Newcastle Disease Immunization Studies

54

R. A. RASMUSSEN, P. W. LUTHY, J. M. VAN LANEN AND C. S. BORUFF

Scott, H. M., W. D. Morrison and P. Griminger, 1955. Studies on unknown growth factors in distillers dried solubles. Poultry Sci. 34: 14461447. Stokstad, E. L. R., and T. H. Jukes, 1950. Further observations on the "animal protein factor." Proc. Soc. Exptl. Biol. Med. 73:523-528. Sunde, M. L., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1950. An unidentified factor re-

quired by chicks fed practical rations. Poultry Sci. 29: 204-207. Tamimie, H. S., 1955. The response of chicks to an unidentified growth factor in fish products. Poultry Sci. 34:1224. Titus, H. W., 1955. The Scientific Feeding of Chickens. Energy values of feedstuffs for poultry. Revision of 2nd Ed. with Addendum. The Interstate, Danville, Illinois, pp 258-261.

Newcastle Disease Immunization Studies 2. THE IMMUNE RESPONSE OF CHICKENS VACCINATED WITH Bl NEWCASTLE DISEASE VIRUS ADMINISTERED THROUGH THE DRINKING WATER* R. W. WINTERFIELD AND E. H. SEADALE Department of Veterinary Science, University of Massachusetts, Amherst, Mass. (Received for publication July 9, 1956)

T

HIS investigation was undertaken to further study and to determine the efficacy of mass vaccination of chickens for Newcastle disease (ND) via the drinking water (Luginbuhl et al., 1954; Winterfield and Seadale, 1955; Luginbuhl et al., 1955). Factors having a possible influence on immunity induced from this route of administration were studied, and the results are presented. MATERIALS AND METHODS

Bl strain Newcastle disease virus (NDV) (Hitchner and Johnson, 1948) vaccines, designated A, B, and C, used in all trials, were obtained from three sources. Vaccine A was a lyophilized product of commercial origin. It was supplied in 500dose vials accompanied by 15 ml. diluent per vial in separate, plastic containers. It was suggested that chicks 3 to 21 days of age be vaccinated after being deprived of water for 4 hours. The dried virus was reconstituted with the diluent and im* Contribution No. 1042 of the Massachusetts Agricultural Experiment Station.

mediately added to the drinking water in the ratio of 1:315.3. Vaccine B, also of commercial origin, was a glycerinated virus preparation supplied in 60-ml., 1,000dose quantities per bottle with an accompanying package of dried, nutrient stabilizer. One ml. of the vaccine was added to 315.3 ml. of water after a premixture had been made with 30 ml. of water and .41 gram of stabilizing material. Vaccine C represented Bl—NDV lyophilized in the authors' laboratory in 3-ml. quantities per vial from a 1:1 mixture of 10 percent skim milk and infected amnioallantoic fluid. It was reconstituted and diluted in the drinking water in the manner described with vaccine A. A stabilizer composed of two-thirds gelatin and one-third dried skim milk was added in the ratio of 1.3 grams to 315.3 ml. of drinking water. Previous studies (Winterfield and Seadale, 1956) demonstrated the ability of a stabilizer to maintain higher vaccine titers in water under simulated conditions of field administration. Single distilled water was used in all trials. Titrations of vaccines and vaccine-

NEWCASTLE DISEASE IMMUNIZATION

55

drinking water mixtures were made in ten-day old chicken embryos. End points were calculated by the method of Reed and Muench (1938) from mortalities and also by the hemagglutinative activity of the allantoic fluid in live embryos 5 days postinoculation. Log embryo infective doses6o (e.i.d.6o) per milliliter of water were determined. From the quantity of water consumed during vaccination, average e.i.d.6o administered per bird were established. In those trials where the vaccine was given by the ocular route, the e.i.d.50 per bird were estimated from the vaccine titer, and the quantity of vaccine delivered by the drop method with a 19 gauge needle applicator.

ferent ages, 4 days and 4 | weeks, originating from a parent flock free from chronic respiratory disease (CRD) and previously vaccinated with Newcastle disease and infectious bronchitis (IB) dust vaccines (Markham et al., 1955) were used. In all lots in each trial, division as to sex was approximately equal. The majority of trials were conducted with the older birds because of their increased immunogenic capacity and lack of ND antibodies (Brandly et al., 1946). Recently hatched chicks completely devoid of congenitally acquired ND antibodies were difficult to obtain in Massachusetts because of the extensive ND vaccination procedures carried out.

In experiments where the vaccine was administered through the water during a 2-hour consumption period, the chickens were deprived of water for 4 hours immediately before vaccinating. Critical vaccine dosage studies were accomplished with vaccine C in this manner. No prior waterfasting period was given those birds vaccinated during an 8- or 24-hour waterconsumption period. Chickens were obtained by random selection for each experimental lot, and all lots were held in similar but separate and isolated quarters during the trial. All chickens were wing-banded, vaccinated, and maintained in conventional batteries. In two trials, nonvaccinated contact controls were placed with the vaccinated chickens immediately after vaccination. Studies with graded or different dosages of virus were carried out in modified Horsfall-Bauer units. Where duration of immunity was the object of study, control and vaccinated chickens were placed together on the floor 5 weeks after vaccination. At the time of challenge the birds were again .divided into their respective lots.

An assessment of passive serum ND antibodies in chicks vaccinated at 4 days of age was made at the beginning of each trial by sampling the hatch and subjecting more than 20 chicks to challenge. Also, individual serum neutralization (SN) tests were made on 20 or more additional hatchmates. Existence of low or partial immunity was shown by the fact that more than two-thirds succumbed to challenge each time, and approximately the same proportions of serums were unable to neutralize 102 e.i.d.50 of NDV. An average neutralization index of 2.1 was recorded on all individual serum samples tested from chicks 4 days old.

Rhode Island Red chickens of two dif-

Prior to conducting SN tests, serum samples were inactivated for 30 minutes at 56° C. The neutralization test procedure described by Cunningham (1952) was employed and significance was attached to neutralization indices of 102 or greater. Hemagglutination inhibition (HI) tests (beta) were conducted according to the method of Markham et al. (1954). A titer exceeding 16 was indicative of an H I positive serum in our study. The GB-Texas-1948 NDV strain (Boney, 1951) was used for challenge with

56

R . W . WlNTERFIELD AND E . H . S E A D A L E

the virus pool titrated in 4j-week-old chickens. Approximately 106 chicken lethal doses80 (c.l.d.Bo) were administered per bird by the intratracheal route to vaccinated and control lots with the exception of 4-day-old chicks where the challenge virus was administered intranasally. Failure to resist challenge was based on the criteria of mortality, paralysis or central nervous system disturbance, morbidity, and effect on egg production. RESULTS Response of chickens vaccinated at 4\ weeks of age through the drinking water and by the ocular route. The data presented (Table 1) reveal a slightly higher serological response 21 days after vaccination among those birds vaccinated by the ocular route in trial 1. However, no significant difference was noted between the lots vaccinated through the drinking water and the chickens vaccinated by the ocular method in trial 2. In either trial, there was no significant or consistent difference in the response of those lots vaccinated through the drinking water during the different consumption periods with the vaccine e.i.d.50 used in the experiment. The contact controls (trial 2) possessed HI titers 21 days postvaccination, which were comparable to those of the vaccinated birds. As will be subsequently pointed out in other trials, the vaccine virus showed less tendency to spread when given at a lower dosage. Duration of response in chickens vaccinated at 4\ weeks of age through the drinking water and by the ocular route. Vaccinated and control lots (Table 1, trial 2) were held for 18 weeks after vaccination, and serological and challenge data were obtained (Table 2). It is evident that HI antibody titers dropped considerably after 12 to 18 weeks in all vaccinated lots. There was little serological difference be-

tween the two lots vaccinated through the drinking water. Both groups retained slightly higher titers than the birds vaccinated ocularly. After challenge at 22^ weeks of age, all vaccinated birds remained clinically normal. Egg production was approximately 40 percent at this time and continued to rise in the vaccinated lots. In contrast, production ceased in three days in the controls with losses and 100 percent morbidity sustained. The serologic test data obtained 12 weeks postvaccination, however, suggest the need for revaccination prior to sexual maturity. Response of chickens vaccinated at 4\ weeks of age with the virus administered in the drinking water for Z-, 8-, and 24-hour consumption periods. Although the data (Table 3, trial 1) would suggest a possible relationship between differences in the length of consumption time of the virusdrinking water mixture, further study (trial 2) did not substantiate such a conclusion. As observed in previous trials, an adequate vaccine dosage intake for obtaining satisfactory immunity was effected in a comparatively short length of time. Twenty-one days postvaccination, contact controls (trial 1) again possessed titers comparable to those of their vaccinated battery mates. Response of chickens vaccinated at 4 days of age through the drinking water and by the ocular route. Table 4 summarizes the response to early vaccination. The HI antibody titer levels, 4 weeks after vaccination, were not consistently higher with any one particular method (ocular or drinking water) of vaccine administration. Extending the length of time to 24 hours for consumption of the drinking water containing the vaccine again produced no additional antibody stimulation either serologically HI or from the standpoint

23

B

23

—

50 ml.

8 hours

controls

38 ml.

~

2 hours

controls

* H I titers >16. f One nonspecific mortality during trial.

D

24

2. A

23

21

D

0 10 21

8.0X10 5 Vaccine A (ocular route) 0 10 21

0 10 21

1.6X10 7 Vaccine B

Unvaccinated

0 10 21

0 10 21

0 10 21

0 10 21

0 10 21

Days post vaccination

2.4X10 Vaccine A

6

Unvaccinated

8.0X10 5 Vaccine A (ocular route)

—

—

23

C

1.6X10' Vaccine B

51 ml.

8 hours

23 f

B

Avg. e.i.d.6o/bird 2.4X10« Vaccine A

2 hours

23

1. A

Avg. H . 0 consumed/ bird

,38 ml.

Vaccinewater consumption time

No. of birds

Trial No. and lot

23 16 23

23

23

21

21 21 21

22

23 1 1

1

23

0

4 1

1

3

1 1

5 1

3 4

16 32

6 4

7 8

6 3

10 8

3 8

3 5

4 4

2 1

3 4

1 4

0 2

6 4

2

1

1

64 128 256 512 1,024 2,048

7

2

7 5 5 3 1 2 5 9 1 3 Contact Controls: 1/32, 1/512.

1

5 8 3 3 1 5 7 4 3 1 Contact Controls: 2/256

1 2

1 2

8 6 2 2 3 2 10 4 4 1 3 Contact Controls: 2/128 1/256, 1/512

3

1 1

1

1

S

H I titer distribution

0.0 0.0 0.0

0 2 0

0 223 256

0 256 181

1 128 256

0.0 0.0 0.0

0.0 95.7 100.0

0.0 100.0 95.7

0.0 100.0 100.0

100.0

0.0 95.7 100.0

1 104 181

Held— see table 2

Held— see table 2

Held— see table 2

Held— see table 2

4.8

95.7

0.0 78.3 91.3

0 39 79

100.0

lenge

% % Immune HI -4.- * to chalpositive* 0.0 91.3 95.7

0 111 128

Geo. mean

TABLE 1.—Immune response of chickens vaccinated at 4\ weeks of age with Bl Newcastle disease virus administered through the drinking water and by the ocular route

ooo

2

8

>

N

cl

>

H

r w

en H1

>

Si O

3 H

58 TABLE 2.-

R . W . WlNTERFIELD AND E . H . SEADALE -Duration of immune response in chickens vaccinated at 4\ weeks of age with Bl Newcastle disease virus administered through the drinking water and by the ocular route

Lot, vaccine dosage and route administered

Weeks Geo. No. of postmean birds vaccination H I titer

A 2.4XlO«e.i.d.5o/bird in H 2 0 in 2 hours. Vaccine A

23

B 1.6X10 7 e.i.d. 50 /bird in H 2 0 in 8 hours. Vaccine B

23

C 8.0X106e.i.d.51>/bird. Ocular route. Vaccine A D Controls Unvaccinated

*HItiters>16.

22

21

% HI positive*

Avg. SN index

% SN positivef 95.7

12 18

64 17

95.7 47.4

2.6

12 18

42 24

78.2 57.8

2.9

12 18

34 12

72.7 36.8

2.6

12 18

3 1

00.0 00.0

<1.5

Results of challenge

No clinical effect 100.0 No clinical effect 72.7 No clinical effect 00.0 7 died. Egg production ceased. 100% morbidity evidenced

f Neutralization of at least 100 e.i.d.so of ND virus.

of challenge. Actually, there appeared to be a slight advantage among the lots deprived of water for 4 hours before being given the virus as compared to those not water-fasted. After obtaining serum samples 4 weeks postvaccination (trial 2) chickens from each vaccinated lot were randomly separated into 3 groups. One group was challenged at that time; another revaccinated; the remainder was held with the control lot for further study. The distribution of HI titers in each group indicates that the sampling was accurate. Additional serological and challenge data obtained at 7 and 9 weeks reveal little significant change among the vaccinated groups. The immune response of 4-day-old chicks was not as satisfactory as that obtained in the older birds. Many birds possessed negative HI titers at 4 and 7 weeks postvaccination. However, at 4 and 9 weeks, 94.6 to 100 percent resisted chal-

lenge. It was of interest that a few vaccinated birds lacked detectable serum antibodies at 4 weeks, both HI and SN, yet resisted challenge. Unvaccinated controls from which similar serologic test data were obtained completely failed to resist challenge. Response of chickens revaccinated at 4\ weeks of age through the drinking water and by the ocular route. Random samples of 20 chickens from the respective lots (Table 4, trial 2) were obtained. The pre-revaccination and post-revaccination data on HI titers and neutralization indices are recorded (Table 5). Pre-revaccination results were similar among those chickens vaccinated ocularly (lot C) and in those vaccinated through the drinking water in a 24-hour, vaccine-water consumption period (lot B). These lots possessed lower HI antibody levels than existed in the group (lot A) vaccinated through the water in 2 hours. Each group responded in a positive

20

21f

20f

20

2. A

B

C

D

controls

24 hours

8 hours

2 hours

controls

.

s

w

100 ml.

43 ml.

33 ml.

Unvaccinated

—

0 10 21

2.7X10 6 Vaccine A 0 10 21

0 10 21

1.4X10 7 Vaccine A

Unvaccinated

0 10 21

1.1X10' Vaccine A

0 10 21

10 10 21

3.3X10 6 Vaccine A

126 ml.

0 10 21

Days postvaccination

0 10 21

1.2X10 7 Vaccine A

Avg. e.i.d.60/bird

1.6X10? Vaccine A

48 ml.

34 ml.

blrd

V

„

Avg. tt2u con ume<

* H I titers >16. t One nonspecific mortality during trial.

19

D

24 hours

20

21

8 hours

19

1. A

C

2 hours

No. of birds

Trial No. and lot

Vaccinewater consumption time

20 20 20

20 10 2

21 9 1

20 7

19 19

21

20 1

19

0

5 2

4

4

16

8

3 1

5' 3

6

3

1 3

2 4

3 6

1 9

1 4

5

1

2

7

5

1

1

1

1

4 8 3 1 5 5 3 4 Contact Controls: 1/64, 1/256

1 5

5 5 3 1 1 2 1 4 5 2 5 Contact Controls: 1/32, 1/2048

4

3

64 128 256 512 1,024 2,048

6 2 2 3 4 9 2 1 2 Contact Controls: 1/128, 1/256

3

16 32

H I titer distribution

0 0 0

0 4 32

0 5 64

0 6 85

9

79 388

0 45 274

0 79 181

Geo. mean

TABLE 3.—Immune response of chickens vaccinated at 4\ weeks of age with Bl Newcastle disease virus administered through the drinking water in 2, 8, and 24 hour consumption periods

00.0 00.0 00.0

00.0 10.0 73.7

00.0 14.2 80.0

00.0 15.0 100.0

00.0 00.0 00.0

95.2 100.0

75.0 100.0

0.0 84.2 100.0

%

HI positive*

%

00.0

100.0

100.0

100.0

00.0

100.0

100.0

100.0

immune to challenge

z

H O

>

N

M

K c!

>

>

en H

H

2

17

17 ml.

Controls

30 * H i titers > 1 6 . t One death during trial. t Randomly selected and challenged. § Randomly selected and placed with controls. II Randomly selected, isolated a n d revaccinated—see table 5.

30

I lot i

40

2011 §

19

19

19§

Unvaccinated

40

5

2

30

10

6

1

2

4

5

2

4

11

K

3

1

4

101*

20||§ 1.8X10' Vaccine C (Ocular route)

7

19

19

6

23

9

6.5X106 Vaccine C

198

49

'20||

I

24 hours

3

2

2

1

I 2

9

2

8

4

1

2

2

1

3

1

2

2

6

6

12

6

3

7

64

1

1

3

2

1

5

7

1

5

128

2 1

3

2

1

1

256

1

2

512

H I titer distribution 32

3

4

4

3

16

17

10

10

2

8

175

4

4

4

0

3

2.2X106 Vaccine C

4.9X106 Vaccine B

4

Weeks postvaccination

3

to\t

2 hours

Unvaccinated

—

5 ml.

Controls

10

48t

D

—

—

20

C

2. A

1.3X10= Vaccine A (Ocular route)

20 ml.

24 hours

20

B

2.3X106 Vaccine A

7 ml.

2 hours

19

1. A

Avg. e.i.d.Bo/bird

Avg. H , 0 consumed/ bird

Vaccine-water consumption time

N o . of birds

Trial N o . a n d lot

1

1,024

2,048

64.7

18

13

4

2

58.8

24

36.8

10.5

10.0

36.8

26.3

0

22.4

70.0

70.0

26

0 66.6

0

90.0

65.0

84.2

%

HI+*

26

69

23

60

Geo. mdan

TABLE 4.—Immune response of chickens vaccinated at 4 days of age with Bl Newcastle disease virus administered through the drinking water and by the ocular route

16.7

100

100

100

100

10.0

100

100

94.7

%

immune to challenge

en w > a > w

a > a W

H

M M

w

5! H

Vi

o

NEWCASTLE DISEASE IMMUNIZATION

61

TABLE 5.—Secondary immune response of chickens revaccinated at 4\ weeks of age with Bl Newcastle disease virus administered through the drinking water and by the ocular route Lot A—20 birds

Lot B—20 birds

Lot C—20 birds

Lot D—15 birds

Revaccinated with 1.4X10 8 e.i.d.,o/bird bird consumed via the drinking water in 2 hours

Revaccinated with 2.0X10 8 e.i.d.so/bird bird consumed via the drinking water in 8 hours

Revaccinated with 1.4X10' e.i.d.Vbird bird via the ocular route

Controls, unvaccinated

Pre-revaccination: (4£ weeks of age) Geo. mean H I titer % HI+* Average N I % SN+t

26 70 2.1 90

7 30 2.4 100

13 45 2.5 100

0 0 <1.0 0

Post-revaccination: ( 7 i weeks of age) Geo. mean H I titer %HI+ Avrage N I %SN+

79 95 3.3 95

49 80 2.9 75

56 85 3.8 100

0 0 <1.0 0

% birds failing to respond ( H I ) : Geo. mean H I (pre-revaccination) of birds not responding

55

35

25

91

52

85

% birds failing to respond (SN): Average N I (pre-revaccination) of birds not responding

25

40

20

2.6

2.3

—

3.0

* H I titers > 1 6 . t Neutralization of at least 100 e.i.d. BO of N D virus.

manner when tested 3 weeks after revaccination as indicated by higher geometric mean HI titers and average neutralization indices. However, a study of individual birds reveals an erratic response with 25 to 55 percent lacking an increase in HI titers. Likewise, 20 to 40 percent showed no subsequent rise in SN antibody titers. It is apparent that these birds did not necessarily possess a high pre-revaccination antibody level, although the titers were above the average or geometric

mean for the respective lots. Although a greater increase in titer occurred among the chickens revaccinated ocularly, an irregular pattern of response was still evident. The group serologic response in each lot was actually of a modest rather than a pronounced nature. Response of chickens vaccinated through the drinking water with different dosages of Bl Newcastle disease virus. The relationship of vaccine dosage to immune response is given (Table 6, trials 1, 2, and 3). Ad

TABLE 6.—Immune response of chickens vaccinated with different dosages of Bl Newcastle disease virus administered through the drinking water Avg. N . I . per bird

Bird no. and age at vaccination

Avg. H , 0 consumed/ bird

Avg. e.i.d.so/bird

1. A B

20-4J weeks 20-41 weeks 20-41 weeks 10-41 weeks

30 ml. 29 ml. 26 ml. controls

1.1X10' 6.7X101 4.0XW

71 71 71 71

wks. wks. wks. wks.

3.4 <1.0 <1.0 <1.0

25 ml. 32 ml. 36 ml. controls

1.0X10 8 1.2X10' 9.0X106

71 71 71 71

wks. wks. wks. wks.

5.2 4.5 4.5 <1.0

3.2X10' 2.9X10« 2.3X10* 1.8X10*

41 wks. 41 wks. 41 wks. 41 wks. 41 wks.

2.9 2.7 2.5 1.9 <1.0

c D 2. A B C D 3. A B

c

D E

llll

Trial No. and lot

23-4 20-4 22-4 23^ 15-4

days days days days days

7 ml. 8 ml. . 8 ml. 7 ml. controls

* Neutralization of at least 100 e.i.d.a of ND virus. t HI titers >16.

Age tested

%

SN+* 95.0 20.0 0 0 100 100 100 0 88.2 85.0 72.7 52.2 0

Geo. mean HI titer

%

%

HI+t

Immune to Challenge

158 3 2 0

95.0 15.0 10.0 0

100 50.0 60.0 10.0

338 137 338 0

95.0 100 100 0

37 32 32 18 0

73.7 75.0 72.7 52.2 0

100 100 100 0 100 100 100 91.3 0

62

R . W . WlNTERFIELD AND E . H . SEADALE

ministration of 6.7 X104 and 4.0X102 e.i.d.6o to 4^-week-old chickens resulted in unsatisfactory immunity as indicated by challenge, SN and HI tests (trial 1). Intake of 1.8X 104 e.i.d.50 in 4-day-old chicks likewise gave a low immune response (trial 3). An inadequate effect was also demonstrated in a trial with a vaccine lot obtained from a commercial source. Four different serial lots of Bl vaccine were obtained for drinking water administration, in which the titers were 10~60, 10 - 6 0 , 10~6-6, and 10~6 8 per ml., respectively. One of the lots with a 10~6 ° titer was diluted according to directions, giving a final titer of 10-3-6 in the water. This vaccine was administered to 13 ND susceptible chickens, 4 | weeks of age. An average quantity of 7.1 X104 e.i.d.50 was consumed per bird in a period of 8 hours. Three weeks later, only 3 chickens demonstrated positive HI titers and 6 failed to resist challenge. In each trial, the average neutralization index was highest in the lot receiving the highest dosage (1.1 X10 7 to 1.0X108 e.i.d.6o) per bird, although 9.0X105 e.i.d.50 (trial 2) gave a satisfactory response. Vaccinated chickens in previous trials (Table 1) also responded well to 8.0X105 e.i.d.50 per bird. With 2.3 X10 5 e.i.d.50 per bird (Table 6, trial 3) and less (trials 1 and 3), lower, and often marginal, protective antibody levels were induced. Though virus multiplication apparently occurred in some birds (as indicated by positive SN and HI tests) where vaccine dosage intake was low (Table 6, trials 1 end 3), other individuals in contact with them failed to demonstrate evidence of infection. In contrast, all unvaccinated contact controls became infected in trials where dosage levels were adequate or high (Tables 1 and 3). Other aspects of the spreading potential of Bl NDV have previously been studied and reported (Markham et al., 1951).

Postvaccination observations. Clinical respiratory signs were slight to absent after vaccination through the drinking water or by the ocular route regardless of age. When signs were observed, they generally occurred 5 to 10 days after vaccinating. Postvaccination difficulties were not encountered with as high as 1.0X108 e.i.d.50 (Table 6, trial 2, lot A) and 2.0X 108 e.i.d.50 (Table 5, lot B) per bird. Intercurrent disease problems, e.g. CRD and IB, were not observed during the course of the trials. An occasional death resulted, however, from cannibalism or undetermined causes. DISCUSSION The principal advantage in mass administration of B1 Newcastle disease virus via the drinking water is in saving of labor together with simplicity of administration. Data presented show that the immunity may be similar to that resulting from ocular administration within an e.i.d.50 range of 8.0X105 to 1.8X10* per bird. Data from this study suggest the importance of an adequate vaccine dosage with the latter largely dependent on the initial titer of the vaccine to be given through the drinking water. When given in the water, most commercial Bl ND preparations will be diluted between 10 -2 and 10~3. If a vaccine containing 106 e.i.d.50 per ml. is diluted in the drinking water so that the latter contains 103 and 104 e.i.d.5o, the consumption of water and vaccine will generally be sufficient to administer between 104 and 105 e.i.d.50 per bird. Our data indicate this dosage to be unsatisfactory. Kohn (1955) found that 20,000 embryo lethal dosesso (e.l.d.50) of a highly pathogenic NDV strain (HP) were necessary to infect chickens by the alimentary route. When vaccination is accomplished through the drinking water, however, the virus also comes in contact with the mu-

NEWCASTLE DISEASE IMMUNIZATION

cosae of the upper respiratory system. Even considering this fact, a higher dosage of Bl ND virus is apparently necessary for obtaining maximum immune response. Luginbuhl et al. (1955) indicate that chickens drinking water containing 106 e.i.d.50 of Bl NDV per 0.2 ml. developed satisfactory levels of protection. From the data presented, consumption of approximately 106 e.i.d.60 per bird and above would seem desirable to insure a satisfactory immune response under highly variable field conditions. This, in turn, emphasizes the desirability of the original vaccine having a minimum titer of 10 -7 per ml. to obtain effective Bl NDV titers of 10-4-5 and higher in the drinking water. As was demonstrated, a vaccine with a titer of 10~60 was unsatisfactory at dilutions generally employed. As the necessary dosage of virus can be administered in a period of less than 2 hours after water deprivation, such a procedure, together with the use of a virus stabilizer, seems justified to minimize the unfavorable effects of environmental factors on the vaccine in the water (Winterfield and Seadale, 1956). It has been recognized that chicks vaccinated during the first few days of life may possess sufficient active immunity at the time of revaccination to prevent a satisfactory response (Brandly, 1955). As a consequence, breaks may occur shortly after revaccination. The erratic response to revaccination 4 weeks after primary vaccination in our studies would seem to substantiate such an observation. The relationship of secondary immune response to interval of time between vaccinations with inactivated Newcastle disease virus has been explored (Brandly et al., 1946; Hofstad, 1954). However, little work of this nature has been done with Bl or other ND live virus vaccine strains. Although chicks 4 days of age responded

63

satisfactorily to vaccination in these trials, it should be emphasized that passive ND antibody levels were low. Results under field conditions, however, may well be conditioned by extremes and gradations of parental immunity existing from flock to flock (Brandly et al., 1946; Brandly, 1955). SUMMARY

The immune response of chickens vaccinated through the drinking water with Bl Newcastle disease virus (NDV) has been studied and data presented. 1. Administration of 8.0X105 to 1.8 X10 7 embryo infective dosesso (e.i.d.so) of Bl NDV per chicken through the drinking water gave a comparable response to that elicited among chickens vaccinated ocularly with a similar dosage. 2. Marginal protective antibody levels were obtained with 2.3X10 6 e.i.d.Bo per chicken. The immune response was unsatisfactory below this dosage intake. Vaccines possessing titers of 10 - 6 0 or less when diluted over 1:100 in the drinking water failed to induce satisfactory immunity. 3. Vaccine dosage administered in the drinking water during a 2-hour period after 4 hours of water-fasting resulted in antibody levels similar to those obtained when the dosage was consumed in 8 or 24 hours without previous water deprivation. 4. Chickens vaccinated at 4 | weeks of age responded serologically more satisfactorily than chicks 4 days of age. All the older chickens withstood challenge 18 weeks later as compared to 94.6 to 100 percent survival among the younger lots 9 weeks postvaccination. 5. Antibody response to revaccination at 4 | weeks of age after primary vaccination at 4 days of age was of an erratic nature. Many birds not possessing high hemagglutination inhibition titers and neutralization indices failed to respond.

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6. Adverse postvaccination difficulties were not observed after administration of as much as 2.OX 108 e.i.d.50 per chicken. Respiratory signs were slight or absent in the various lots after vaccination through the drinking water and by the ocular route, irrespective of age. REFERENCES Boney, W. A., Jr., 1951. The isolation of a neurotropic strain (GB) of Newcastle disease virus. Southwestern Vet. 5:19-21. Brandly, C. A., H. E. Moses, E. E. Jones and E. L. Jungherr, 1946. Immunization of chickens against Newcastle disease. Am. J. Vet. Res. 7: 307-332. Brandly, C. A., H. E. Moses and E. L. Jungherr, 1946. Transmission of Antiviral Activity via the egg and the role of congenital passive immunity to Newcastle disease in chickens. Am. J. Vet. Res. 333-342. Brandly, C. A., 1955. Diagnosis and control of respiratory diseases of poultry. No. Am. Vet. 36: 644-647. Cunningham, C. H., 1952. Methods employed in the diagnosis and investigation of infectious bronchitis and Newcastle disease. Proceedings Book 89th Annual Meeting American Vet. Med. Assoc.: 250-256. Hitchner, S. B., and E. P. Johnson, 1948. A virus of low virulence for immunizing fowls against Newcastle disease. Vet. Med. 43: 525-530. Hofstad, M. S., 1954. The secondary immune response in chickens revaccinated with inactivated Newcastle disease virus vaccine. Am. J. Vet. Res. 15:604-^06.

Kohn, A., 1955. Quantitative aspects of Newcastle disease virus infection—effect of route of infection in the susceptibility of chicks. Am. J. Vet. Res., 16:450-457. Luginbuhl, R. E.. E. L. Jungherr and T. W. Chomiak, 1954. Administration of Newcastle disease and infectious bronchitis vaccines through the drinking water. Poultry Sci. 33: 1068. Luginbuhl, R. E., E. L. Jungherr and T. W. Chomiak, 1955. Administration of Newcastle disease and infectious bronchitis vaccines through the drinking water. Poultry Sci. 34:1399-1403. Markham, F. S., C. A. Bottorff and H. R. Cox, 1951. Conjunctival application of Newcastle disease vaccine (intranasal type) in parentally immune and susceptible chicks. Cornell Vet. 3: 267-282. Markham, F. S., H. R. Cox and C. A. Bottorff, 1954. Newcastle disease: A serologic study in vaccination and revaccination. Cornell Vet. 44: 324-345. Markham, F. S., A. H. Hammar, P. Gingher, H. R. Cox and J. Storie, 1955. Vaccination against Newcastle disease and infectious bronchitis. 1. Preliminary studies in mass vaccination with live virus dust vaccines. Poultry Sci. 34: 442-448. Reed, L. J., and H. Muench, 1938. A simple method of estimating 50 percent end points. Am. J. Hyg. 27:493-497. Winterfield, R. W., and E. H. Seadale, 1955. Newcastle disease immunization studies. Proceedings of the Twenty-Seventh Annual Meeting of the Northeastern Conference of Laboratory Workers in Pullorum Disease Control, Durham, N. H. Winterfield, R. W., and E. H. Seadale, 1956. Newcastle disease immunization studies. I. Viability of Bl Newcastle disease virus as administered as a vaccine in the drinking water. Am. J. Vet. Res. 17:5-11.

NEWS AND NOTES (Continued from page 33) Federation. He is studying transmission of inNEW HAMPSHIRE NOTES fectious sinusitis of turkeys. W. C. Skoglund, Chairman of the Poultry Department, University of New Hampshire has reNPB & E ASSOCIATION NOTES turned after a year's leave for graduate study at At the golden anniversary convention of the Pennsylvania State University. National Poultry, Butter and Egg Association, held in Chicago, October 5-7, the following officers SALSBURY FELLOWSHIP were elected: President—T. E. Schluderberg; First Dr. O. H. Osborn, University of Minnesota, has Vice-President—C. S. Christoffersen; Second VicePresident—V. C. Dauber; Secretary—A. L. been awarded the Dr. Salsbury Research Fellowship for another year, as announced by M. C. Myrick; Treasurer—G. H. Hiller; and Chairman of the Board—M. J. Goodrich, Jr. Small, Executive Secretary of the National Turkey {Continued on page 103)