Laboratory Experiments with Living Newcastle Disease Vaccine

Laboratory Experiments with Living Newcastle Disease Vaccine CARL F. CLANCY,* HERALD R. COX AND CHARLES A. BOTTORFF

With the Technical Assistance of Miss Anne Merrill, Lederle Laboratories Division, American Cyanamid Co., Pearl River, New York (Received for publication June 28, 1948)

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was the second egg passage of the Isele strain which was isolated in this laboratory from a field case. A supply pool designated Isele 2D, having an LD50 of 10~ 81 in chick embryos was kept in the dry-ice chest. A 10~8 dilution of Isele 2D given intravenously in 0.5 cc dose killed 5 of 6 six-week old chickens and was uniformly fatal in all concentrations greater than 10- 8 . Preparation of vaccine. Vaccine A. After 6 consecutive passages in chick embryos strain MK 107 was serially passed in duck embryos. The 11th duck embryo passage (MD 17B) was harvested and processed as follows. Pooled allantoic, amniotic and yolk fluids plus the yolk sacs, chorioallantoic membranes and embryos were homogenized in a Waring blendor. The resulting tissue-fluid suspension was distributed in r cc amounts in glass ampoules and then frozen and dried in vacuo. The rehydrated residue had an LD50 titer of 10 -6 - 7 in chick embryos. METHODS AND MATERIALS Administration of Vaccine. Vaccine A Virus strains. The MK 107 strain of was reconstituted to its original volume Newcastle disease virus** isolated from a before the preparation of ten-fold dilufield case in Massachusetts was selected tions in liver-extract broth. The vaccine for duck embryo passage. was administered by wing-web puncture Challenge virus for all immunity tests as commonly performed in fowl pox vaccination. Two heavy sewing machine * Present address: Pennsylvania Hospital, Phila- needles attached to a holder were dipped delphia, Pennsylvania. in the vaccine suspension and then passed ** Isolated by Dr. Henry Van Roekel and obtained through the courtesy of Dr. L. T. Giltner of through the wing-web. the U.S.B.A.I., Washington, D. C. In vitro virus titrations and hemagT VARIOUS attempts to produce a prac* tical and effective killed vaccine for Newcastle disease having failed in our hands, attention was devoted to the development of a live virus vaccine of the type reported by Iyer and Dobson (1940), Brandly and his colleagues (1946) and more recently by Komarov and Goldsmit (1946), Van Roekel, el al. (1948), all of whom found this kind of vaccine to be a safe and efficient immunizing agent. In view of the successful modification by Komarov and Goldsmit of a MiddleEastern strain of Newcastle disease virus by brain to brain passage in ducklings, it was decided to attempt serial passage in duck embryos as a means of attenuating American strains. The present report describes the method of preparing and testing a live virus vaccine containing a modified strain of virus derived in this manner. Komarov and Goldsmit (1947) have subsequently reported the attenuation of the Haifa strain by this technique.

LABORATORY EXPERIMENTS WITH LIVING NEWCASTLE DISEASE VACCINE

glutination-inhibition tests. Serum antibody titrations were done by a modification of Salk's (1944) method for influenza virus antibody titrations. Equal volumes (0.5 cc.) of washed, 0.25 percent chicken red blood cells (RBC) and serial two-fold dilutions of virus were mixed and incubated for 90 minutes at room temperature. The highest dilution of virus giving

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ough mixing the tubes were incubated as in the virus titration. The endpoint of hemagglutination-inhibition was expressed as the highest final dilution of serum giving complete inhibition of agglutination as evidenced by a sharply defined brick red button of RBC comparable to the deposit in the RBC-saline control tubes.

CHALLENGE

VACCINATION

Age week 1

Dilution of vaccine 10-4 10- 5

Number of birds 10 10

Mortality

Challenge Dose*

Number of birds

Number of deaths

Survival

percent 50 50

10" 10"

5 5

0 2

percent 100 60

104

14

14

0

104 104

7 10

0 1

100 90

104

Controls 2

10-" 10- 5

10 10

30 0 !

Controls 3

4

1010-*

30 30

0 3

Controls 4

10"3 10- 3

20 20

0 0

Controls 4

IO-4 10-6

10 10

0 0

Controls

11

10

9

5

10 10*

30 29

2 1

93 97

105

20

20

0

107 106

20 20

0 0

100 100

**

46

43

7

104 104

10 10

0 2

JOO 80

104

15

12

20

|

* Dose expressed as the log of the number of chick embryo ZAo doses 104=10,000; 107=10,000,000 LDM doses. ** Titration of Isele 2D from 10 -2 through 10 - 8 ; 5 of 6 birds receiving 10 - 8 died.

a complete and uniformly diffuse deposit of agglutinated red cells over the bottom curvature of the tube was designated as one agglutinating unit of virus. All hemagglutination-inhibition (H-I) tests were set up by making serial two-fold dilutions of serum in a virus suspension containing 8 units per cc. To 0.5 cc of the serum-virus mixture an equal volume of washed 0.25 percent red cells was added. After thor-

EXPERIMENTAL OBSERVATIONS

Relationship of age to vaccination and challenge. The results of three experiments employing chicks of various ages are summarized in Table I. New Hampshire Reds 1, 2, 3 and 4 weeks of age were used and 10 -3 , 10~4 and 10 -8 dilutions of Vaccine A were administered by wing-web puncture. Prevaccination bleedings from all 4-week old and from representative 3-week old

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TABLE I.—Newcastle Disease live virus vaccine. Vaccination and challenge mortality in chicks of various ages.

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CARL F. CLANCY, HERALD R. COX AND CHARLES A. BOTTORFF

strain of virus. To secure this information 100 serologically negative New Hampshire Reds, 3 to 5-weeks of age, were banded and vaccinated by wing-web puncture with Vaccine A in a dilution of 10~3. The birds were kept in a house provided with feed-room and outdoor pen. Five days after vaccination of the flock, 17 serologically negative 3-week old birds were banded and placed in contact with the 100 vaccinated birds. Fifteen days later the contact birds were bled and were all found to be H-I positive. Their titers were as follows: Four birds had titers of 1:2560, three 1:5120, six 1:10,240 and four 1:20,480. Forty-one days after the introduction of the first group of contacts a second group of 20 banded and serologically negative birds was put into the pen. During this 41-day interval, litter in the shelter house was changed but no other sanitary or disinfection procedures were employed. These birds and a third group of contacts introduced 83 days after the first group remained H-I negative on repeated bleedings taken during a period of 272 days or approximately 9 months. The persistent absence of antihemagglutinins for Newcastle disease virus in these contact birds is doubly noteworthy: It indicates that vaccination with the attenuated virus did not result in a carrier state either in vaccinated birds or in those which acquired infection by contact with vaccinated birds; and secondly, it serves to establish the fact that Newcastle virus was not fortuitously introduced into the experimental group during the observation period. The latter fact is important because it eliminates the possibility that the persistence of the H-I titers in the original group of vaccinated birds may have been due to subsequent stimulating exposures to infection. The serologic data relating to the vaccinated group are summarized in Table II. It jyill be seen

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birds were H-I negative at the time of vaccination. All birds under 4 weeks of age were challenged by intramuscular injection of 0.5 cc of the Isele 2D challenge virus pool while the 4-week old birds received 0.5 cc (intravenously. The concentration of challenge virus shown under "dose" in Table I is expressed as the log of the number of chick embryo LD50 doses: 10,000 and 10,000,000 being indicated by 104 and 107 respectively. In these experiments all vaccinated birds were challenged 13 days after vaccination. It will be seen in Table I that half of the one-week old chicks were unable to tolerate the modified virus in either dilution employed and that 30 percent of the two-week old birds also succumbed. However 89 percent of the surviving birds in these two age groups successfully withstood challenge with 10,000 LDBO doses of virulent field strain—a dose which killed all but one of the 25 non-vaccinated controls of the same age. Among the three and four week old birds there was but one death prior to challenge in 120 chicks vaccinated with various dilutions of vaccine. Upon challenge with doses as high as 10,000,000 LD6o doses all but five of these birds remained healthy and survived. Serum samples collected from vaccinated birds on the day of challenge showed that the majority had developed good H-I antibody titers by the 13th post vaccination day. The geometric mean titer for the 199 birds was 1:2344. The carrier state and duration of immunity. In addition to the questions of safety and effectiveness, it was of immediate practical importance to determine whether or not the inapparent vaccination infection was associated with the development of a carrier state and also to know the duration of the immunity conferred by vaccination with an attenuated

LABORATORY EXPERIMENTS WITH LIVING NEWCASTLE DISEASE VACCINE TABLE II.

Date

100 94 91 89 77 63 46

-Newcastle Disease live virus vaccine. Antibody titrations at intervals following vaccination Hemagglutination-inhibition titers* Geometric mean titer

Number of sera with each titer <160 100 11 0 2

rl l

160

320

640

1,280

2,560

4 0 0 3 3 5

1 1 4 6 3 5

4 0 19 15 9 11

4 11 22 21 6 16

16 10 21 17 19 5

5,120 10,240 20,480 14 26 12 6 14 2

17 25 6 3 7 0

23 18 3 4 1 1

Neg. 1 3,060 1 6,194 1- 1,380 1 1,400 1 2,075 1:851

* Expressed as the reciprocal of the serum dilution.

from the geometric mean titers that the collective titer of the vaccinated group reached a peak 10 days after vaccination and thereafter slowly declined during the 9 months' observation period. One hundred and ten days after vaccination 10 of the vaccinated birds were removed from the flock, bled and challenged by intravenous injection of 107 chick embryo LD50 doses of Isele 2D virus. Six normal non-vaccinated adult birds were similarly injected at the same time. Two of the vaccinated birds developed muscular weakness and one died on the 13 post-challenge day; the other recovered and the remainder of the vaccinated birds continued to be symptom-free during an observation period of 16 days. Of the adult controls only one remained symptom-free, two died and the other three developed varying degrees of paralysis. Final evidence as to the character and duration of the immunity conferred by vaccination with the duck embryo adapted strain is that obtained from serologic examination of the progeny of vaccinated dams. Eggs collected on May 17, 1948 from birds vaccinated June 13, 1947 were incubated. Upon hatching, 5 serum pools representing 3 chicks each were prepared and titrated. Four of the pools had H-I titers of 1:80 and the 5th

had a titer of 1:640. Thus it is most probable that these chicks coming from birds vaccinated approximately one year previously would have enjoyed a considerable measure of immunity during their first 3 or 4 weeks of life and at a time when mortality from Newcastle disease is likely to be highest. Reactions to vaccination: In preliminary experiments with a live virus vaccine of chick embryo origin a small area of erythema and swelling was noted at the site of vaccination. However, such reactions were very uncommon in birds vaccinated with the duck embryo preparations and this reaction is therefore unreliable as an evidence of a successful take. Between the Sth and 10th post-vaccination day some 4-week old birds show slight depression but they continue to feed and to grow normally. In the laboratory trials respiratory symptoms have not been observed following the vaccination of 4-week old birds by wing-web puncture with vaccine of duck embryo origin. DISCUSSION Live virus vaccines for laryngotracheitis and fowl pox have been accepted and successfully employed on a large scale in the poultry field. It is well recognized that when properly employed the use of these

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6/13/47 6/23/47 7/23/47 8/27/47 10/8/47 12/3/47 3/16/48

Number of birds vaccinated

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CARL F. CLANCY, HERALD R. COX AND CHARLES A. BOTTORFF

doses of virulent challenge virus far in excess of any likely to be encountered in the field. Serological evidence suggests that this immunity is of considerable duration. REFERENCES

1. Iyer, S. G., and H, Dobson, 1940. A successful method of immunizing against Newcastle disease of fowls. Vet. Rec. 52: 889-894. 2. Brandley, 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. 3. Komarov, A., and L. Goldsmit, 1946. Preliminary observations on the modification of a strain of Newcastle disease virus by intracerebral passage through ducklings. Vet. J. 102:212-218. 4. Komarov, A., and L. Goldsmit, 1947. The use of live viruses in Palestine for the vaccination of poultry against Newcastle disease. Cornell Vet. 37: 368-372. 5. Salk, J. E., 1944. A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49: 87-89. 6. Van Roekel, H., F. G. Sperling, K. L. Bullis, and O. M. Olesiuk, 1948. Immunization of chickens against Newcastle disease. J.A.V.M.A. 112: 131-132.

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vaccines is attended by little risk and that they give rise to an enduring immunity. The modified strain of Newcastle disease virus used in the present experiments while not ideal in all respects is one which has the properties requisite in a practical field vaccine. It is well tolerated by healthy birds of four weeks of age and older. Due to the widespread distribution of Newcastle disease in America and the consequent carryover of antibody from immune dams to baby chicks the vaccination of very young stock is likely to be ineffective under any circumstance. Moreover a strain which is attenuated to a point where it is completely avirulent for the youngest and most susceptible animal often sacrifices a considerable part of its immunizing potency for older and more valuable members of the species. The laboratory trials summarized here demonstrate that when administered by the simple, rapid and familiar "stick-method" the duck embryo propagated strain of Newcastle virus is capable of inducing a degree of immunity which withstands