Comparison of Two Challenge Strains Used to Test the Potency of Brucella Abortus Vaccines

Br. vet .}. ( 19 77), 133, 554

COMPARISO N OF TWO CHALLENGE STRAINS USED T O TEST THE POTENCY OF BR UCELLA ABOR TUS VACCINES By C . GARCiA -C ARRILLO AND

R. CASAS-OLASCOAGA

Pan American Zoonoses Center, Pan American H ealth Organization/ World Health Organization, Casilla3092-Correo Central, 1000 Buenos Aires, Argentina

SUMMARY

The virulence of Brucella abortus strain W-544, used as the international reference for biotype I, was compared with the American B. abortus strain 2308 in guinea pIgs. One major group of animals was inoculated with strain 2308 and the other with W-544 using 5 s , 5\ 5 3 ,5 2 ,5 and I viable cells for each subgroup respectively. Calculations by the Spearman-Karber method gave 36 cells for the ro so of'the standard strain 544 and II cells for the IDso of strain 2308. The infected animals inoculated with B. abortus 23 08 presented a greater spleen weight, width, length and area as compared to those inoculated with strain W-544 . Animals infected with strain W-544 did not show any significant difference from non-infected animals nor from the non-inoculated controls in spleen weight, le ngth or area. Based on the results obtained, it is suggested that changing the challenge strain to the more virulent 2308 would be of value to the improved criti cal measurement of potency of B. abortus vaccines. INTROD UCT IO N

Brucella abortus W -544 is the international reference strain for biotype I. This strain has

been exte nsively used by British authors for challenge in immunity experiments (Stableforth , 1959 ), while the B . abortus 2308 strain is widely used in the U.S.A. The latter strain has also been emp loyed by workers in other countries (Rasoo ly, Oli tzki & Suli tzeanu, 19 67; Garcia -Carrillo & Trenchi, 1974). In the United Kingdom, a protection test in laboratory animals is the accepted method used to measure the potency of strain 19 vaccines. Failures of the test have been attrib uted to low challenge doses (Thornton & Muskett, 197 2), although these could also have been due to low virulence of the challenge strain. In the authors' experience, potency tests in guinea pigs are good indicators of the immunity exp ected in large animals. Diffic ulties have been found when strain W-544 was used fo r challenge b ecause of variations in results of repeated experiments. 0 ther studies (a uthors ' unpublished data) have shown lower infectivity of strain W-544 when

POTE CY OF BR UCEL LA ABO RTUS VACCI

ES

555

compared to strain 2308 and more reproducib le results were obtained with the latter strain. The purpose of this study was to compare the virulence of these two strains in gUinea pigs. MATERIALS AND METHODS

To determine the 50% infective dose (ID 50 ) for the two strains of B. abortus, 2308 and 544, 120 female American Short Hair gu inea pigs, weighing approximately 550 g each, were randomly divided into two major groups, each containing six subgroups of 6, 6, 12, 18, 12 and 6 animals. This arrangement was used to minimize the variance of the 1D50 accord ing to the modified method of Spearman-Karber. One major group was ino culated with strain 2308 and the other with strain 544; in both cases, 55, 54, 53, 52, 5 and I viable cells were used for each subgroup. A group of 24 guinea pigs, also randomly selected, was maintained as uninoculated controls. Lyo philized cu lture of B. abortus W-544 was obtained in 1970 from Dr W. J. B. Morgan, Central Veterinary Laboratory, Weybridge, England and strain 2308 was received from the National Animal Diseases Laboratory, United States Department of Agriculture. The strain W-544 is less virulent than the American 2308 strain (Stableforth, 1959). Both strains were stored in liquid nitrogen. At the time of inoculation of the guinea pigs, cultures for viable cell counts were mad e to ensure that the inoculation doses were correct. All suspensions were brought to a volume of I ml and inoculated intramuscularly. All animals were weighed at the beginning and end of the experiment. All guinea pigs, including the controls, were killed eight weeks after inoculation. Note was taken of the presence of lesio n- in the liver attributable to brucellosis. The parameters of weight, width, length and area of the spleen were also recorded and these were related to gross body weight. The information thus obtained was compared with normal range values as established in previous studies (Garcia-Carrillo, 1977 ). Cervical, precrurallymph nodes and samples of macerated spleen were inoculated on brucella albimi agar and incubated at 37 a C with 10% CO 2, An individual animal was considered to be infected if any brucella organisms were recovered Ii-om tissues, irrespective of the number of colonies obtained. RES U LTS

Infection Table I shows the number of infected animals at each dose level. Calculations by the Spearman-Karber method are 36 cells for the 1D5o of the standard strain W-544, with a 95% co nfidence interval from 14 to 90 cells and II cells for the 1D5o of strain 2308, with a 95% co nfidence interval from six to 21 cell s. The difference was significant at P
Hepatic lesions In the group inoculated with strain W-544, one out of 33 infected animals showed lesions while 17 out of 38 animals infected with strain 2308 showed macroscopic lesions attributable to brucella infection . This was significant at P < 0·00 I.

TABLE I

c.;-. c.;-.

I NfECT ION AND MACROSCOP I C PATHOLOG ICAL CHANGES Of GUINEA PIGS I N O CU LATED WITH BR UC ELLA ABORTUS STRA IN 2308 OR W-544

0>

No. of animals per group

6 6 12 18 12 6

No . of cells inoculated

Strain 544 No. infected animals

3 125 625 125 25 5

S train 2308 No. infected animals

H epatic lesions

Splenic changes

6 4 10

0 0

I I

6 6

II

0 0 0

5 2 0 0

II II

2 0

6 0

H epatic lesions

Splenic changes

5 2 6 4 0 0

6 4 10 9 4 0

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Z

-<

'TABLE I I

Parameters of the spleen (mean ± s.d.)

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z

;J>

..r uo uo 0'>

Strain inoculated

Bacteriological results

No . ofanimals

Weight (g)

Width (mm)

Length (mm)

Area (mm 2 )

Body weight (gain (g)

544

I nfected Negative Infected Negative

33 27 40 20 24

1·348 ± 0·490 1· 155±0·22 1 3·03 1± 1·5 98 1·123±0·228 1·223±0· 192

16·9± 1·75 15·7 ± 1·06 20·814·03 15 ·3 ± 1· 00 16·3± 1·37

33· 9± 362 33·4± 2·83 430± 8·33 33·6 ± 287 35·5±3·27

443 ± 95 428 ± 90 6931 24 1 407 ± 56 436± 73

124± 60 13 7 ± 5 1 871 11 5 168±103 145± 54

s. d.: standard deviation.

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COMPAR I SON OF CHANGES I N THE SPLEEN AND WE I GHT - GA I N IN GU I NEA P I GS I NOCU LATED W ITH B . ABORTUS STRAIN W-544 OR 2308

Controls

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•

2308

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POT ENCY OF BR UCE LLA ABORTUS VACCINE

557

Splenic changes In the group inoculated with strain 544, nine out of 33 infected guinea pigs had a heavier sp leen than the expected upper 95% limi t in normal guinea pigs, while 33 out of the 38 animals infected with strain 2308 had a heavier sp leen than expected in normal animals. Table II shows the changes of the various parameters of the spleen in both groups of inoculated animals and in the controls. The infected animals inoculated with B. abortus 2308 had greater spleen weight, wid th, length and area compared to those inoculated with strain W-544. The calculated Student t statistics for the differences between the means, gave values of 5·82, 5·22, 5· 78 and 5·59 for weight, width, length and area, respectively. The above values were all significant at P < 0·001. Animals infected with strain W-544 did not show any significant difference from non-infected animals nor from the non-inoculated controls in sp leen weight, length or area. Spleen width showed a significant difference at P < 0·01. Weight gain Table II shows weight gains during the eight-week period of the experiment (from inoculation to slaughter). Animals infected with strain 2308 gained less weight than any other group. This difference was significant at P< 0·01 as compared with the noninfected or the control group . The difference in animals infected with W-544 was not significant at P< 0 ·05 (0 ·05 < P < 0·10 ). Serologic response All guinea pigs with positive cultures developed high agglutinin titres irrespective of the infective strain.

DISCUSSION

The present study demonstrates that there is a considerable difference in the virulence for guinea pigs of the two strains of B. abortus studied . The worldwide recognized standard strain 544 was shown to be less virulent than strain 2308 . In a few cases guinea pigs infected with strain 544 developed macroscopic liver lesions and/or splenic changes attributable to brucella infection, while the majority of those infected with strain 2308 consistently developed such a pathology. Thornton & Muskett (1972 ) have found that the spleen/body weight ratios of animals infected with W-544 were greater than those of non-infected animals, but the differences were not statistically significant. Our findings agree with these results . Other pathological changes produced by this strain were also minimal. The ID50 of 36 cells for B. abortus 544 is similar to that of the 49·4 cells reported by Hulse & Carnaghan ( 1970) and is in accordance with an earlier estimate made in our laboratory of ID50 =4 7 (unpublished data). The average number of colonies recovered from cervical and precrurallymph nodes and from the spleen of animals infected with strain 2308 was higher than the average obtained from tissues of animals infected with strain W-544. Only exceptionally the animals infected with strain 2308 yielded a limited number of colonies. The fact that

558

BRITISH VETERINARY JOURNAL, 133, 6

large numbers of colonies can usually be recovered from guinea pigs inoculated with strain 2308 is of advantage in defining infected animals. I t has been suggested by some auth ors (Thornton & M uskett, 1972) that the challenge with 5000 organisms of strain W-544 would permit a poor vaccine to pass the test. Consequently, it was proposed that the number of cells should be increased above 5000 to make the test more critical. Our results suggest that it would be more advantageous to use the more virulent strain 2308 as challenge. A further advantage of strain 2308 is that infection in guinea pigs can be determined by gross observation of lesions thus avoiding the need for brucella culture. B. abortus strain 2308 maintained in liquid nitrogen consistently produces uniform infectivity (Garcia-Carrillo, unpublished data). In a previous study (Garcia-Carrillo & Trenchi, 1974) 15 cells infected 75% and 150 cells infected 100% of25 non-immunized guinea pigs. The results presented here suggest that a dose of 1000 cells of strain 2308 would be an appropriate immunity challenge for guinea pigs. REFERENCES

GARCiA-CARRILLO, C. (l977).Lab . Anim. ll , 17 5. GARCiA-CARRILLO, C. & TRENCH I, A. ( 1974). Revta Med. vet., B. Aires 55, 3. HULS E, E. C. & CARNAGHAN, R. B. A. (1970 ). International Symposium on Brucellosis, Tunis 196 8; Symposia series in immunobiological standardization 12, I. Basel and New York : Karger. RASOOLY, G., OLITZKI, A. L. & SULITZEANU, D. ( 1967). Israel}. med. Sci. 3, 814. STABLEFORTH, A. W. (1959 ). In Infectious Diseases oj Animals, ed . Stableforth, A. W. & Galloway, I. A. London : Butterworths Scientific Publications. THORNTON, D. H . & MUSKETT,J. C. (l972).j. compo Path. 82, 201.

( Accepted for publication JJ January 19 77)