Inactivated infectious bronchitis vaccine

J.

COMP.

PATH.

1965.

INACTIVATED

VOL.

75.

409

INFECTIOUS

BRONCHITIS

VACCINE

BY

D. M.

BERRY

Glaxo Laboratories

Ltd.,

Greenfmd

INTRODUCTION

In 1957 Christian and Mack described a &propiolactone inactivated infectious bronchitis vaccine, pointing out that live virus vaccines had several disadvantages. They might spread infection to neighbouring susceptible stock, predispose vaccinated birds to chronic respiratory disease, depress egg production and be contaminated with leucosis agents and other pathogens. Christian and Mack assessed the potency of their inactivated vaccine by the respiratory symptoms shown in vaccinated and control chicks after challenge. Woernle (1961) described a formalin-inactivated infectious bronchitis virus vaccine and assessed its potency by preciptin antibody response fn vaccinated and control chicks after challenge. Chu (personal communication) described a &propiolactone inactivated infectious bronchitis virus vaccine prepared by ultra-centrifuging virus-infected allantoic fluid. The vaccine was assessed by its ability to prevent a decrease in egg production in infected hens after challenge. This paper describes an inactivated infectious bronchitis vaccine produced on a commercial scale and the trials carried out to demonstrate its efficacy. The economic importance of infectious bronchitis is associated more with its effect on egg production than with its ability to predispose chickens to respiratory diseases, and so this vaccine has been evaluated on the former basis.

MATERIALS

AND

METHODS

Vaccine Production Virus source. Seed virus was prepared in embryonated eggs from a field strain of virus isolated by H. P. Chu at the School of Veterinary Medicine, Cambridge. This strain is of the Massachusetts tyPe, as are all the strains of infectious bronchitis (T.B.) isolated in England to date. Vaccine virus production. Eggs embryonated for 10 days were inoculated in the allantoic cavity with virus. The allantoic fluids were harvested after 2 days further incubation ahd the virus harvests titrated. The fluids were examined by haemagglutination

tests to verify the absence of Newcastle disease virus. /3-propiolactone inactivation was carried out, after which a sample was withdrawn to check that inactivation was complete and that the fluids were sterile. Sterility tests. Sterility tests were made in glucose broth and Robertson’s cooked-

Inactivation.

meat medium, selenite broth and Albimi broth at 37°C. and room temperature. Safety tests. Twenty g-day embryonated eggs from a flock free from infectious bronchitis were inoculated with 0.5 ml. of undiluted inactivated fluid in the allantoic cavity. Twenty eggs from the same supply were used as uninoculated controls. After seven days incubation, allantoic fluid was harvested from each egg and passaged twice in a further twenty eggs. At the third passage, the embryos were removed and

410

INACTIVATED

INFECTIOUS

BRONCHITIS

VACCINE

examined for dwarfing, and allantoic fluids at a dilution of 1 : 5 were checked by haemagglutination test for absence of Newcastle disease virus . Any egg showing dwarfing or dying before seven days was checked for sterility and 0.5 ml. of allantoic fluid harvested from such eggs was inoculated into further eggs, If the embryos from these eggs after seven days incubation were similar in size to the embryos from the control eggs, the material was passed. If all eggs were dwarfed, the material was rejected. In the event of an indeterminate result, further similar passages were made until a clear positive or negative result was obtained. Addition of adjutant. After completing sterility and safety tests, the inactivated virus fluids were blended with aluminium hydroxide gel. Sterility and safety tests on final vaccine. In media and in eggs these tests were similar to those carried out on the inactivated virus fluids. A further safety test was carried out by inoculating six 3 week old chickens intramuscularly with 3 ml. of final vaccine and then inspecting them for signs of clinical disease during the 14 days after inoculation. Vaccine Evaluation Antigenicity test. Twenty 6 week old chicks were each inoculated intramuscularly with 0.5 ml. of final vaccine, and a further 0.5 ml. was inoculated into each of the same birds 14 days later. Blood samples were taken at the time of the first. vaccination and 28 days after the first vaccination, both from the vaccinated chickens and from ten uninoculated controls, to determine the neutralising antibody levels of the sera from each group. The neutralisation indices of the sera were assayed by the method described by Cunningham (1951). Challenge test. The intramuscular or intranasal inoculation (or both) of 10s EID,, of U.K. strains of virus into susceptible growing chickens in isolated accommodation provoked little or no clinical respiratory disease and no mortality. The evaluation of inactivated T.B. vaccines by the method of Christian and Mack (1957) with a U.K. virus isolate was thus not feasible, and the efficacy of the vaccine was therefore tested by observing the effect of challenge on vaccinated hens. At 10 to 14 weeks old and again at 16 to 20 weeks, two groups (A and B) of LB. free chickens (20 pullets and 2 cockerels per group) were inoculated intramuscularly with O-5 ml. of vaccine. Other groups of 20 pullets and 2 cockerels were left as uninoculated controls. These birds were reared in isolation. Egg production, weight and quality were recorded from the onset of lay to the end of experiment. At 20 weeks of age all groups were bled to determine their immune status and transferred to individual pens in a challenge area. Vaccinated and unvaccinated groups were challenged with lo6 EID,, of Cambridge virus intranasally and lo6 EID,, of Strain 91 I.B. virus (isolated by L. A. Hemsley) intramuscularly when the birds were at different periods in lay. In the first and second experiments, egg production of two vaccinated (A and B) groups and one unvaccinated group was compared before and after challenge, In the third experiment susceptible chickens in a fourth group were neither vaccinated nor challenged. Egg-shell quality was examined by checking all the eggs laid by each group in each experiment for haircracks, soft shells and other faults. In the third experiment eggs from each group were graded by a packing station. Egg fertility was investigated by incubating eggs from each group each week and recording fertile eggs, early deaths and infertile eggs by candling after ten days incubation. Virus isolations were carried out by the inoculation of suspensions of tracheal epithelium into ten day embryonated I.B.-free eggs. The tracheas were obtained from two birds in each group, which were sacrificed ten days after challenge. Gross and microscopic reproductive tract changes were investigated in the birds in each group sacrificed ten days after challenge in the manner described by Sevoian and Levine (1957).

D.

M.

411

BERRY

RESULTS

Antigenicity

Tests on Vaccine Batches

The neutralisation indices obtained as described above of five batches of vaccine are shown in Table 1. Each index is for pooled aliquots of serum from five birds. Acceptable neutralisation index levels were based on the findings of Raggi (1961). It will be seenthat the indices obtained with inactivated Massachusetts strain I.B. vaccine are similar to those obtained with live Massachusetts strain I.B. vaccine by Hitchner, Appleton and Winterheld (1964). Antigenic Variation During the period of vaccine development, isolates of I.B. virus from infections in various parts of Britain, were analysed for antigenic variation by cross neutralisation tests on sera obtained from I.B.-free chickens after their artificial infection with individual virus isolates. No significant variations have been observed to date, and all field virus isolates were neutralised by antiserum of the Massachusetts strain of virus.

TABLE NEUTRALISATION

INDICES

OF SBRA

FROM

CHICKENS

1 ON ROUTINE

POTENCY

TEST OF VACCINE

BATCHES

Vaccine batch

Index

Average

of separate

index

pools

of batch

TABLE AVERAGE

Vaccinated

NUMBER

A

B

c

D

E

5.9 4.9

4.0 1.7

j:qo

34:;

;:2”

3.9 4.9

4.4 3.5

3:;

2.8 3.5

;::

4,9

3.4

4.7

3.6

4.4

2

OF EGGS PER

HEN-DAY

and unvaccinated chickens before and during after challenge with live I.B. virus

six weeks

Controls

Vaccinated (group 4

Vaccinated (PUP B)

Controls ( unchnllcngcd)

Expt. 1 before challenge For six weeks after challenge

0.64 0.33

0.53 0.42

0.57 0.42

N.D.’

Expt. 2 before challenge For six weeks after challenge

04

0.42

0.33 0.47

0.47 0.48

N.D.

Expt. 3 More challenge For six weeks after challenge

0.51

0.61 0.77

0.56 0.77

0.53 0.67

* Not

determined

0.57

412

INACTIVATED

Efect

INFECTIOUS

BRONCHITIS

VACCINE

of Challenge on Vaccinated Hens

Clinical disease. Mild respiratory symptoms, characterised by sneezing and continual shaking of the head and accompanied by a fall in food consumption, were observed on the third to the fifth days after challenge in the unvaccinated chickens in the first and third experiments. Egg production. The egg production of each group in each experiment, before and six weeks after challenge is summarised in Table 2. The pattern in the third experiment is shown in Fig. 1. Within seven weeks after challenge, the average FIGURE

1

RESULT OF CHALLENGE WITH l.B.VlRUS ON EGG PRODUCTION OF VACCINATED AND UNVACClbjATED PULLETS

% EGG PRODUCTION

80 -

70 -

60 . . . . CHALLENGED . . . . . ..“......I......... CONTROLS 50

....-----C

-

40 -

CHALLENGED 30 -

I

I 1

I 2

I 3

1 4

L 5

I 6

I 7

1 8

I 9

WEEKS

egg yield per hen was 28, i.e. 9 eggs lessin the control group than in either of the vaccinated groups (Table 3). The total egg yields in this experiment are recorded in Table 4. Statistical analysis of Table 4 with x2, shows that there is no significant difference between the groups before challenge, but that after challenge the differences between the groups are significant at the 1 per cent. level x2 = 77 with 3 d.f. Egg quality. No significant difference in egg-shell quality was observable between the groups in any experiment. Soft-shell eggs and eggs with haircracks associated with this infection were not greatly in evidence.

D.

3

TABLE AVERAGE

NUMBERS

413

BERRY

M.

AND MONETARY VALUES OF EGG FOR 7 WEEKS AFTER CHALLENGE

YIELDS

PER

HEN

Awrage number eggs/hen during 7 weeks after challenge

Value of egg yield/hen during this period* (pence)

Vaccinated

(group

A)

38

125

Vaccinated

(group

B)

37

122

Controls

(challenged)

28

92

Controls

(unchallenged)

33

109

* Based on wholesale

prices

of eggs in 1963, as recorded

Recorded

Before challenge Hen

days with

I%rtidays

EGG

as hen days with

eggs eggs

3 eggs and hen days without

93

105

190 97

1;;

Vaccinated (Group B)

egg Controls (unchallenged)

86

Vaccinated (PUP 4

Controls

After challenge

Board.

IN EXPERIMENT

Vaccinated (group 4

Controls

without

YIELDS

Egg Marketing

4

TABLE TOTAL

by the British

Total

100

384

1;:

1:

346 730

Vaccinated @UP 4

Controls (unchalbznged)

Total

Hen days with eggs Fmdays without eggs

452 290 742

576 756 180

556 699 143

628 307 935

2212 3132 920

Proportion

.609

.762

.795

.672

.706

eggs/total

TABLE ANTIBODY

Vaccination

with

inactivated

5 RESPONSE

vaccine and challenge neutralising indices.

with

live I.B. virus

expressed

as

Vaccinated !iw@ A )

Vaccinated (group 4

Controls

Exp. 2. before challenge 8 weeks after challenge

53:;

4.0 5.6

;:i

-

Exp. 3. before challenge 8 weeks after challenge

3.9 5.1

;::

::;

0.7 1.6

Unchallenged controls

414

INACTIVATED

INFECTIOIiS

BRONCHITIS TABLE

HISTOLOGICAL

Vaccinated

and unvaccinated

Otqv

Vaccinated

Unvaccinated

FINDINGS

weight is.)

6

IN THE REPRODUCTIVE

chickens

Ovidict length (inches)

VACCINE

10 days after

Mean

TRACTS

challenge

with

height of ouiduct epithelium (64

I.B. Histological evidence of increased blood supplv to o&duct

38

26

13

No

i:

26 25

20 18

i::

2

;:

28

30

26

:i

Yes YeS Yes

Egg fertility. In the third experiment no significant differences in fertility of incubated eggs were observable between groups during six weeks after challenge. Antibody response. Neutralisation indices immediately before challenge and 8 weeks after challenge are shown in Table 5. Virus recovery. It had been planned that in the third experiment there should be a group of unchallenged control birds, but I.B. virus was isolated from the trachea of at least one of the two birds in each group, including the unchallenged controls, indicating that transmission had in fact occurred between groups. Reproductive tract histopathology. The gross and microscopic findings of the reproductive tracts of the chickens killed 10 days after challenge are summarised in Table 6, and are similar to those reported by Broadfoot, Pomeroy and Smith (1954) and Sevoian and Levine (195 7). Microscopic examination of the oviducts of the unvaccinated chickens after challenge showed hyperaemia and epithelial hypertrophy, unlike those of vaccinated chickens or unvaccinated chickens of the same breed and age in an I.B.-free flock kept in isolation. DISCUSSION

In three separate experiments, the fall in egg production associated with infection of susceptible laying chickens was reproduced. This depression lasted 6 to 8 weeks in each test. Previously vaccinated chickens were apparently unaffected. This is exemplified in Fig. 1, where it is seen that egg production for both vaccinated groups was 77 per cent. compared with 57 per cent. for the controls in the weeks after challenge. In this same experiment, the value of the egg yield per hen was 2/6d to 2/9d more from the vaccinated than from the unvaccinated hens over the critical 7 week period after I.B. infection in laying stock. In an 80,000 bird unit this would result.in an increased profit of ~10,000. This 20 per cent. greater production may mean the difference between profit and loss for the poultry farmer whose flocks are infected with I.B. or the difference between a good profit and a marginal one for the poultry farmer whose flocks are infected with a mild strain of the virus. The nature of I.B. infection in laying birds as reflected by a prolonged fall in egg production and an increase in the proportion of poor quality eggs, suggests

D.

M.

415

BERRY

that a carrier state develops in which the virus affects the reproductive system of the hen during this period. If this hypothesis is correct, live I.B. vaccines would appear to be contra-indicated for the maintenance of egg production and egg quality. The potential tr ansmission of the agents of leucosis and other diseases represents another contra-indication of the use of live vaccines (Poultry Biologics, 1963), especially for the immunisation of chickens which are to become laying stock and therefore have a lifespan in which leucosis will have time to appear. Preliminary data of the inactivated vaccine from several trials, involving over 50,000 laying chickens on different premises, suggest that results similar to the experimental findings reported here may be obtained in the field. CONCLUSIONS

The production and evaluation of an infectious bronchitis (I.B.) vaccine inactivated with P-propiolactone are described. The antibody response was similar to that obtained with live I.B. (Massachusetts) vaccine. In three experiments, the inactivated vaccine appeared to protect laying chickens from the 6 to 8 week depression in egg production shown by unvaccinated susceptible layers after exposure to a heavy infection with living I.B. virus. The resulting financial benefit is illustrated and it is suggested that the higher level of egg production shown by the vaccinated chickens after challenge, compared with that of the controls, might enable the poultry farmer to operate profitably on sites where exposure to infection and subsequent depression in egg production have made previous operations financially unrewarding. ACKNOWLEDGMENTS

Mrs. Barbara Hay was responsible for the laboratory work, and Mr. Brendan Halpin, M.R.C.V.S., Veterinary carried out the histological examinations. I am indebted yield data.

to Mr.

H. Gresley-Grey

procedures College

for the statistical

involved in this of Ireland,

examination

kindly

of the egg

REFERENCES

Broa;lzlg

I., Pomeroy,

B. S., and Smith,

W. M. (1954). J. Amer.

Christian, R.-T., and Mack, W. (1957). Poultry Sci., 37, Cunningham, C. H. (1951). Amer. J. vet. Res., 12, 129. Hitchner, S. B., Appleton, G. G., and Winterfield, R. W. Poultry Biologics (1963). 134. Nat. Acad. Sci. publication Raggi, L. J. (1961). J. Amer. vet. med. Assoc., 138,387. Sevoian, M., and Levine, P. P. (1957). Avian Diseases, 1, Woernle, H. (1961). Mh. Tierhk., ,13, 136. [Received

for publication,

February

vet. med. Assoc.,

1177. (1964). 1038. 136.

12th, 19651

Aviun

Dis., 8, 151.