Vaccination of Chickens against Newcastle Disease with Live and Inactivated Newcastle Disease Virus

Vaccination of Chickens against Newcastle Disease with Live and Inactivated Newcastle Disease Virus R. W. WINTERFIELD, A. S. DHILLON, and L. J. ALBY School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907 (Received for publication February 27, 1979)

1980 Poultry Science 59:240-246

INTRODUCTION

MATERIALS AND METHODS

Vaccines and i m m u n i z a t i o n p r o c e d u r e s for protecting chickens against Newcastle disease (ND) have been in existence for m a n y years and have varied considerably (Hanson, 1 9 7 8 ) . Different live virus vaccines have been e m p l o y e d extensively over this period (Eidson et al., 1 9 7 7 ; Hanson, 1 9 7 8 ; Winterfield et al, 1 9 5 7 ) Occasionally, inactivated vaccines were used ( A p p l e t o n et al, 1 9 6 3 ; Hanson, 1 9 7 8 ; Hofstad, 1 9 5 5 ) . However, inactivated vaccine preparations could n o t b e applied successfully o n a mass, a u t o m a t e d basis and t h u s were n o t as economical and practical t o apply as were live virus vaccines. Also, t h e efficacy of s o m e inactivated vaccines was often in d o u b t . More recently, a renewed interest in t h e inactivated vaccines has been s h o w n , especially o u t s i d e t h e United States (Cessi a n d Nardelli, 1 9 7 4 ; R o b e r t s o n et al, 1 9 7 8 ) . Because of this i n t e r e s t and a possible i m p r o v e m e n t in immunizing capability (Brugh and Siegel, 1 9 7 8 ) , t h e a u t h o r s c o m p a r e d t h e primary and secondary i m m u n e response of t w o inactivated ND vaccines, which currently are available, with a LaSota strain live virus vaccine. T h e results are presented in this paper.

Vaccines. Two commercially-prepared , oil emulsion t y p e inactivated ND vaccines were o b t a i n e d and designated as vaccines A and B. T h e live Newcastle disease virus ( N D V ) , LaSota strain, originated from a commercial source and has b e e n retained in t h e a u t h o r s ' l a b o r a t o r y as a seed virus for p r o p a g a t i o n and use since 1 9 6 6 . For challenge of i m m u n i t y in t h e various trials, a velogenic N D V strain, GB-Texas, was administ e r e d . This strain r o u t i n e l y has caused 100% d e a t h loss or nervous signs in fully susceptible chickens. T h e same strain also was used in virus-serum neutralization (VN) tests in some experiments. Chickens and Embryos. Specific-pathogenfree chickens ( S P A F A S R ) were employed in t h e s t u d y . T h e y were reared and maintained in isolation until being distributed into isolation units at t h e start of each trial. All were free of N D , Mycoplasmagallisepticum, and Mycoplasma synoviae antibodies. E m b r y o s from t h e same source were used for t h e propagation of LaSota and GB-Texas N D V . Virus Propagation. All live N D V preparations were propagated in 9- or 10-day-old chicken e m b r y o s and t h e chorioallantoic fluids were

240

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ABSTRACT Chickens were vaccinated and revaccinated with inactivated Newcastle disease (ND) vaccines from 2 different sources and also with LaSota strain, live Newcastle disease virus (NDV). Both inactivated vaccines induced higher virus neutralizing (VN) and hemagglutination-inhibition (HI) titers than the LaSota virus. One of the inactivated preparations was found superior to the other by both the VN and HI tests. However, poor protection from apparent virus replication, virus shed, and transmission occurred after challenge with a velogenic NDV strain in those vaccinated with each of the inactivated vaccines. In contrast, LaSota virus, given by the eye drop route, produced excellent protection by the same criteria. In one of the groups of chickens, gross lesions of airsacculitis were seen after vaccination with an inactivated vaccine and subsequent challenge. Revaccination with inactivated vaccines did not enhance the protection of the respiratory tract but did result in an anamnestic serological response (VN and HI). In the post-challenge period, the use of tracheal swabs proved more sensitive as an indicator of virus shed than did cloacal swabs with the velogenic NDV strain used. The practical implications of observations made from the trials are discussed.

VACCINATION AGAINST NEWCASTLE DISEASE

Virus-Neutralization and HemagglutinationInhibition Tests. In all VN tests and titrations, 9- to 10-day-old chicken embryos were used. Titrations were done with tenfold dilutions of virus suspensions in nutrient broth. Neutralization tests were conducted with serums inactivated for 30 min in a water bath at 56 C. Each virus dilution was mixed 1:1 with serum and incubated at room temperature for 30 min. In tritration and neutralization tests, 5 embryos were inoculated per dilution, .1 ml per embryo. Inoculated embryos were held up to 7 days postinoculation. Deaths occurring within 24 hr postinoculation were regarded as nonspecific. Other deaths were recorded and the titer calculated according to Reed and Muench (1938). Log 10 VN indices were determined from serum antibody assays. Hemagglutinationinhibition (HI) tests were performed using the beta microtest method with 8 hemaglutinating units of NDV (AAAP, 1975). Experimental Procedure. In trials 1 and 2 (Table 1), the immune response to vaccination and revaccination was determined by applying vaccine A and LaSota NDV. In trial 1, 20 2-week-old chickens were vaccinated with each vaccine. The VN and HI titers were determined 3 weeks later and the challenge virus was given to 10 chickens in each experimental lot. In trial 2, 10 chickens from trial 1, which were not tested, were revaccinated at 5 weeks of age using the LaSota NDV in the chickens vaccinated initially with vaccine A. At this time, vaccine A was also given to those vaccinated initially with

LaSota NDV. From trials 3 and 4 (Table 2), the immune response was determined by vaccinating and revaccinating chickens with inactivated vaccines A, B, and LaSota NDV. In trial 3, 10 chickens were vaccinated at 2 weeks of age with vaccine B and the immunity was tested 3 weeks later. Trial 4 represented 10 chickens in each lot vaccinated at 2 weeks of age with A, B, and LaSota vaccines and then revaccinated 3 weeks later with the same vaccines. Their immunity was assessed 3 weeks later. In trials 5 and 6 (Table 3), the immune response was studied in 2-week-old chickens after administering A, B, and LaSota vaccines and also by determining the virus shed and transmission from the respiratory tract and cloaca. Vaccine A and LaSota NDV were compared in trial 5 using 10 birds per group while vaccines A, B, and LaSota were compared in trial 6 with 15 per group. In the latter trial, 4 or 5 ND susceptible chickens were placed in the vaccinated lots 4 days postchallenge. Five vaccinated birds were necropsied from each lot 120 hr postchallenge. RESULTS

Table 1 gives the results from trials 1 and 2. Where inactivated vaccine A was compared with LaSota vaccine in trial 1, it is noted that vaccine A resulted in higher VN and HI titers. In this experiment, which was of short duration, no deaths occurred from challenge. However, vaccine A failed to prevent infection of the respiratory tract while, in contrast, the LaSota vaccine induced excellent protection when assessed by virus isolation attempts from tracheal swabs. In trial 2, Table 1, where chickens in lot 1 were vaccinated initially with A vaccine and then revaccinated with LaSota NDV, an excellent anamnestic response was obtained. The VN and HI titers were very high and the protection of the respiratory tract increased from 0 to 100%. In the second lot of trial 2, where the chickens were vaccinated initially with LaSota virus but revaccinated with vaccine A, a comparable, high VN and HI response was obtained. In both lots, where the LaSota vaccine was used for either primary or secondary vaccination, excellent respiratory tract protection was apparent. Data from trials 3 and 4 are presented in Table 2. In trial 3, inactivated vaccine B induced VN titers varying from 3.0 to 4.7 with HI

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harvested in 24 hr postinoculation. Fluids from pools of the respective viruses were stored in 1-ml lots in screw-cap vials at —65 C. Titrations and testing for bacterial contaminants were accomplished before use. Vaccination and Challenge, The inactivated vaccines, A and B, were given subcutaneously in the neck, .5 ml per bird. LaSota NDV was given by eye drop, 10 6 embryo infective doses per bird, after dilution in nutrient broth. The ND challenge virus was also administered by the same route and at a similar dosage. Vaccinated and nonvaccinated groups were maintained in separate but adjoining isolators for the duration of the experiment. Four days postchallenge, the trachea or cloacas were swabbed from randomly selected birds in each group. Each swab was suspended in 3 ml of broth containing penicillin, dihydrostreptomycin, neomycin, and bacitracin and was then held at —65 C until attempted virus isolatio n (AAAP ,1975).

241

A vaccine

LaSota vaccine Unvaccinated

LaSota vaccine A vaccine

Unvaccinated

1-1

1-2

2-1

2-2

2-3

S=3.7, »3.9, >4.0, > 4 . 1 , >4.6, >4.6, >4.6, >4.8, >5.2 >5.4, >4.6, >4.6, >4.8 : >4.8 >4.9, >4.9, >4.9, >4.9, >4.9 0, .4, .5, • 5, 0,

2.9, 2.9

4, 128 0,

0,

0,

0,

0,

0,

0,

0,

0,

>2048 >2048 >2048 >2048

32,

512,

32,

128,

32,

128, 1024

512, 512, 512, >2048, >2048, >2048, >2048, »2048, >2048, >2048, >2048, >2048, >2048, >2048, >2048, >2048,

4, 64, 0,

512,

64,

128, 512,

3.8

2.8, 3.1, 3.2, 3.2, »4.0, >4.0, >4.0, >4.0 1.3, 1.3, 1.5, 2.6, 3.1, 3.3, 3.6, 3.7 .2, .3, .3, •2, 3.3,

Hemagglutination-inhibition titers of individual birds

Neutralization indices of individual birds (logi 0 )

2,2,4,4,

2, 2, 4

64

512

10/10

0/10

0/10 0/10

0/10

10/10

0/10 0/10

0/10

10/10

Tracheal infection from challenge 0

0/10

0/10

Deaths from challenge 0

Birds positive for virus/birds tested.

Birds died/birds given challenge virus.

In trial 1, chickens were vaccinated at 2 weeks of age with the immunity determined in one half of the lot 3 weeks later. In trial 2, one-half of the chickens from trial 1, which were not tested, were revaccinated at 5 weeks of age using the LaSota vaccine in those vaccinated initially with vaccine A and vaccine A in those vaccinated initially with LaSota vaccine; the immunity was determined 3 weeks later.

1-3

Vaccine applied

Trial, lot no.

TABLE 1. Immune response to vaccination (trial 1) and revaccination (trial 2) against Newcastle disease with inactivated vaccine A and the LaSota strain live virus"

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> r

m H

m r a

H tn

z

to

B vaccine

Unvaccinated controls

A vaccine

B vaccine

LaSota vaccine Unvaccinated controls

3 -- 1

3 -- 2

4 --1

4 --2

4 -- 3

4.7,

4.7,

2.7, 2.9, 3.7, 4.5, 4.5 2.0, 2.7, 2.7, 3.8 «1.5,«1.5,«1.5

4.3,

3.0, 3.2, 3.3, 4.7 <1.5, <1.5, <1.5

2.7, 3.3, 3.3

4.3,4.4,4.4

4.7, 5.0, 5.2

3.5, 3.5, 3.7,

Neutralization indices of individual birds (log l0 )

0,

0, 0,

0, 0,

8, 0

8,

8, 16, 16, 32, 32

4, 0,

4, 0,

0,

8, 0,

8, 0

8,

8, 16, 32,

32,64

32, 1 2 8 , 1 2 8 , 2 5 6 , 2 5 6 , 2 5 6 , 2 5 6 , 256, 1024 16, 16, 16, 16, 16, 32, 32, 32, 32, 32

0,

0,

Hemagglutination-inhibition titers of individual birds

10/10

0/10

0/10

0/10

10/10

0/10

Deaths from challenge 0

Birds positive for virus/birds tested.

Birds died/birds given challenge virus.

In trial 3, chickens were vaccinated at 2 weeks of age and the immunity was determined 3 weeks later. In trial 4, chickens were vaccinated at 2 weeks of age and the respective groups were revaccinated 3 weeks later with the same vaccine.

4 --4

Vaccine applied

Trial, lot no.

10/10

0/10

10/10

10/10

10/10

UJ

>

M w

r

VI

>

n

Pi

H Z

z > > z

H O

z > 10/10

n o

< > Tracheal infection from challenge 0

TABLE 2. Immune response to vaccination (trial 3) and revaccination (trial 4) against Newcastle disease with inactivated vaccines B and A and the LaSota strain live virus"

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6-8

6-7

6-5 6-6

Birds positive for virus/birds tested.

0, 0

0, 0, 0,

Unvaccinated contact controls placed with each lot 96 hours postchallenge.

0/4

0/10

16,32

16,16,16,

4/4

10/10

4/4

3/5

3/5

5/5 0/5 5/5

0/10 5/6

0/6

Virus iso lationb Trachea

16, 32, 64, 512

6/6

0/10

0/10 0/10 7/10

Deaths from challen

4/4

0/4

0/5

0/5

0/5 0/5 5/5

Cloaca

p

r

DETA

Birds died/birds given challenge virus.

32,32,64,

A vaccine Unvaccinated contact controls 0 B vaccine Unvaccinated contact controls LaSota vaccine Unvaccinated contact controls Unvaccinated noncontact controls Unvaccinated contact controls

6-1 6-2

64,128

32,64,64, 64,128,128,256,512,512 1 6 , 1 6 , 1 6 , 16, 32, 32, 32, 32, 3 2 , 6 4 0, 0, 0, 0, 0, 0, 2, 2, 2, 8

A vaccine LaSota vaccine Unvaccinated noncontact controls

5-1 5-2 5-3

Hemagglutination-inhibition titers of individual birds

Vaccine applied

Trial, lot no.

6-3 6-4

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TABLE 3. Immune response of 2-week-old chickens vaccinated against Newcastle disease with inactivated vaccines A and B and the LaSota strain live virus and the immunity determined 3 weeks later, including the determination of virus shed and transmission from the respiratory tract and cloaca

WINTE-RFI

VACCINATION AGAINST NEWCASTLE DISEASE

Table 3 demonstrates the immune response of chickens vaccinated at 2 weeks of age with vaccines A, B, and LaSota NDV with the immunity determined 3 weeks later, including the challenge-virus-shed and transmission from the respiratory tract and cloaca. It is seen from trial 5 that inactivated vaccine A resulted in higher HI titers than did LaSota vaccine. However, 5 of 5 birds from the A-vaccinated lot yielded challenge virus from tracheal swabs whereas all of the individuals given LaSota vaccine proved negative and demonstrated that virus isolation attempts from the trachea were more sensitive indicators of infection. In trial 6, Table 3, where vaccines A, B, and LaSota were compared, it is again seen that inactivated vaccines A and B incited somewhat higher HI titers from an initial, or one-time, vaccination than did the LaSota virus. However, it is also apparent that the virus was being shed from the respiratory tract where both inactivated vaccines were employed. Challenge virus was recovered from all the birds swabbed; it also spread to the susceptible contact controls. Neither of these events occurred where LaSota virus was used as the vaccine. Upon necropsy of 5 birds from each lot 5 days after challenge, 3 individuals from B-vaccinated

chickens had airsacculitis whereas no gross lesions were present in those given vaccine A and LaSota NDV. All 5 controls had lesions. DISCUSSION For many years, live virus ND vaccines have been used to a much greater extent than inactivated vaccines. It was found early that the relatively apathogenic, lentogenic ND virus strains were more economically feasible to use on a mass basis than were the inactivated vaccines, and, furthermore, they induced substantially better protection. However, in recent years, it has been suggested that improvements have been made in the preparation of ND inactivated vaccines through the process of inactivation, use of different adjuvants, and emulsifying procedures. The series of experiments reported in this study demonstrate that inactivated vaccines, from two different sources and used globally, were able to stimulate VN and HI titers which were higher than where LaSota strain NDV was used. In contrast, LaSota NDV induced much better protection of the respiratory tract which, in turn, minimized transmission of velogenic challenge NDV to other chickens. The shedding and transmission rates of challenge NDV were exceedingly high (100%) where the inactivated vaccines were used and non-existent with LaSota NDV. Also, where one of the inactivated vaccines was applied, failure to protect against lesions in the respiratory tract occurred. If the LaSota virus was applied before or after the use of an inactivated vaccine, respiratory tract protection was evident. In these experiments, with each of the vaccines, chickens were vaccinated individually. In intensive vaccination programs, the lentogenic NDV strains, usually LaSota or BlHitchner, are used frequently by the aerosol method. This results generally in even higher VN and HI titers with improved protection over that obtained by eye drop or drinking water administration. A labor-saving method of vaccine application on a routine basis, and with the protection obtained, would most often negate the use of individual vaccine application in this country unless the birds were to be handled individually for some other reason. While improvements may have been made in the preparation of inactivated ND vaccines in recent years, the justification for their use may be selective. For example, when little or no stress can be tolerated because of intercurrent

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titers of 0 to 32. There were no deaths from challenge but 100% were infected by the challenge virus as determined from tracheal swabs. Trial 4 compares A, B, and LaSota vaccines when the chickens were vaccinated initially at 2 weeks of age and then revaccinated with the same respective vaccines 3 weeks later. Vaccine A stimulated higher VN and HI titers than vaccine B and the latter induced higher titers than the LaSota virus vaccine. However, vaccination and revaccination with LaSota virus resulted in no apparent tracheal infection from challenge as compared to 100% infection in those chickens given A and B vaccines. Revaccination with the latter had no apparent effect on increasing the resistance to NDV infection. Upon necropsy of 5 birds from each lot 5 day postchallenge, none had gross evidence of airsacculitis when vaccine A and LaSota NDV were given but 3 of 5 chickens had airsacculitis where vaccine B alone was administered. All 5 controls had airsacculitis. Thus a positive VN and HI response was not necessarily indicative of complete immunity, or a wide spectrum of protection to virulent NDV challenge.

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WINTERFIELD ET AL.

respiratory infections in certain flocks a n d labor is n o t a factor, these vaccines m a y be indicated. At least with certain velogenic N D V isolants, tracheal swabs m a y b e preferred t o those from cloacas as was suggested in this s t u d y . Alternatively, b o t h tracheal and cloacal swabs should be taken t o assure greater success in isolating N D V strains which possess different degrees of tissue t r o p i s m .

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REFERENCES American Association of Avian Pathologists, 1975. In Isolation and identification of avian pathogens. S. B. Hitchner, C. H. Domerermuth, and J. E. Williams, Arnold Printing Corp., Ithica, NY. Appleton, G. S., S. B. Hitchner, and R. W. Winterfield, 1963. A comparison of the immune response of chickens vaccinated with formalin and betapropiolactone inactivated Newcastle disease vaccines. Amer. J. Vet. Res. 2 4 : 8 2 7 - 8 3 1 . Brugh, Max, Jr., and H. S. Siegel, 1978. Inactivated Newcastle disease vaccines: Influence of virus concentration on the primary immune response. Poultry Sci. 57:892-896. Cessi, D., and L. Nardelli, 1974. Vaccination against

Newcastle disease: Efficacy of an oil emulsion vaccine. Avian Pathol. 3:247—253. Eidson, C. S., J. J. Giambrone, B. O. Barger, and S. H. Kleven, 1977. Comparison of the efficacy and transmissibility of conventional NDV vaccines and vaccines prepared after back-passage through chickens. Poultry Sci. 56:19—25. Hanson, R. P., 1978. Newcastle disease. In Diseases of poultry. 7th ed. M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W. Yoder, Jr., ed. Iowa State University Press, Ames. Hofstad, M. S., 1955. The immune response in chickens following the use of three different types of inactivated Newcastle disease vaccine. Amer. J. Vet. Res. 16:608-612. Reed, L. J., and H. Muench, 1938. A simple method of estimating 50 percent endpoints. Amer. J. Hyg. 27:493-497. Robertson, W. W., D. Worden, and J. Kerr, 1978. Persistence of antibodies in laying hens following vaccination with Newcadin emulsion. Vet. Rec. 102:216. Winterfield, R. W., C. L. Goldman, and E. H. Seadale, 1957. Newcastle disease immunization studies. 4. Vaccination of chickens with Bl, F, and LaSota strains of Newcastle disease virus administered through the drinking water. Poultry Sci. 36: 1076-1088.