, Omaha, Chicago, Cleveland, Cincinnati, a n d D e troit. M r . Echterling has completed all of the requirements toward his P h . D . with a major in agricultural economics other t h a n the completion of his thesis. TEXAS NOTES D r . J. P . Delaplane has been appointed H e a d of the D e p a r t m e n t of Bacteriology and Hygiene, Texas A and M College, College Station. He was born in Greenville, Ohio and obtained a D.V.M. degree at Ohio State in 1929 and a M.S. in 1931.
{Continued on page 586)
A
CALIFORNIA strain of Newcastle disease virus (No. 11,914) adapted to the Syrian hamster (Reagan et al., 1947) has proved effective, in the form of both hamster brain suspension (Lillie et al., 1948) and allanto-amniotic fluid from embryonated chicken eggs infected with hamster-adapted virus (Reagan et al., 1950), in immunizing chickens against the virulent non-modified virus of this California strain. These successful immunizations prompted further experiments to determine the value, as vaccines, of various components of embryonated chicken eggs infected with hamster-adapted Newcastle disease virus. Twenty 5-week-old Syrian hamsters (average weight 40 grams) were injected intracerebrally with 0.05 ml. of a 10% brain suspension from infected hamsters of the 78th passage. Hamsters were sacrificed in forty-eight to seventy-two hours when typical symptoms of central nervous system involvement appeared. The brains of these hamsters were removed aseptically, ground with alundum, and diluted to a 10% suspension with pysiological saline. Fifteen-hundredths ml. of this suspension was injected into the allantoic sac of each of twenty 8-day 575
embryonated White Leghorn eggs. All eggs were incubated at 37.5°C. None of the embryos died within the first twentyfour hours post inoculation, but all were dead by ninety-six hours. The allantoic and amnionic fluids were harvested and pooled from 10 of the dead eggs for vaccine No. 1; the embryos were removed and pooled for vaccine No. 2; and the yolk sacs were removed and pooled for vaccine No. 3. Yolk sacs and embryos were ground aseptically in large mortars using alundum as an abrasive. Vaccine No. 4 was prepared from the other 10 eggs by emulsifying the entire eggs without the shells in a Waring blender. All vaccine preparations were diluted to 10% suspensions using physiological saline. After storage for one week in a dry ice locker at — 40°C, all four vaccines were titrated in 9-day embryonated eggs. The l.d.so's, as calculated by the Reed and Muench (1938) method, were as follows: vaccine No. 1, 10~2-5; vaccine No. 2, 10~2-5; vaccine No. 3, 10-3-6; vaccine No. 4, 10-3-6. Healthy 8-week-old New Hampshire chickens, identified by wing bands, were used in the experiment. Hemagglutination-inhibition tests performed with serum
576
R. REAGAN, D. SCHENCK, H. WERNER AND A. BRUECKNER TABLE 1.—Results of vaccination and challenge Vaccination
Grou
P
No. of birds
Vaccine
Date
Challenge Symptoms and deaths Typical
Atypical
No. of' birds*
Date
Died
Survived
1
50
Allantoamniotic fluid
6-5-50
6
0
44
7-5-50
1
43
1 RC
6
—
—
0
0
6
7-5-50
5
1
6-5-50
2
0
51*
7-5-50
0
51
—
0
0
6
7-5-50
4
2
6-5-50
10
0
38*
7-5-50
0
38
—
0
0
6
7-5-50
4
2
6-5-50
3
0
46*
7-5-50
3
43
2
54 . Embryo
—
2 RC
6
3
51
3 RC
6
4
52
4 RC
6
—
—
2
0
4
7-5-50
4
0
Control
—
—
—
—
—
54
7-5-50
40
14
Yolk sac
— Whole egg
* In some groups the number of birds challenged does not correlate with the number surviving the vaccination period. This is due to the fact that these birds lost their bands and could not be definitely identified and were discarded before challenge. RC = Room control.
collected in pooled lots before vaccination were negative. The chickens were divided into four groups, and each group was vaccinated with one of the above vaccines. In all instances the dose consisted of 0.25 ml. injected into the base of the left wattle. Six non-vaccinated chickens were placed with each vaccinated group as contact controls. Careful observations of all birds were made twice daily. Results of the 30-day observation period before challenging are shown in Table 1. After thirty days birds of all four vaccinated groups were challenged in the base of the right wattle with 0.5 ml. of a 10_1 dilution of virulent chicken embryopropagated Newcastle disease virus, California strain, No. 11,914 (24th embryo passage). Fifty-four normal control birds of corresponding age were challenged at the same time with the same inoculum. All birds were examined twice daily for fourteen days. The response of the chick-
ens to the challenge is given in Table. 1 The challenge material titrated 10~6-2 in 9-day embryonated chicken eggs and 10~3-2 in 12-week-old chickens. DISCUSSION
The pathogenicity of vaccines 1, 2, 3, and 4 was 12.0%, 3.7%, 19.6% and 5.8%, respectively. Although vaccines No. 2 and No. 4 were less pathogenic than vaccines No. 1 and No. 3, their immunizing power was of slightly lower order as indicated in Table 1. Of the normal room controls added to the vaccinated chickens, 2 chickens out of 6 from vaccine contact in the whole egg vaccine group (No. 4) developed Newcastle disease before challenge. Although no contact controls from the, other 3 vaccine groups showed evidence of Newcastle disease, 16.6 to 33.3% were resistant to challenge. It is possible that these birds had developed some
577
N E W S AND N O T E S
immunity through contact with the vaccinated chickens. SUMMARY Various components;—allanto-amnionic fluid, embryo, yolk sac, and whole egg—• of embryonated eggs infected with hamster-adapted N D V were studied to determine their pathogenicity and immunological value as vaccines for chickens. T h e embryo and whole egg virus proved to be the least pathogenic. T h e immunizing power of the allanto-amnionic fluid and the whole egg was of slightly lower order t h a n t h a t of the other components.
REFERENCES Lillie, M. G., R. L. Reagan, J. E. Hauser and N. J. Kincaid, 1948. Early immunological response of young chickens to modified Newcastle virus. Poultry Sci. 27: 798-801, Reagan, R. L., M. G. Lillie, L. J. Poelma and A. L. Brueckner, 1947. Transmission of the virus of Newcastle disease to the Syrian hamster. Am. J. Vet. Res. 8: 136-138. Reagan, R. L., J. W. Hartley, H. O. Werner and A. L. Brueckner, 19S0. Immunological studies of embryo-propagated hamster-adapted Newcastle virus in chickens. Proc.'Soc. Exptl. Biol. Med. 73:241-243. Reed, L. J., and H. Muench, 1938. A simple method of estimating the 50% end points. Am. J. Hyg. 27: 493-497.
News and Notes (Continued from page 574)
Irving I. L u t s k y received his Master of Science degree from P u r d u e University in J a n u a r y , with a major in poultry physiology. He received his bachelor's degree from Rutgers University and is now continuing in graduate work a t Purdue University in the area of endocrine physiology and the physiology of disease resistance. C. J. Echterling (B.S.A., M.S., P u r d u e University), who has served as part-time graduate research assistant in the P u r d u e Poultry D e p a r t m e n t for the past three a n d a half years, was shifted to junior assistant in agricultural economics (with rank of instructor) in the Agricultural Extension Service a t P u r d u e University, effective F e b r u a r y 1, 1951. M r . Echterling is being employed halftime b y the P u r d u e D e p a r t m e n t of Agricultural Economics and half-time through P.M.A. to develop a regional poultry extension marketing project. H e will be officed a t Lafayette, Indiana, b u t 'willcover the twelve north central states a n d
K e n t u c k y in coordinating the poultry marketing extension program. T h e spring m o n t h s of 1951 will be spent in laying the ground work for t h e program which is of regional n a t u r e . T h e program will be built around the marketing and merchandising of poultry and eggs in the principal metropolitan markets within the area, since a number of these states contribute to such i m p o r t a n t markets as St. Louis, Omaha, Chicago, Cleveland, Cincinnati, a n d D e troit. M r . Echterling has completed all of the requirements toward his P h . D . with a major in agricultural economics other t h a n the completion of his thesis. TEXAS NOTES D r . J. P . Delaplane has been appointed H e a d of the D e p a r t m e n t of Bacteriology and Hygiene, Texas A and M College, College Station. He was born in Greenville, Ohio and obtained a D.V.M. degree at Ohio State in 1929 and a M.S. in 1931.
{Continued on page 586)