- Email: [email protected]
Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA)
Veterinary Microbiology, 8 (1983) 187--198
187
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ANTIGENIC RELATIONSHIPS BETWEEN THE SEROTYPES OF PASTE URELLA H A E M O L YTICA DEMONSTRABLE BY ENZYMELINKED IMMUNOSORBENT A S S A Y (ELISA)
C. BURRELLS, H.B. EVANS* and A. McL. DAWSON
Moredun Research Institute, 408 Gilrnerton Road, Edinburgh EH17 7JH (Gt. Britain) * Present address: Department of Rheumatology, University of Manchester, Oxford Road, Manchester M13 9PT (Gt. Britain) (Accepted 4 November 1982)
ABSTRACT Burrells, C., Evans, H.B. and Dawson, A. McL., 1983. Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA). Vet. Microbiol., 8: 187--198. Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.
INTRODUCTION
The subdivision of Pasteurella haemolytica species into several distinct serotypes was achieved by the agglutination reaction between bovine red blood cells (RBC) sensitised with soluble antigens from P. haemolytica and antisera raised in rabbits against selected strains (Biberstein et al., 1960). Fourteen serotypes are now recognised and these can be divided into 2 biotypes, A and T (Smith, 1961). Most biotype A strains ferment arabinose but not trehalose, whereas all biotype T strains ferment trehalose. Clinically, biotype A strains are associated with pneumonias in sheep of all ages, and with septicaemia in lambs under 2 months of age. The biotype T strains are associated with the septicaemic form of pasteurellosis in older sheep (Gilmour, 1978). Serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13 and 14 belong to biotype A, and serotypes 3, 4 and 10 to biotype T. Other methods have also been used in the classification of P. haemolytica. A
0378-1135/83/$03.00
© 1983 Elsevier Science Publishers B.V.
188 study of nucleic acid homologies between biotypes A and T showed a high degree of homology between two biotype A strains, whereas the 30-50% homology between a biotype A and a biotype T strain was less than that often obtained for interspecies hybridisation (Biberstein and Francis, 1968). Electrophoresis in polyacrylamide gels of the constituent proteins of each serotype of P. haemolytica gave patterns which could be used to subdivide the species into two groups which broadly corresponded to the biotypes A and T (Thompson and Mould, 1975). As the indirect haemagglutination (IHA) test became more widely used to serotype larger numbers of strains, occasional strains were found to give low-titre cross-reactions with the typing antisera of other serotypes (Biberstein, 1965). Although rare, reactions have been recorded between serotypes 1 and 6; 3 and 10; 4 and 10; 3 and 7; 7 and 8; and 7 and 12. Recently, the enzyme-linked immunosorbent assay (ELISA) was shown to be at least 160 times more sensitive than the IHA test in measuring antibody to P. haemolytica type A1 in the sera of vaccinated specific pathogen-free (SPF) lambs (Burrells et al., 1979). This study was carried out to determine if the greater sensitivity of ELISA would reveal antigenic relationships between serotypes n o t detected b y IHA. MATERIALS AND METHODS
Antigen preparations The outer cell wall layers (considered to contain capsule material) of a number of A serotypes obtained by extraction with sodium salicylate (Wells et al., 1979a) have previously been shown to be immunogenic when tested as vaccines either singly or in combination (Gilmour et al., 1979) and protection in sheep against aerosol challenge with live organisms was serotype-specific. Antigens were derived from A biotypes 1, 2, 5, 6, 7, 8, 9, 11 and 12 and T biotypes 3, 4 and 10 using a modification of the m e t h o d of Wells et al. (1979a) in which sodium salicylate extracts (SSE) of 12 serotypes o f P . haemolytica were prepared. Nutrient broth (50 ml) (Oxoid Nutrient Broth No. 2) was seeded from a lyophilised aliquot of bacteria and incubated at 37°C overnight. Two 1.5 1 volumes of sterile broth were each inoculated with 15 ml of the overnight broth culture and incubated at 37°C with continuous shaking for 6 h. The cultures were then centrifuged at 2200 g for 30 rain and the bacterial deposit resuspended to 300 ml in 1 M sodium salicylate. The suspension was shaken for 3 h at 37°C, centrifuged in an angle-rotor for 30 min at 23000 g and the supernatant finally clarified in a swinging bucket rotor at 40000 g for 30 min. The clarified SSE preparation was concentrated by ultrafiltration through a Diaflo XM 100A membrane (Amicon Corporation, Lexington, MA, U.S.A.) to a volume of approximately 20 ml. The concentrated SSE was dialysed for 48 h at 4°C against 3 changes
189
of dilute phosphate buffered saline (0.02 M sodium phosphate, 0.03 M sodium chloride, pH 7.6) followed by dialysis against 3 changes of distilled water over 3 days at 4°C, and finally, each SSE was diluted to a final dry weight concentration of 1.5 mg ml -~ using as diluent a dialysed filtrate from the Diaflo concentration step.
Antigens Inocula were prepared from each of the SSE antigens using an aluminium hydroxide/off adjuvant (AHO) of proven efficacy in stimulating an antibody response to P. haemolytica in sheep (Wells et al., 1979b). The final vaccine contained 0.341 mg m1-1 of SSE.
Immunisation o f animals Mice Groups of C57 black mice (10--20 mice for each preparation) were inoculated subcutaneously with 0.1 ml of antigen per mouse (34.1 pg of SSE) on two occasions, with a 2-week interval between each. Two weeks after the second injection, the mice were anaesthetised with CO2 and bled out. The blood from all mice in a single group was pooled and the resultant sera stored in 0.5-ml aliquots at -20°C. Pooled serum from a group of untreated mice was obtained and stored in the same way. Rabbits New Zealand White rabbits were each inoculated intramuscularly with 1 ml of vaccine (0.341 mg of SSE). A second injection was given 8 weeks after the first. Blood samples were taken from the marginal ear vein preimmunization and again 4 weeks after the second injection. The serum was stored in 5-ml aliquots at -20°C. Serum from an unvaccinated rabbit was obtained and stored in a similar way. Micro-ELISA procedures The technique was basically a microplate modification (Voller and BidweU, 1975} of the ELISA method (Engvall and Perlmann, 1972) as previously described for the quantitation of antibodies to P. haemolytica type A1 (Burrells et al., 1979}. In this work, however, a rabbit IgG antimouse IgG conjugate and a sheep IgG anti-rabbit IgG conjugate were used where appropriate.
Conjugates Anti-mouse IgG The IgG fraction of rabbit anti-mouse IgG was obtained commercially (Miles Laboratories, Slough, Gt. Britain).
190
Anti-rabbit IgG The IgG fraction was purified from rabbit serum over protein A-Sepharose (Goding, 1976), and was used to raise hyperimmune sheep serum. The IgG fraction of the antiserum was prepared using a DE 52 cellulose column as previously described (Burrells et al., 1979). These two IgG preparations were then conjugated with alkaline phosphatase, Sigma type VII (Sigma Chemical Company, Poole, Dorset) by the m e t h o d of Engvall and Perlmann (1972). Standardisation o f the conjugates These standardisations were carried o u t as previously described (Evans, 1979). Briefly, a checkerboard titration of type A1 SSE against its mouse or rabbit IgG antibody using a conjugate at an arbitrary dilution yielded dilutions of SSE and antiserum which were considered to be optimal. These optimal dilutions were then used in a test in which the conjugates were titrated to establish suitable working dilutions. These were determined as 1/200 for the anti-mouse IgG conjugate, and 1/2000 for the anti-rabbit IgG conjugate.
Determination o f the optimal dilutions of SSE antigens and mouse and rab bit antisera Using the anti-mouse IgG conjugate at its predetermined working dilution, 12 checkerboard titrations were carried out to determine the optimal coating dilution of each SSE which would react with the optimal dilution of its respective mouse antiserum to give an optical density at 400 nm (OD400) of 0.7, the highest c o m m o n maximum for all serotypes. Twelve checkerboard titrations were carried o u t in a similar fashion using the optimal dilution of anti-rabbit conjugate, and the predetermined optimal dilutions of rabbit antisera. Cross-ELISA tests were then performed by reacting each antiserum at the dilution determined to be optimal for its homologous SSE against the optimal dilutions of each of the other SSEs. Results were corrected for any background colouration by subtraction of the OD400 of an "antigen alone" control from the OD400 of the test. To assess antigenic relationships between strains in terms of antibody reactivity, a m e t h o d described by Archetti and Horsfall (1950) and Gois et al. (1974) was used. In it, the geometric mean of the ratios (r) is given by the formula r = vr-~l-~K-r-~2 where
191
heterologous titre (strain 2) r 1 =
. . . . . . . . . . . . . . . . . . . .
homologous titre (strain 1) and heterologous titre (strain 1) r2
homologous titre (strain 2) In this assay, OD400 was substituted for titre. The value r measures similarities between serological reactions to different strains. When the homologous and heterologous values are equal, r is maximal at 1. (i.e., rl X r2 = 1/1). The greater the denominator of the expression r, X r2 the greater the difference between the strains. Conversely, as the value of r tends towards unity, the more closely related are t w o strains antigenically.
Indirect haemagglutination (IHA ) tests IHA tests were performed by the m e t h o d of Carter (1955) as modified by Burrells et al. (1979). RESULTS
The results of cross-IHA tests are shown in Tables I and II. Titres of < 1/8 were considered negative. In mice (Table I), antibody to homologous antigen was evoked with the SSE of types A1, T3, T4, A8 and T10. In addition, mice immunised with type A2 reacted with type T3; those immunised with type A5 reacted with type A9 and those immunised with type T3 reacted with type T10. Pooled serum from unvaccinated control mice gave negative reactions with all 12 antigens. In rabbits, antibody to homologous antigen was produced by the SSE of all the types with the exception of types A2 and A12. Heterologous reactions were evident where rabbit anti-A1 serum reacted with type A6 SSE: anti-T3 serum reacted with t y p e T4, and a n t i - A l l serum reacted with type T4. Serum from the unvaccinated control rabbit and pre-immunisation sera from the vaccinated rabbits gave negative reactions with all 12 serotypes. The lack of response b y 7 of the 12 serotypes in mice and 2 of the 12 serotypes in rabbits observed with the IHA test is contrasted by the measurable responses of all 12 homologous serotypes when the sera from both species were tested by ELISA. Figures 1 and 2 illustrate for mouse and rabbit sera, respectively, the degree of "relatedness" of each SSE t y p e to the other 11 when tested by ELISA. With mice, some degree of cross-reactivity occurred between all the 12 types. T y p e A9 cross-reacted strongly with other types of P. haemolytica, notably types A1, A2, A5, A6, A7, A8 and A12, whilst type
192 TABLE I Indirect haemagglutination (IHA) reactions o f m o u s e antisera tested against h o m o l o g o u s and heterologous s o d i u m salicylate extract ( S S E ) Mouse antiserum
A1
P. haemolytica s o d i u m salicylate extract ( S S E ) ............................................................ A1 A2 T3 T 4 A 5 A6 A7 A8 A9 32*
T10
All
A12
--
m
T3
8 8
T4
-
A2
16
-
8
-
-
-
w
-
A5 A6
8 m
A7
A8 A9
16
T10
16
All A12
w
Control
- - = Negative reactions at less than 1 / 8 . * Titres expressed as reciprocals o f end-point dilution.
T A B L E II Indirect haemagglutination ( I H A ) reactions o f rabbit antisera tested against h o m o l o g o u s and heterologous s o d i u m salicylate extract ( S S E ) Rabbit
P. haemolytica s o d i u m salicylate extract ( S S E )
antiserum A1 A1 A2 T 3
A2
128" .
T3
--
T4
A5
--
A6
.
128 --
A5
--
A8
--
--
8
.
T4
A7
32 512
T10
All
--
--
--
16
--
A6
--
A7
--
128
A8
--
A9 T10
--
--
--
--
All
--
AI2
--
--
Control
--
--
--
-64
---
32
8
--
- - = negative reactions at less t h a n 1/8. * Titres expressed as reciprocals o f end-point dilution.
--
A12
m
.
--
--
A9
512 --
16
-8
193
123456789101112 A1
=
u_
~
123456789101112 A2
T4
A5
A7
A8
123456789101112 T3
A6
0"8
0-2 n" LLI
1 2 3 4 5 6 7 8 9 101112 A9
¢'= 1.0
0-6 8"4 0'2
123456789101112 T10 SODIUM
123456789101112 A11 SALICYLATE
123456789101112 A12
EXTRACT
(SSE)
Fig. 1. Cross-relatedness (r) between serotypes of PasteureUa haemolytica using antisera prepared in mice. • = Homologous type ( r = 1 ) ; [] = A b i o t y p e s ; [] = T biotypes.
A l l was the least cross-reactive. In general, strong cross-relationships existed between the A biotypes on the one hand, and between the T biotypes on the other, although there was a considerable degree of shared antigenic relatedness throughout. Rabbit antisera presented clearer extremes of antigenic relatedness between A biotypes and T biotypes, although there was a surprisingly high cross-reactivity between type A2 and type T3 (Fig. 2). Type T10 was the least cross-reactive whilst types A2, A5 and A12 possessed a high degree of cross-reactivity with the other A biotypes. The T biotypes were less cross-reactive than the A biotypes. Of the A biotypes, type A l l showed the least cross-reaction with the other types. One outstanding difference between mouse and rabbit antisera in ELISA was that with the former, T biotypes reacted more strongly with the other T biotypes than with
::LLL
194
0"6
0.4
0-2
0,8-t
o.6-1 H
AI
123456789101112 A2
123456789101112 T3
T4
123456789101112 A5
123456780101112 A6
123456789101112 A8
123456789101112 A9
|
0 0.2 n,.,
0 192 314 0 516 71 1 ~ A7
°i LIJd 123456789101112 T10
123456789101112 A11
123456789101112 A12
SODIUM SALICYLATE EXTRACT {SSE) Fig. 2. C r o s s - r e l a t e d n e s s (r) b e t w e e n s e r o t y p e s o f Pasteurella haernolytica u s i n g a n t i s e r a p r e p a r e d in r a b b i t s . • = H o m o l o g o u s t y p e (r = 1); ~ = A b i o t y p e s ; ~ = T b i o t y p e s .
A biotypes, whilst with rabbit antisera the T biotypes showed only limited cross-reactions. Control sera from unvaccinated mice, the unvaccinated rabbit and pre-immunization rabbit sera all gave negative ELISA reactions with the 12 serotypes. DISCUSSION
In IHA tests the titres of the sera from mice tended to be low (1/8-1/32) whilst titres obtained with rabbit sera confirmed the findings of Biberstein (1965) that, where cross-reactions occurred, the heterologous titres were of a much lower order than the homologous titres. In ELISA tests with mouse sera there appeared to be stronger relationships between the A biotypes on one hand, and T biotypes on the other.
195 It is interesting also that type A9 cross-reacted as strongly with each T serotype as did the T serotypes with each other. By polyacrylamide-gelelectrophoresis Thompson and Mould (1975) subdivided the 12 serotypes into 2 groups corresponding to biotypes A and T, b u t type A9 showed only minor band differences from types T3, T4 and T10. The strong reaction observed between type A2 and type T3 in ELISA was also observed in IHA tests on the sera from mice (Table I). In the original classification of P. haemolytica types (Biberstein et al., 1960) t y p e 2 was grouped with types 3, 4 and 10 on the basis of biochemical tests, as was type 11 which cross-reacted poorly in ELISA even with the other A biotypes. Differences observed in the cross-ELISA tests between mouse and rabbit antisera could conceivably be due to the fact that each mouse serum was a pool from 10--20 animals whereas only 1 rabbit antiserum was used for each serotype. Thus individual variation of response would be masked with the mouse antisera, b u t may affect the results with the rabbit sera. Cross IHA tests with the sera from immunised rabbits indicated the probable existence of a small number of minor antigens responsible for the low-titred cross-reactions between P. haemolytica types which do n o t seriously interfere with the typing procedure (Biberstein, 1965). The results obtained from the sera of mice vaccinated with bacterins of various strains (Biberstein and Thompson, 1965) suggested that capsular antigens play the major role in determining the specificity of the immune response whilst somatic antigens are of secondary importance. Further studies in cross protection using bacterins in mice (Knight et al., 1969) confirmed the importance of the capsular antigens, b u t anomalies were found. Mice vaccinated with bacterins from strains which were serologically distinct from the challenge strain showed a better degree of protection than those vaccinated with homologous bacterins. As a result of these findings it is suggested that although capsular and somatic antigens are important, other factors may exist which determine the specificity of conferred immunity. No such cross-protection is apparent in mice immunised with SSE antigens (capsular preparations). In vaccination-challenge experiments, a trivalent vaccine containing SSEs of P. haemolytica serotypes A1 and A6, and heat-killed organisms of type A2 gave protection against subsequent challenge with types A1 and A6 organisms, b u t not against challenge with types A2 and A9 organisms (Evans and Wells, 1979). Similar findings have been obtained using the same vaccine in SPF lambs (Gilmour et al., 1979). Since the establishment of the ELISA technique for P. haemolytica (Burrells et al., 1979), it has become evident that wider serological cross reactivity exists between serotypes. Sera from conventional ewes and lambs which had been vaccinated with a trivalent vaccine, containing type A1 and A6 SSEs and heat-killed organisms of type A2 when examined by ELISA, exhibited antibody titres to t y p e A2 SSE which were comparable
196
to t y p e A l l SSE which was n o t included in the vaccine (N.J.L. Gilmour and C. Burrells, 1980, unpublished observations). This could conceivably be due to the pre-existence of antibody to type A l l in the ewes and hence their lambs. Alternatively the vaccine evoked the production of antibodies which cross-reacted with type A l l . Evidence for the latter hypothesis is provided by data from experiments with serum from SPF lambs immunised solely with type A1 SSE (Wells et al., 1979a). When tested in ELISA against types A1 and A l l SSE a pool of SPF lamb serum gave strong reactions with both, indicating the presence of c o m m o n group antigenic components. Absorption of this serum pool with SSEs of P. haemoly tica serotypes other than that of type A1 removed antibody activity to type A l l leaving antibody reacting only with t y p e A1 SSE (Donachie et al., 1983). Of those cross-reactions in mice and rabbits demonstrated b y IHA, only that between types A5 and A9 in m i c e was apparent in the ELISA crossreactions. The cross-reactions demonstrated by ELISA could be due to the greater sensitivity of the technique over IHA allowing detection of " g r o u p " reactivity and inter-type relationships n o t recognised by IHA. Alternatively, it may be that only certain antigenic components are bound to the RBCs used in IHA tests whereas a larger repertoire of antigens in the SSE preparations are capable of binding to the polystyrene plates used in ELISA. In IHA tests, sensitised RBCs are more readily agglutinated by immunoglobulin M (IgM) than by IgG. The ELISA technique employed in this study detected only IgG. It has previously been shown in SPF lambs that intramuscular (i/m) immunisation with antigen in oil-based adjuvant stimulates very high levels of circulating IgG compared to IgM. The IgG content of the total circulating antibody is further increased by a second i/m inoculation of the same preparation (Smith et al., 1976). The animals used in this study were immunised in a similar fashion, which suggests that the ELISA is a more efficient method of detecting the bulk of antib o d y stimulated b y this m e t h o d of immunisation. The cross-reactivity illustrated by ELISA suggests the presence of common antigenic components among the serotypes of P. haemolytica which can be detected b y ELISA and n o t by IHA. Further work has been done using the ELISA technique to determine if a type-specific, non cross-reacting serum such as that described above (Donachie et al., 1983) can be used to prepare purified type-specific antigens of P. haemolytica serotypes. These antigens would then allow the ELISA technique to be employed for the measurement of the immune responses in an animal to the individual serotypes of a multivalent vaccine. ACKNOWLEDGEMENTS
H.B. Evans was supported by a research studentship awarded by the Agricultural Research Council. The authors thank Dr. I.D. Aitken for his assistance in the preparation of this manuscript.
197 REFERENCES Archetti, I. and Horsfall, F.L., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralisation in ovo with heterologous i m m u n e serum. J. Exp. Med., 92: 441--462. Biberstein, E.L., 1965. Cross-reactions between types of Pasteurella haemolytica. Cornell Vet., 55: 495--499. Biberstein, E.L. and Francis, C.K., 1968. Nucleic acid homologies between the A and T types of Pasteurella haemolytica. J. Med. Micro., 1: 105--108. Biberstein, E.L. and Thompson, D.A., 1965. Type specificity of immunity to Pasteurella haemolytica infection in mice. J. Comp. Pathol., 75: 331--337. Biberstein, E.L., Gills,M.G. and Knight, H., 1960. Serological types of Pasteurella haemolytica. Cornell Vet., 50: 283--300. Burrells, C., Wells, P.W. and Dawson, A. McL., 1979. The quantitative estimation of antibody to Pasteurella haemolytica in sheep sera using a micro enzyme-linked immunosorbent assay (ELISA). Vet. Microbiol., 3: 291--301. Carter, G.R., 1955. Studies on Pasteurella multocida. 1. A haemagglutination test for the identification of serological types. A m . J. Vet. Res., 16: 481--484. Donachie, W., Burrells, C. and Dawson, A. McL., 1983. Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haernolytica antigens. Vet. Microbiol., 8: 199-205. Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen coated tubes. J. Immunol., 109: 129--135. Evans, H.B., 1979. A study of the mechanisms of immunity to PasteureUa haemolytica infection. P h . D . Thesis, University of Edinburgh, pp. 154--171. Evans, H.B. and Wells, P.W., 1979. A mouse model of Pasteurella haemolytica infection and its use in assessment of the efficacy of P. haemolytica vaccines. Res. Vet. Sci., 27: 213--217. Gilmour, N.J.L., 1978. Pasteurellosis in sheep. Vet. Rec., 102: 100--102. Gilmour, N.J.L., Martin, W.B., Sharp, J.M., Thompson, D.A. and Wells, P.W., 1979. The development of vaccines against pneumonic pasteurellosis in sheep. Vet. Rec., 104: 15. Goding, J.W., 1976. Conjugation of antibodies with fluorochromes: modifications to the standard methods. J. Immunol. Meth., 13: 215--216. Gois, M., Kuksa, F., Franz, J. and Taylor-Robinson, D., 1974. The antigenic differentiation of seven strains of Mycoplasma hyorhinis by growth-inhibition, metabolisminhibition, latex-agglutination and polyacrylamide-gel-electrophoresis tests. J. Med. Microbiol., 7 : 105--114. Knight, N.D., Biberstein, E.L. and Allison, M., 1969. The role of capsular and somatic antigens in immunisation of mice against Pasteurella haemolytica infection. CorneU Vet., 59: 55--64. Smith, G.R., 1961. The characteristics of two types of Pasteurella haemolytica associated with different pathological conditions. J. Pathol. Bacteriol., 81 : 431--440. Smith, W.D., Dawson, A. McL., Wells, P.W. and BurreUs, C., 1976. Immunoglobulins in the serum and nasal secretion of lambs following vaccination and aerosol challenge with parainfluenza 3 virus. Res. Vet. Sci., 21 : 341--348. Thompson, D.A. and Mould, D.L., 1975. Protein electrophoretic pattern of Pasteurella haemolytica. Res. Vet. Sci., 18: 342--343. VoUer, A. and Bidwell, D.E., 1975. A simple method for detecting antibodies to Rubella. Br. J. Exp. Pathol., 56: 338--339.
198 Wells, P.W., Evans, H.B., Burrells, C., Sharp, J.M., Gilmour, N.J.L., Thompson, D.A and Rushton, B., 1979a. Inability of passively acquired antibody to protect lamb~ against experimental pasteurellosis. Infect. Immun., 26: 25--29. Wells, P.W., Gilmour, N.J.L., Burrells, C. and Thompson, D.A., 1979b. A serologica comparison of Pasteurella haemolytica vaccines containing different adjuvants. Res Vet. Sci., 27: 248--250.
187
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ANTIGENIC RELATIONSHIPS BETWEEN THE SEROTYPES OF PASTE URELLA H A E M O L YTICA DEMONSTRABLE BY ENZYMELINKED IMMUNOSORBENT A S S A Y (ELISA)
C. BURRELLS, H.B. EVANS* and A. McL. DAWSON
Moredun Research Institute, 408 Gilrnerton Road, Edinburgh EH17 7JH (Gt. Britain) * Present address: Department of Rheumatology, University of Manchester, Oxford Road, Manchester M13 9PT (Gt. Britain) (Accepted 4 November 1982)
ABSTRACT Burrells, C., Evans, H.B. and Dawson, A. McL., 1983. Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA). Vet. Microbiol., 8: 187--198. Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.
INTRODUCTION
The subdivision of Pasteurella haemolytica species into several distinct serotypes was achieved by the agglutination reaction between bovine red blood cells (RBC) sensitised with soluble antigens from P. haemolytica and antisera raised in rabbits against selected strains (Biberstein et al., 1960). Fourteen serotypes are now recognised and these can be divided into 2 biotypes, A and T (Smith, 1961). Most biotype A strains ferment arabinose but not trehalose, whereas all biotype T strains ferment trehalose. Clinically, biotype A strains are associated with pneumonias in sheep of all ages, and with septicaemia in lambs under 2 months of age. The biotype T strains are associated with the septicaemic form of pasteurellosis in older sheep (Gilmour, 1978). Serotypes 1, 2, 5, 6, 7, 8, 9, 11, 12, 13 and 14 belong to biotype A, and serotypes 3, 4 and 10 to biotype T. Other methods have also been used in the classification of P. haemolytica. A
0378-1135/83/$03.00
© 1983 Elsevier Science Publishers B.V.
188 study of nucleic acid homologies between biotypes A and T showed a high degree of homology between two biotype A strains, whereas the 30-50% homology between a biotype A and a biotype T strain was less than that often obtained for interspecies hybridisation (Biberstein and Francis, 1968). Electrophoresis in polyacrylamide gels of the constituent proteins of each serotype of P. haemolytica gave patterns which could be used to subdivide the species into two groups which broadly corresponded to the biotypes A and T (Thompson and Mould, 1975). As the indirect haemagglutination (IHA) test became more widely used to serotype larger numbers of strains, occasional strains were found to give low-titre cross-reactions with the typing antisera of other serotypes (Biberstein, 1965). Although rare, reactions have been recorded between serotypes 1 and 6; 3 and 10; 4 and 10; 3 and 7; 7 and 8; and 7 and 12. Recently, the enzyme-linked immunosorbent assay (ELISA) was shown to be at least 160 times more sensitive than the IHA test in measuring antibody to P. haemolytica type A1 in the sera of vaccinated specific pathogen-free (SPF) lambs (Burrells et al., 1979). This study was carried out to determine if the greater sensitivity of ELISA would reveal antigenic relationships between serotypes n o t detected b y IHA. MATERIALS AND METHODS
Antigen preparations The outer cell wall layers (considered to contain capsule material) of a number of A serotypes obtained by extraction with sodium salicylate (Wells et al., 1979a) have previously been shown to be immunogenic when tested as vaccines either singly or in combination (Gilmour et al., 1979) and protection in sheep against aerosol challenge with live organisms was serotype-specific. Antigens were derived from A biotypes 1, 2, 5, 6, 7, 8, 9, 11 and 12 and T biotypes 3, 4 and 10 using a modification of the m e t h o d of Wells et al. (1979a) in which sodium salicylate extracts (SSE) of 12 serotypes o f P . haemolytica were prepared. Nutrient broth (50 ml) (Oxoid Nutrient Broth No. 2) was seeded from a lyophilised aliquot of bacteria and incubated at 37°C overnight. Two 1.5 1 volumes of sterile broth were each inoculated with 15 ml of the overnight broth culture and incubated at 37°C with continuous shaking for 6 h. The cultures were then centrifuged at 2200 g for 30 rain and the bacterial deposit resuspended to 300 ml in 1 M sodium salicylate. The suspension was shaken for 3 h at 37°C, centrifuged in an angle-rotor for 30 min at 23000 g and the supernatant finally clarified in a swinging bucket rotor at 40000 g for 30 min. The clarified SSE preparation was concentrated by ultrafiltration through a Diaflo XM 100A membrane (Amicon Corporation, Lexington, MA, U.S.A.) to a volume of approximately 20 ml. The concentrated SSE was dialysed for 48 h at 4°C against 3 changes
189
of dilute phosphate buffered saline (0.02 M sodium phosphate, 0.03 M sodium chloride, pH 7.6) followed by dialysis against 3 changes of distilled water over 3 days at 4°C, and finally, each SSE was diluted to a final dry weight concentration of 1.5 mg ml -~ using as diluent a dialysed filtrate from the Diaflo concentration step.
Antigens Inocula were prepared from each of the SSE antigens using an aluminium hydroxide/off adjuvant (AHO) of proven efficacy in stimulating an antibody response to P. haemolytica in sheep (Wells et al., 1979b). The final vaccine contained 0.341 mg m1-1 of SSE.
Immunisation o f animals Mice Groups of C57 black mice (10--20 mice for each preparation) were inoculated subcutaneously with 0.1 ml of antigen per mouse (34.1 pg of SSE) on two occasions, with a 2-week interval between each. Two weeks after the second injection, the mice were anaesthetised with CO2 and bled out. The blood from all mice in a single group was pooled and the resultant sera stored in 0.5-ml aliquots at -20°C. Pooled serum from a group of untreated mice was obtained and stored in the same way. Rabbits New Zealand White rabbits were each inoculated intramuscularly with 1 ml of vaccine (0.341 mg of SSE). A second injection was given 8 weeks after the first. Blood samples were taken from the marginal ear vein preimmunization and again 4 weeks after the second injection. The serum was stored in 5-ml aliquots at -20°C. Serum from an unvaccinated rabbit was obtained and stored in a similar way. Micro-ELISA procedures The technique was basically a microplate modification (Voller and BidweU, 1975} of the ELISA method (Engvall and Perlmann, 1972) as previously described for the quantitation of antibodies to P. haemolytica type A1 (Burrells et al., 1979}. In this work, however, a rabbit IgG antimouse IgG conjugate and a sheep IgG anti-rabbit IgG conjugate were used where appropriate.
Conjugates Anti-mouse IgG The IgG fraction of rabbit anti-mouse IgG was obtained commercially (Miles Laboratories, Slough, Gt. Britain).
190
Anti-rabbit IgG The IgG fraction was purified from rabbit serum over protein A-Sepharose (Goding, 1976), and was used to raise hyperimmune sheep serum. The IgG fraction of the antiserum was prepared using a DE 52 cellulose column as previously described (Burrells et al., 1979). These two IgG preparations were then conjugated with alkaline phosphatase, Sigma type VII (Sigma Chemical Company, Poole, Dorset) by the m e t h o d of Engvall and Perlmann (1972). Standardisation o f the conjugates These standardisations were carried o u t as previously described (Evans, 1979). Briefly, a checkerboard titration of type A1 SSE against its mouse or rabbit IgG antibody using a conjugate at an arbitrary dilution yielded dilutions of SSE and antiserum which were considered to be optimal. These optimal dilutions were then used in a test in which the conjugates were titrated to establish suitable working dilutions. These were determined as 1/200 for the anti-mouse IgG conjugate, and 1/2000 for the anti-rabbit IgG conjugate.
Determination o f the optimal dilutions of SSE antigens and mouse and rab bit antisera Using the anti-mouse IgG conjugate at its predetermined working dilution, 12 checkerboard titrations were carried out to determine the optimal coating dilution of each SSE which would react with the optimal dilution of its respective mouse antiserum to give an optical density at 400 nm (OD400) of 0.7, the highest c o m m o n maximum for all serotypes. Twelve checkerboard titrations were carried o u t in a similar fashion using the optimal dilution of anti-rabbit conjugate, and the predetermined optimal dilutions of rabbit antisera. Cross-ELISA tests were then performed by reacting each antiserum at the dilution determined to be optimal for its homologous SSE against the optimal dilutions of each of the other SSEs. Results were corrected for any background colouration by subtraction of the OD400 of an "antigen alone" control from the OD400 of the test. To assess antigenic relationships between strains in terms of antibody reactivity, a m e t h o d described by Archetti and Horsfall (1950) and Gois et al. (1974) was used. In it, the geometric mean of the ratios (r) is given by the formula r = vr-~l-~K-r-~2 where
191
heterologous titre (strain 2) r 1 =
. . . . . . . . . . . . . . . . . . . .
homologous titre (strain 1) and heterologous titre (strain 1) r2
homologous titre (strain 2) In this assay, OD400 was substituted for titre. The value r measures similarities between serological reactions to different strains. When the homologous and heterologous values are equal, r is maximal at 1. (i.e., rl X r2 = 1/1). The greater the denominator of the expression r, X r2 the greater the difference between the strains. Conversely, as the value of r tends towards unity, the more closely related are t w o strains antigenically.
Indirect haemagglutination (IHA ) tests IHA tests were performed by the m e t h o d of Carter (1955) as modified by Burrells et al. (1979). RESULTS
The results of cross-IHA tests are shown in Tables I and II. Titres of < 1/8 were considered negative. In mice (Table I), antibody to homologous antigen was evoked with the SSE of types A1, T3, T4, A8 and T10. In addition, mice immunised with type A2 reacted with type T3; those immunised with type A5 reacted with type A9 and those immunised with type T3 reacted with type T10. Pooled serum from unvaccinated control mice gave negative reactions with all 12 antigens. In rabbits, antibody to homologous antigen was produced by the SSE of all the types with the exception of types A2 and A12. Heterologous reactions were evident where rabbit anti-A1 serum reacted with type A6 SSE: anti-T3 serum reacted with t y p e T4, and a n t i - A l l serum reacted with type T4. Serum from the unvaccinated control rabbit and pre-immunisation sera from the vaccinated rabbits gave negative reactions with all 12 serotypes. The lack of response b y 7 of the 12 serotypes in mice and 2 of the 12 serotypes in rabbits observed with the IHA test is contrasted by the measurable responses of all 12 homologous serotypes when the sera from both species were tested by ELISA. Figures 1 and 2 illustrate for mouse and rabbit sera, respectively, the degree of "relatedness" of each SSE t y p e to the other 11 when tested by ELISA. With mice, some degree of cross-reactivity occurred between all the 12 types. T y p e A9 cross-reacted strongly with other types of P. haemolytica, notably types A1, A2, A5, A6, A7, A8 and A12, whilst type
192 TABLE I Indirect haemagglutination (IHA) reactions o f m o u s e antisera tested against h o m o l o g o u s and heterologous s o d i u m salicylate extract ( S S E ) Mouse antiserum
A1
P. haemolytica s o d i u m salicylate extract ( S S E ) ............................................................ A1 A2 T3 T 4 A 5 A6 A7 A8 A9 32*
T10
All
A12
--
m
T3
8 8
T4
-
A2
16
-
8
-
-
-
w
-
A5 A6
8 m
A7
A8 A9
16
T10
16
All A12
w
Control
- - = Negative reactions at less than 1 / 8 . * Titres expressed as reciprocals o f end-point dilution.
T A B L E II Indirect haemagglutination ( I H A ) reactions o f rabbit antisera tested against h o m o l o g o u s and heterologous s o d i u m salicylate extract ( S S E ) Rabbit
P. haemolytica s o d i u m salicylate extract ( S S E )
antiserum A1 A1 A2 T 3
A2
128" .
T3
--
T4
A5
--
A6
.
128 --
A5
--
A8
--
--
8
.
T4
A7
32 512
T10
All
--
--
--
16
--
A6
--
A7
--
128
A8
--
A9 T10
--
--
--
--
All
--
AI2
--
--
Control
--
--
--
-64
---
32
8
--
- - = negative reactions at less t h a n 1/8. * Titres expressed as reciprocals o f end-point dilution.
--
A12
m
.
--
--
A9
512 --
16
-8
193
123456789101112 A1
=
u_
~
123456789101112 A2
T4
A5
A7
A8
123456789101112 T3
A6
0"8
0-2 n" LLI
1 2 3 4 5 6 7 8 9 101112 A9
¢'= 1.0
0-6 8"4 0'2
123456789101112 T10 SODIUM
123456789101112 A11 SALICYLATE
123456789101112 A12
EXTRACT
(SSE)
Fig. 1. Cross-relatedness (r) between serotypes of PasteureUa haemolytica using antisera prepared in mice. • = Homologous type ( r = 1 ) ; [] = A b i o t y p e s ; [] = T biotypes.
A l l was the least cross-reactive. In general, strong cross-relationships existed between the A biotypes on the one hand, and between the T biotypes on the other, although there was a considerable degree of shared antigenic relatedness throughout. Rabbit antisera presented clearer extremes of antigenic relatedness between A biotypes and T biotypes, although there was a surprisingly high cross-reactivity between type A2 and type T3 (Fig. 2). Type T10 was the least cross-reactive whilst types A2, A5 and A12 possessed a high degree of cross-reactivity with the other A biotypes. The T biotypes were less cross-reactive than the A biotypes. Of the A biotypes, type A l l showed the least cross-reaction with the other types. One outstanding difference between mouse and rabbit antisera in ELISA was that with the former, T biotypes reacted more strongly with the other T biotypes than with
::LLL
194
0"6
0.4
0-2
0,8-t
o.6-1 H
AI
123456789101112 A2
123456789101112 T3
T4
123456789101112 A5
123456780101112 A6
123456789101112 A8
123456789101112 A9
|
0 0.2 n,.,
0 192 314 0 516 71 1 ~ A7
°i LIJd 123456789101112 T10
123456789101112 A11
123456789101112 A12
SODIUM SALICYLATE EXTRACT {SSE) Fig. 2. C r o s s - r e l a t e d n e s s (r) b e t w e e n s e r o t y p e s o f Pasteurella haernolytica u s i n g a n t i s e r a p r e p a r e d in r a b b i t s . • = H o m o l o g o u s t y p e (r = 1); ~ = A b i o t y p e s ; ~ = T b i o t y p e s .
A biotypes, whilst with rabbit antisera the T biotypes showed only limited cross-reactions. Control sera from unvaccinated mice, the unvaccinated rabbit and pre-immunization rabbit sera all gave negative ELISA reactions with the 12 serotypes. DISCUSSION
In IHA tests the titres of the sera from mice tended to be low (1/8-1/32) whilst titres obtained with rabbit sera confirmed the findings of Biberstein (1965) that, where cross-reactions occurred, the heterologous titres were of a much lower order than the homologous titres. In ELISA tests with mouse sera there appeared to be stronger relationships between the A biotypes on one hand, and T biotypes on the other.
195 It is interesting also that type A9 cross-reacted as strongly with each T serotype as did the T serotypes with each other. By polyacrylamide-gelelectrophoresis Thompson and Mould (1975) subdivided the 12 serotypes into 2 groups corresponding to biotypes A and T, b u t type A9 showed only minor band differences from types T3, T4 and T10. The strong reaction observed between type A2 and type T3 in ELISA was also observed in IHA tests on the sera from mice (Table I). In the original classification of P. haemolytica types (Biberstein et al., 1960) t y p e 2 was grouped with types 3, 4 and 10 on the basis of biochemical tests, as was type 11 which cross-reacted poorly in ELISA even with the other A biotypes. Differences observed in the cross-ELISA tests between mouse and rabbit antisera could conceivably be due to the fact that each mouse serum was a pool from 10--20 animals whereas only 1 rabbit antiserum was used for each serotype. Thus individual variation of response would be masked with the mouse antisera, b u t may affect the results with the rabbit sera. Cross IHA tests with the sera from immunised rabbits indicated the probable existence of a small number of minor antigens responsible for the low-titred cross-reactions between P. haemolytica types which do n o t seriously interfere with the typing procedure (Biberstein, 1965). The results obtained from the sera of mice vaccinated with bacterins of various strains (Biberstein and Thompson, 1965) suggested that capsular antigens play the major role in determining the specificity of the immune response whilst somatic antigens are of secondary importance. Further studies in cross protection using bacterins in mice (Knight et al., 1969) confirmed the importance of the capsular antigens, b u t anomalies were found. Mice vaccinated with bacterins from strains which were serologically distinct from the challenge strain showed a better degree of protection than those vaccinated with homologous bacterins. As a result of these findings it is suggested that although capsular and somatic antigens are important, other factors may exist which determine the specificity of conferred immunity. No such cross-protection is apparent in mice immunised with SSE antigens (capsular preparations). In vaccination-challenge experiments, a trivalent vaccine containing SSEs of P. haemolytica serotypes A1 and A6, and heat-killed organisms of type A2 gave protection against subsequent challenge with types A1 and A6 organisms, b u t not against challenge with types A2 and A9 organisms (Evans and Wells, 1979). Similar findings have been obtained using the same vaccine in SPF lambs (Gilmour et al., 1979). Since the establishment of the ELISA technique for P. haemolytica (Burrells et al., 1979), it has become evident that wider serological cross reactivity exists between serotypes. Sera from conventional ewes and lambs which had been vaccinated with a trivalent vaccine, containing type A1 and A6 SSEs and heat-killed organisms of type A2 when examined by ELISA, exhibited antibody titres to t y p e A2 SSE which were comparable
196
to t y p e A l l SSE which was n o t included in the vaccine (N.J.L. Gilmour and C. Burrells, 1980, unpublished observations). This could conceivably be due to the pre-existence of antibody to type A l l in the ewes and hence their lambs. Alternatively the vaccine evoked the production of antibodies which cross-reacted with type A l l . Evidence for the latter hypothesis is provided by data from experiments with serum from SPF lambs immunised solely with type A1 SSE (Wells et al., 1979a). When tested in ELISA against types A1 and A l l SSE a pool of SPF lamb serum gave strong reactions with both, indicating the presence of c o m m o n group antigenic components. Absorption of this serum pool with SSEs of P. haemoly tica serotypes other than that of type A1 removed antibody activity to type A l l leaving antibody reacting only with t y p e A1 SSE (Donachie et al., 1983). Of those cross-reactions in mice and rabbits demonstrated b y IHA, only that between types A5 and A9 in m i c e was apparent in the ELISA crossreactions. The cross-reactions demonstrated by ELISA could be due to the greater sensitivity of the technique over IHA allowing detection of " g r o u p " reactivity and inter-type relationships n o t recognised by IHA. Alternatively, it may be that only certain antigenic components are bound to the RBCs used in IHA tests whereas a larger repertoire of antigens in the SSE preparations are capable of binding to the polystyrene plates used in ELISA. In IHA tests, sensitised RBCs are more readily agglutinated by immunoglobulin M (IgM) than by IgG. The ELISA technique employed in this study detected only IgG. It has previously been shown in SPF lambs that intramuscular (i/m) immunisation with antigen in oil-based adjuvant stimulates very high levels of circulating IgG compared to IgM. The IgG content of the total circulating antibody is further increased by a second i/m inoculation of the same preparation (Smith et al., 1976). The animals used in this study were immunised in a similar fashion, which suggests that the ELISA is a more efficient method of detecting the bulk of antib o d y stimulated b y this m e t h o d of immunisation. The cross-reactivity illustrated by ELISA suggests the presence of common antigenic components among the serotypes of P. haemolytica which can be detected b y ELISA and n o t by IHA. Further work has been done using the ELISA technique to determine if a type-specific, non cross-reacting serum such as that described above (Donachie et al., 1983) can be used to prepare purified type-specific antigens of P. haemolytica serotypes. These antigens would then allow the ELISA technique to be employed for the measurement of the immune responses in an animal to the individual serotypes of a multivalent vaccine. ACKNOWLEDGEMENTS
H.B. Evans was supported by a research studentship awarded by the Agricultural Research Council. The authors thank Dr. I.D. Aitken for his assistance in the preparation of this manuscript.
197 REFERENCES Archetti, I. and Horsfall, F.L., 1950. Persistent antigenic variation of influenza A viruses after incomplete neutralisation in ovo with heterologous i m m u n e serum. J. Exp. Med., 92: 441--462. Biberstein, E.L., 1965. Cross-reactions between types of Pasteurella haemolytica. Cornell Vet., 55: 495--499. Biberstein, E.L. and Francis, C.K., 1968. Nucleic acid homologies between the A and T types of Pasteurella haemolytica. J. Med. Micro., 1: 105--108. Biberstein, E.L. and Thompson, D.A., 1965. Type specificity of immunity to Pasteurella haemolytica infection in mice. J. Comp. Pathol., 75: 331--337. Biberstein, E.L., Gills,M.G. and Knight, H., 1960. Serological types of Pasteurella haemolytica. Cornell Vet., 50: 283--300. Burrells, C., Wells, P.W. and Dawson, A. McL., 1979. The quantitative estimation of antibody to Pasteurella haemolytica in sheep sera using a micro enzyme-linked immunosorbent assay (ELISA). Vet. Microbiol., 3: 291--301. Carter, G.R., 1955. Studies on Pasteurella multocida. 1. A haemagglutination test for the identification of serological types. A m . J. Vet. Res., 16: 481--484. Donachie, W., Burrells, C. and Dawson, A. McL., 1983. Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haernolytica antigens. Vet. Microbiol., 8: 199-205. Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen coated tubes. J. Immunol., 109: 129--135. Evans, H.B., 1979. A study of the mechanisms of immunity to PasteureUa haemolytica infection. P h . D . Thesis, University of Edinburgh, pp. 154--171. Evans, H.B. and Wells, P.W., 1979. A mouse model of Pasteurella haemolytica infection and its use in assessment of the efficacy of P. haemolytica vaccines. Res. Vet. Sci., 27: 213--217. Gilmour, N.J.L., 1978. Pasteurellosis in sheep. Vet. Rec., 102: 100--102. Gilmour, N.J.L., Martin, W.B., Sharp, J.M., Thompson, D.A. and Wells, P.W., 1979. The development of vaccines against pneumonic pasteurellosis in sheep. Vet. Rec., 104: 15. Goding, J.W., 1976. Conjugation of antibodies with fluorochromes: modifications to the standard methods. J. Immunol. Meth., 13: 215--216. Gois, M., Kuksa, F., Franz, J. and Taylor-Robinson, D., 1974. The antigenic differentiation of seven strains of Mycoplasma hyorhinis by growth-inhibition, metabolisminhibition, latex-agglutination and polyacrylamide-gel-electrophoresis tests. J. Med. Microbiol., 7 : 105--114. Knight, N.D., Biberstein, E.L. and Allison, M., 1969. The role of capsular and somatic antigens in immunisation of mice against Pasteurella haemolytica infection. CorneU Vet., 59: 55--64. Smith, G.R., 1961. The characteristics of two types of Pasteurella haemolytica associated with different pathological conditions. J. Pathol. Bacteriol., 81 : 431--440. Smith, W.D., Dawson, A. McL., Wells, P.W. and BurreUs, C., 1976. Immunoglobulins in the serum and nasal secretion of lambs following vaccination and aerosol challenge with parainfluenza 3 virus. Res. Vet. Sci., 21 : 341--348. Thompson, D.A. and Mould, D.L., 1975. Protein electrophoretic pattern of Pasteurella haemolytica. Res. Vet. Sci., 18: 342--343. VoUer, A. and Bidwell, D.E., 1975. A simple method for detecting antibodies to Rubella. Br. J. Exp. Pathol., 56: 338--339.
198 Wells, P.W., Evans, H.B., Burrells, C., Sharp, J.M., Gilmour, N.J.L., Thompson, D.A and Rushton, B., 1979a. Inability of passively acquired antibody to protect lamb~ against experimental pasteurellosis. Infect. Immun., 26: 25--29. Wells, P.W., Gilmour, N.J.L., Burrells, C. and Thompson, D.A., 1979b. A serologica comparison of Pasteurella haemolytica vaccines containing different adjuvants. Res Vet. Sci., 27: 248--250.