The use of laboratory animals in the potency test of Brucella abortus S.19 vaccine

J.

COMP.

PATH.

1972. VOL.

82.

201

THE USE OF LABORATORY ANIMALS IN THE POTENCY TEST OF BRUCELLA ABORTUS S.19 VACCINE RESPONSE

OF GUINEA-PIGS TO GRADUATED VACCINE AND CHALLENGE

DOSES

OF

BY DENISE Central

H. THORNTON Veterinary

and J.

Luboratar~, Minishy Weybndge

C. MUSKETT of Agriculture,

INTRODUCTION Protection tests in laboratory animals are the usual method for measuring the potency of Brucella vaccines. Three principal methods are available for evaluating protection tests. The first is the use of a constant challenge dose and a constant vaccinal dose; this should only be used if experience permits (Tootill, 1969). The second is the use of a fixed challenge dose against graduated vaccinal doses. With other vaccines, this is the usual method of carrying out potency tests (Knight, 1969). However, with Brucella vaccines, this method is said to give a poor dose response curve (Smith, Keppie, Pearce and Witt, 1962; Ellwood, Keppie and Smith, 1967; Rasooly, Olitzki and Sulitzeanu, 1967). The third is the use of graduated challenge doses against a constant vaccinal dose; Gargani (1960) found this method tended to give a better dose response curve. The object of the work described here is to examine the last two methods of carrying out potency tests using guinea-pigs vaccinated with Brucella abortus $19 vaccine, and to compare these methods with the routine test procedure (Anon., 1970). Immunity against challenge was taken to be the criterion of protection, but it was also determined whether the response of the serum agglutination titre (SAT) and the skin sensitivity test were related to immunity. MATERIALS

AND

METHODS

Guinea-pigs. Female guinea-pigs of the Dunkin Hartley strain weighing between 200 and 400 g. were used; female guinea-pigs had previously been shown to be more sensitive than males to infection by BY. abortus (Hulse and Carnaghan, 1970). Vaccines. Two types of S.19 vaccine were used in these experiments. One was a standard vaccine which is used as a control in all potency tests in guinea-pigs of production batches of S.19 at the Central Veterinary Laboratory, Weybridge. The standard is a freeze-dried preparation of S.19 grown on potato agar in Roux flasks and standardised at 8 x lOlo organisms per ampoule, the minimum number of organisms in one cattle dose. The seed material was obtained from the U.S. Department of Agriculture. Other vaccines were taken at random from freeze-dried batches of S.19 vaccine produced routinely by continuous culture methods at this laboratory (Boyce and Edgar, 1966). Before use, the vaccine was reconstituted in phosphate-buffered saline pH 6.8 (PBS) to contain the required dose in 1 ml. The number of organisms in the inoculum

202

POTENCY TESTS OF Br. abortus S. 19 VACCINE

was determined by plating decimal dilutions onto serum dextrose agar (SDA) plates. Guinea-pigs were vaccinated by intramuscular inoculation into the left hind leg. The vaccinal strain grows aerobically on suitable media and can therefore be distinguished from the challenge strain which requires additional CO, for growth. Challenge. The strain of Br. abortus used was W.544 provided by Dr. W. J. Brinley Morgan which has been used in routine potency tests at the Central Veterinary Laboratory, Weybridge, since 1968; it is maintained in a freeze-dried state. The virulent characteristics of this strain have been described by Hulse and Carnaghan (1970). Before use, a fresh ampoule was reconstituted, seeded onto glycerine dextrose agar plates and incubated at 37OC. in an atmosphere of 10 per cent. CO, for 3 days. SDA slopes were then seeded and incubated as above. The growth was washed off in PBS and adjusted to the required concentration using Brown’s opacity tubes. The number of organisms in the inoculum was determined by plating decimal dilutions onto SDA plates. Guinea-pigs were challenged by the intramuscular inoculation of 1 ml. of the freshly-prepared suspension into the right hind leg. Post-mortem procedures. At the conclusion of the experiments, the guinea-pigs were weighed and killed, and individual blood samples were collected from them. The spleens were removed aseptically into pre-weighed sterile Petri dishes and individually weighed. In order that the number of splenic organisms could be assessed, a 1 /lO (w/w) dilution of the spleen was made by suspending with PBS in Griffiths’ tubes. Further dilutions were made from the initial 1 /lO dilution and 0.2 ml. of each dilution was plated onto two SDA plates which were incubated at 37OC. for 5 days. If the animals had been inoculated only with S.19, the plates were incubated aerobically. However, if vaccinated animals had been challenged with strain W.544, the plates were incubated in an atmosphere of 10 per cent. CO,, and if Brucella were present, colonies were subcultured and incubated aerobically to determine whether S.19 or W.544 had been recovered. The SAT of each serum was determined as described by Morgan (1967), using Weybridge standard Br. abortus antigen, and expressed in international units (I.U.). Skin sensitivity test. Delayed hypersensitivity was measured by the intradermal inoculation of a cell-free extract of S.19 prepared by a modification of the method of Ottosen and Plum (1949). S.19 cells (25 g. wet weight) were suspended in 50 ml. saline solution and adjusted to pH l-4 with 0.1 N-HCI. The mixture was boiled gently for 40 min. and allowed to cool, after which the cell debris was separated by centrifugation and discarded. The supernatant liquid was adjusted to pH 4.5 in order to obtain the maximum amount of precipitate which was harvested by centrifugation, resuspended in PBS and adjusted to pH 7.5 with 0.1 N-NaOH. The final volume was 108 ml., and the protein content was 3.5 mg./ml. The allergenic properties of the extract were retained for over a year when kept at 4OC. The preparation was inoculated intradermally into the depilated flanks of guineapigs in 0.1 ml. amounts and the reaction measured 24 hours later. No reaction was produced in animals that had not been previously inoculated with immunogenic preparations of Br. abortus. When inoculated into guinea-pigs vaccinated with S.19 vaccine, a reaction was produced that did not vary significantly in size after the 15th day after vaccination (Thornton, unpublished). Readings could, therefore, be taken at any stage in routine assays from the 15th day after vaccination until the animals were challenged at 60 days. As generalised anaphylactic shock sometimes occurred in animals after they had been infected with strain W.544, no measurements of the skin response were taken after challenge in these experiments. The skin response was derived from the formula r-Y, where r is the radius in mm. of the reaction and t the increase in thickness in mm. of the skin fold. It has been shown (Thornton, unpublished) that when expressed in this way, the size of the skin reaction is directly proportional to the dose of allergen injected. The intramuscular inoculation of 1 ml. or intradermal inoculation of O-1 ml. of the allergen did not cause the production of a SAT, neither did it enhance the Qre

DENISE

H.

THORNTON

AND

J.

C.

203

MUSKETT

in unvaccinated animals, nor did it protect animals against challenge with 5,000 organisms of strain W.544. Measurements of delayed hypersensitivity could, therefore, be taken without interfering with the other reactions to vaccination.

RESULTS

Estimation

of Protective

Dose of S.19 in Guinea-pigs

It was shown by de Ropp (1945) that the spleen was the site of collection of S.19 and that no multiplication occurred within the guinea-pig. As the present vaccinal strain of S.19 has been modified over the years, it was considered necessary to confirm this work. It was found that the persistence of S. 19 was independent of the dose. The incidence of S.lS-infected guinea-pigs decreased up to the 40th day after vaccination, when 90 per cent. of guinea-pigs were found to be free of S.19. However, small amounts of S.19 were recovered from 10 per cent. of the animals. This low level of infection persisted until the end of the experiment at 150 days. As no evidence of extensive multiplication of the vaccinal strain was found, it was possible to carry out experiments involving graduated doses of vaccine and expect that a dose response curve would be obtained. This type of assay is not suitable for vaccine strains which multiply in the test animal. Alton (1969b) found that 100 Br. melitensis Rev. 1 organisms conferred as much protection in guinea-pigs as 100 x lo* organisms. He had previously shown that the organism multiplied within the guinea-pig to the same level in each case (Alton, 1969a). The number of S.19 organisms that conferred protection on 50 per cent. of guinea-pigs (PD B0) w h en challenged with a routine challenge dose of 5,000 W.544 organisms (Anon., 1970) was estimated by the following procedure. Guinea-pigs were vaccinated in groups of 12 with doses of standard vaccine ranging from 7 x 10e to 70 S. 19 organisms. Before challenge at 60 days, measurements were made of the skin sensitivity reaction at 27 and 55 days after vaccination. Fortytwo days after challenge, the SAT, spleen-body weight ratios and the number of organisms in the spleen of each animal were determined. The rcsu1t.s for the protection against challenge obtained at various vaccine doses are shown in Table 1. The dose response curve is shown in Fig. 1. The PD50 of S.19 estimated by the method of Probit analysis was 127 organisms. It can be seen from Fig. 1 that vaccine doses up to 7 x 10’ do not confer significantly greater protection than 7 x 10’. Similar unpublished experiments have given protection against challenge of 83 per cent. and 75 per cent. at vaccine doses of 7 x 10’ and 7 x 10” respectively. The routine test dose (approximately 7 x 103 gives protection of 75 to 100 per cent. Other Responses

of the Guinea-pig

to Graduated

Vaccine

Doses

In the vaccine groups 7 x 101, 7 x 102 and 7 x 10’ the SAT of guinea-pigs infected with strain W.544 were greater than uninfected ones and the number of animals with titres above 30 I.U. was greater in W.544-infected animals than in uninfected ones (12/16 and Z/24 respectively; P < @OOl). In the other vaccine groups there were no significant differences in the SAT of infected and uninfected animals (Table 2).

abortus S.19 VACCINE

POTENCY TESTS OF Br.

204

TABLE VARIATION

Dose of vaccine (Number of X19 oreanismr) 7 7 7 7 7 7

x x x x x x

1

OF PROTECTION AGAINST CHALLENGE, SKIN SENSITIVITY WITH VACCINE

BODY DOSE

WEIGHT

Number of guinea-pigs found to be infected with W.544

Mean body weight is?)

2/11* 4110 2/11 4112 5112 7110 10/10

669 700 668 709 722 752 813

109 10s 104 108 lo* 10’ 0

*Although 12 guinea-pigs were used in each group, several course of the work due to causes unrelated to the experiment.

AND

Skin sensitivity test jr2t : mm.) 164 156 163 161 108 75 0 died

throughout

the

gK;p;; 100

~

50

I/-‘

80

40

0 60 fi

30 L skin

reaction 20

40

L weight

20

protection

10

0 10’ Number

lo2 of

10’

10’

lo5

S.19

organisms

lo6

10’

loB

.f ; ; ii g m 2 : %

0 lop

inoculatedb’xlog,o)

Fig. 1. The relationship between skin sensitivity, body weight, protection and vaccine dose. Protection was judged by recovery of challenge organisms. Values for skin reaction, percentage protection and inhibition of body weight gain in unvaccinated control animals was zero.

The spleen-body weight ratios of infected animals were greater than those of uninfected animals, but these differences were not statistically significant (Table 2). The body weights of vaccinated animals weighed prior to killing were found to decrease with increasing vaccine dose, indicating that the vaccine had prevented the normal weight gain of the growing guinea-pigs. These results are shown in Table 1 and Fig. 1. The correlation between body weight and vaccine dose using 76 pairs of individual values is very highly significant (correlation coefficient = -0.484; P < O*OOl).

DENISE

VARIATION

H.

THORNTON

AND

J.

C.

205

MUSKETT

TABLE 2 OF SAT, SPLEEN-BODY WEIGHT RATIO AND SKIN SENSITIVITY WfTH VACCINE S”BSEQ”ENTLY ESTABLISHED STATUS REGARDING IMMUNITY AGAINST CHALLENGE

Dost of vaccine (Number of S.19 organisms)

Number of animals u P

SAT u

Number of animals with SAT> 30 I.U. u P

(I.U.) P

7 x 100

:

9

z;

15

7 x 106 10”

2

5

26

2”

: :: :8” 4

45

87

235 122

::

:

114 76

7 x 0 10’

1:

P = protected against U = unprotected

2

Spleen-body weight ratio u P 0.17 0.26 0.23 O-28 0.39 0.30 0.20

0.19 0.16 o-21 0.17 0.16 0.16 -

DOSE AND

Skin semititi~ test (r*t : mm.) u P 170 164 196 151 129 66 0

162 147 151 163 64 92

challenge

There was also a correlation using individual values between the size of the skin reaction and the vaccine dose (Table 1). The values given are the means of 4 readings taken on 2 occasions. The correlation coefficient using 15 pairs of values for uninfected animals was +0*60 (P < O-05), and for infected animals using 19 pairs of values was +0*47 (P < O-05). However, the skin reaction (Table 2) could not be used to distinguish those animals subsequently shown to be resistant to challenge because these was no significant correlation between skin reactions and the presence or absence of infection by the challenge strain. This confirmed previous unpublished findings that the degree of skin reaction is not related to protection. The variation of protection against challenge, body weight and skin reaction with vaccine dose are shown in Fig. 1. Estimation 5f fL)so 5f Strain W.544 in Guinea-pigs The dose required to infect 50 per cent. of guinea-pigs (IDao) was determined by using groups of 10 inoculated with graduated doses of strain W.544. The IDso in unvaccinated guinea-pigs was 89 organisms and in vaccinated guineapigs was 182 x 10’ and 191 x lo3 in the standard and routine vaccines respectively. A previous experiment (Hulse and Carnaghan, 1970) gave an IDa0 in unvaccinated animals of 49 organisms; this value was also increased 2,000-fold in guinea-pigs previously vaccinated with the standard vaccine. From these results it was calculated that the challenge dose could be increased lo-fold in the routine test to 50,000 W.544 organisms: vaccines would still give 75 per cent. protection, the standard to be attained in the test (Anon., 1970). DISCUSSION

Br. abort2~s S.19 vaccine manufactured for use in Great Britain is currently tested for potency by the method described in detail by Hulse and Carnaghan (1970). T we 1ve g uinea-pigs are vaccinated with 1/15th cattle dose, challenged after 60 days with 5,000 organisms of strain W.544, killed and bled 42 days later and cultures made from the spleen. Recovery of the virulent strain is the

206

POTENCY TESTS OF Br. abortus S.19 VACCINE

sole criterion of infection. If no more than 25 per cent. of the animals are infected the vaccine passesthe test. The delay of 60 days between vaccination and challenge was intended to ensure that the vaccinal strain is no longer present in the guinea-pigs : however, it has been shown that the majority of guinea-pigs are free from S.19 infection after 40 days. Residual infection, when it does occur, may persist far longer than 60 days. The period of 42 days between challenge and autopsy is to permit the optimal growth of the challenge strain in unprotected animals. It has been shown that the peak of infection as judged by the number of organisms recovered, does occur at 42 days (Thornton and Musket& in preparation). However, infection may be detected at an earlier stage, and non-infected animals at this time do not subsequently become infected. These intervals may, therefore, be shortened to 40 and 35 days respectively without altering the validity of the test procedure. This effects a total reduction of 27 days for the test. Using a range of vaccine dilutions, mean values from each vaccine group for skin reaction and protection against challenge showed similar variations with vaccinal dose. Morgan, Littlejohn, MacKinnon and Lawson (1966) using goats vaccinated with various Brucella vaccines, found a correlation between immunising capacity and the extent of skin sensitivity evoked with each vaccine : however, present studies showed no correlation in individual animals between skin reaction and protection. The responseto the vaccine measured by the skin test could not, therefore, be used to determine the immune status of the animal. The use of SAT as a criterion of infection is complicated by the titre produced as a reaction to the vaccine. It has been shown that with vaccine dosesof 7 x lo3 and below, significant titres in uninfected animals at the time of autopsy were uncommon : however, not every infected animal had a significant titre. With higher vaccine doses,antibody level was an even more unreliable guide to infection. The use of a constant vaccine and constant challenge dose will not always determine the protective capacity of vaccines since a low challenge dose will permit poor immunogenic vaccines to pass the test (Gargani, 1960). The use of a high vaccinal dose will have the same effect. If this type of assay is used, the vaccine dose must be such that significant variations in the quality of the vaccine will be detected and the challenge dose be such that it will overcome protection only when the protection afforded by the vaccine is inadequate. In the present work, two types of assay have been carried out and the results compared with those obtained in routine potency tests of the constant vaccine dose/constant challenge dose type. The constant vaccine/graduated challenge assaysshowed that the IDao in unvaccinated animals was 50 to 100 W.544 organisms. The routine challenge dose (5,000 organisms) is, therefore, equivalent to 50 to 100 IDao. The number of organisms required to infect 50 per cent. of guinea-pigs previously vaccinated with l/ 15th cattle dose of S.19 was 100,000 to 200,000 (a 2,000-fold increase). The graduated vaccine/constant challenge assay showed that 127 vaccinal organisms were required to protect 50 per cent. of guinea-pigs against 5,000 challenge organisms. As a linear relationship was obtained at low vaccine doses between the number of S.19 organisms in the dose and protection against challenge, it was calculated that a 2,000-fold increase in protection would be attained

DENISE

H.

THORNTON

AND

J. C.

MUSKETT

207

with 7 x l@+’ ES.19 organisms. Increasing the vaccine dose from 7 x 104’58 organisms to the normal test dose (7 x 10’) will not be expected, therefore, to increase the protection against 5,000 W.544 organisms, and this was found to be the case. The responses of body weight and skin sensitivity also showed no response to varied vaccine doses in this range. Hence both the vaccine dose and challenge dose in the routine test are not suitable. The challenge of 5,000 organisms would permit vaccines with 10 times less protective power than those tested in these experiments to pass the test. The vaccine dose of 1 / 15th cattle dose can be diluted over lO,OOO-fold before protection against 5,000 W.544 organisms will decrease. It is considered that the test could be made more rigorous by using higher challenge doses; this would mean that vaccines that pass the present test, but are of a quality inferior to those tested in these experiments, would fail the test. Alternatively, the test could be made more sensitive by using low vaccine doses at levels where variations in its quality would be detected when challenged with 5,000 virulent organisms. These methods may necessitate three-point assays because there is an inherent risk in biological tests of this type that a satisfactory batch of vaccine will fail the test when a single dose is used. The use of a three point assay using doses of vaccine at dilutions within the range where a correlation between dose of vaccine and protection has been established and challenge with 5,000 virulent organisms is employed would appear to be the method of choice. Besides being a sensitive assay, the growth rate of vaccinated animals is not retarded to the same extent as with the normal vaccinal dose, and the lack of persistence of vaccinal SAT in guinea-pigs vaccinated with high dilutions of vaccine is an additional aid in detecting infected animals at the end of the experimental period. Because of the low slope of the dose response curve, it would eventually be possible, when sufficient assays of this type had been carried out, to select a single vaccine dose in this range in order to reduce the number of animals used in the test. SUMMARY

The three methods which have been described for assaying the potency of Brucella abortus S.19 vaccines by challenge tests in guinea-pigs comprise constant vaccine/graduated challenge, graduated vaccine/constant challenge, and single level vaccine and challenge. A dose response curve was obtained by the first method and it was found that the infective dose (IDao) of the challenge strain W.544 was 89 organisms; this value was increased approximately 2,000-fold by vaccination with S.19 at l/ 15th cattle dose. Using a wide range of vaccine dilutions, a dose response curve was obtained with the second method. The protection dose (I’Dgo) in vaccinated guinea-pigs challenged with 5,000 W.544 organisms was 127 S.19 organisms. It was concluded that in the potency test specified in the British Veterinary Codex Supplement (1970), the vaccinal dose is too high or the challenge dose too low to present a critical test. The results suggest that the single level vaccine and challenge dose method would be satisfactory if used within the range where the protection is proportional to the dose. The number of S.19 organisms present in the spleens of vaccinated guinea-

208

POTENCY TESTS OF Br. abortus S.19 VACCINE

pigs was found to decrease over a period of 40 days. After this time, a small persistent infection was found in a few guinea-pigs. No evidence was found of the multiplication of S. 19 in guinea-pigs. Vaccination with S.19 inhibited the normal weight gain of the guinea-pigs. Attempts to determine the immune status of vaccinated guinea-pigs by measuring delayed hypersensitivity were made. Although the reaction in the skin sensitivity test was related to vaccine dose, it was not possible to distinguish individual animals that were found to be protected after challenge. The use of serum agglutination titres as a criterion of infection was complicated by the serum response due to the vaccine, especially at the higher vaccine levels. At the lower vaccine levels, this was far less evident, but even so, the only unequivocal criterion of infection was recovery of challenge organisms. ACKNOWLEDGMENTS

We wish to thank Miss J. B. Gateland and Mr. M. D. Chandler for their valuable technical

assistance

and Miss C. N. Hebert

for carrying

out the statistical

analyses.

REFERENCES

(1970). British Veterinary Codex Supplement, Pharmaceutical Press; London. Alton, G. G. (1969a). Res. vet. Sci., 10, 326; (1969b). Ibid., 329. Boyce, K. J., and Edgar, A. W. (1966). J. appl. Back, 29, 401. Ellwood, D. C., Keppie, J., and Smith, H. (1967). Brit. J. exp. Path., 48, 28. Gargani, G. (1960). 5th int. Mtg. biol. Standard, Jerusalem, 1959, p. 393. Hu~s~~E; C., and Carnaghan, R. B. A. (1970). Symp. Ser. immunobiol. Standard, Knight: G: J. (1969). Ibid., 10,161. Morgan, W. J. B. (1967). Iret. Rec., 80, 612. Morgan, W. J. B., Littlejohn, A. L., Mackinnon, D. J., and Lawson, J. R. (1966). Bull. Wld. Hlth. Org., 34,33. Ottosen, H. E., and Plum, N. (1949). Amer. J. vet. Res., 10, 5. Rasooly, G., Olitzki, A. L., and Sulitzeanu, D. (1967). Israel J. med. Sci., 3, 814. de Ropp, R. S. (1945). J. camp. Path., 55, 70. Smith, H., Keppie, J., Pearce, J. H., and Witt, K. (1962). Brit. J. exp. Path., 43, 538. Tootill, J. P. R. (1969). Symp. Ser. immunobiol. Standard, 10, 225. [Received

for publication,

June

15th,

19711