INFECTION AND IMMUNITY, Apr. 1991, p. 1470-1475 0019-9567/91/041470-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 59, No. 4
Antibodies to Outer Membrane Proteins but Not to Lipopolysaccharide Inhibit Pulmonary Proliferation of Pasteurella multocida in Mice YUE-SHOUNG LU,* H. N. AGUILA, WAYNE C. LAI, AND S. P. PAKES Division of Comparative Medicine, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 Received 4 September 1990/Accepted 21 December 1990
The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 106 CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P < 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P < 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P > 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs.
Pasteurella multocida causes a common and widespread infection in laboratory rabbits. Disease manifestations range from fatal septicemia, severe pleuritis, and pneumonia to less severe sequelae, such as multiple abscesses, chronic rhinitis, and otitis media. Some rabbits may harbor P. multocida without showing clinical signs of pasteurellosis (8). Unsuspecting investigators may have their research compromised when such rabbits are stressed by an experimental procedure and subsequently show clinical signs and lesions. One of our goals is to develop a vaccine that protects rabbits against P. multocida colonization and disease. Recently, we demonstrated that a KSCN extract of P. multocida and rabbit immune sera against a KSCN extract of P. multocida actively and passively protect rabbits against a homologous challenge, respectively (13, 14). It is also known that KSCN extracts of P. multocida contain outer membrane proteins (OMP), lipopolysaccharides (LPS), capsule, and nucleic acids (14) and that rabbits immunized with KSCN extracts produce antibodies against OMP and LPS of homologous P. multocida (13). We also reported that a vaccine prepared from purified outer membranes of P. multocida protected rabbits against a homologous challenge (12a) and that the resulting immune sera contained antibodies against OMP and LPS. Rabbits immunized with purified P. multocida outer membranes were protected against death and colonization in the lungs and had reduced prevalence and severity of pneumonia, numbers of P. multocida in nasal cavities, and prevalence of P. multocida colonization in nonrespiratory organs, such as the liver, spleen, uterus, and tympanic bullae. The goal of this study was to determine whether antibodies against OMP or LPS or both are respon*
sible for resistance. Studies on the relative roles of LPS antibodies and OMP antibodies in resistance to disease may facilitate the development of an effective vaccine to control rabbit pasteurellosis. In this study, we demonstrated that antibodies against P. multocida OMP but not LPS inhibited pulmonary proliferation of P. multocida in mice. MATERIALS AND METHODS Bacterial strains and culture media. P. multocida (strain UT-1; serotype 3:A) isolated from a rabbit with suppurative rhinitis was used in these experiments. This isolate does not produce P. multocida exotoxin (14). The organisms were grown on blood agar plates and cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) at 37°C. All P. multocida organisms used for experiments were in the log phase of growth. Preparation of P. multocida outer membranes. The outer membranes were prepared as described previously (11). The capsules of the organisms were removed enzymatically with hyaluronidase (11), since type A P. multocida is known to contain hyaluronic acid (3). The cells were disrupted and subjected to sucrose gradient centrifugation. Selected fractions from the gradient were pooled and dialyzed extensively against phosphate-buffered saline (PBS). The amount of hyaluronic acid in the final preparation was determined with purified hyaluronic acid (Sigma Chemical Co., St. Louis, Mo.) as a standard (14). Preparation of rabbit immune sera against P. multocida outer membranes. Pasteurella-free New Zealand White female rabbits weighing 2.7 to 3.6 kg were purchased from Hazleton-Dutchland Laboratories, Denver, Pa. The nasal cavities of all rabbits were determined to be free of P. multocida by culturing and to be negative for serum immunoglobulin G antibodies against P. multocida by an enzyme-
Corresponding author. 1470
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1471
Rabbit Immune Serum
LPS-Sepharose Column Effluent
Adsorption with Outer Membrane Vesicles
LPS antibody-LPS-Sepharose | Elution
Adsorption with intact P.mulitcdacells
LPS antibody
Elution luted antibody
Supematant FIG. 1. Fractionation of rabbit immune sera against P. multocida outer membranes.
linked immunosorbent assay (13). Three rabbits were inoculated intranasally (i.n.) with purified P. multocida outer membranes (1 mg of protein containing 121 ,ug of hyaluronic acid per rabbit per inoculation) in PBS at days 0, 7, 14, and 35. Four weeks later, rabbits were anesthetized intravenously with pentobarbitol sodium to collect sera. Rabbits immunized by this protocol confer resistance to a homologous challenge. Pooled rabbit immune sera were used in the experiments. Experimental design. First, we precipitated the rabbit immune sera with ammonium sulfate and separated the antibodies into OMP antibodies and LPS antibodies. Second, we characterized the various antibodies by Western immunoblot analyses with outer membrane vesicles or purified LPS as antigens and determined the LPS antibody concentrations in various antibody fractions by quantitative precipitin analysis. Third, we tested the efficacy of each fraction of antibody in the inhibition of P. multocida proliferation in mice by passive transfer studies. Separation of rabbit immune sera. Pooled immune sera and preimmunized rabbit sera were precipitated with ammonium sulfate and separated as outlined in Fig. 1. (i) Preparation of P. multocida LPS. Purified LPS was prepared from P. multocida (3:A) by the hot phenol water method (22). (ii) Purification of LPS antibodies by affinity chromatography. LPS'antibodies in the outer membrane immune sera were purified by affinity chromatography (24, 25), which consisted of coupling purified LPS to cyanogen bromideactivated Sepharose 4B (Pharmacia, Piscataway, N.J.) and eluting antibodies from the column. In brief, 4 mg of LPS was suspended in coupling buffer and reacted overnight at 4°C with 1 g of CNBr-activated Sepharose 4B. Residual reactive groups were blocked with Tris-HCl buffer (pH 8.0) for 2 h. The matrix was washed extensively with coupling buffer to remove noncovalently bound LPS before equilibration with PBS containing 0.5% bovine serum albumin. Immune sera were adsorbed in this matrix, and LPS antibodies were eluted with 3.5 M MgCl2. The eluted fraction was dialyzed extensively against PBS at 4°C, checked for sterility, and used for experiments. (iii) Removal of antibodies against OMP of P. multocida. The effluent immune sera (sera from adsorption through the LPS-Sepharose column) were incubated with an equal volume of outer membrane 'Vesicles (14) of homologous P.
multocida (1 mg/ml) for 3 h at 37°C and centrifuged at 106,000 x g for 60 min. The supernatants were saved. This procedure was repeated four times. Final supernatants were tested for sterility and used for experiments. (iv) Purification of antibodies against P. multocida OMP. For determination of whether antibodies against OMP were the protective components of the immune sera, the effluent immune sera were adsorbed with intact P. multocida cells and the antibodies were eluted from the cell-antibody complex (11). In brief, freshly cultured homologous P. multocida cells (108 CFU in 0.5 ml of PBS) were mixed with an equal volume of serum and incubated for 1 h at 4°C with gentle agitation. The mixture was centrifuged, and the pellet was saved. An equal volume of fresh effluent immune serum was added to the pellet, and the procedure was repeated six times. The final pellet was washed with PBS and suspended in 1 ml of 0.2 M glycine-HCl buffer (pH 2.8) to dissociate the antibodies and P. multocida cells. The suspension was centrifuged, and the supernatant containing the antibodies was collected and adjusted to pH 7.0 with Tris buffer. The eluted antibodies, containing 0.1% bovine serum albumin, were stored at -20°C until used. Preimmunized rabbit sera, used as a'control in the passive transfer experiments, were treated in the same manner as the rabbit immune sera. Characterization of separated rabbit antibodies. (i) Identification of antibodies against P. multocida OMP. The various fractions of rabbit immune sera were analyzed by Western blotting (11, 13) with P. multocida outer membrane vesicles (14) as antigens. (ii) Identification of antibodies against P. multocida LPS. Purified P. multocida LPS were separated in a 15% separating gel containing 4 M urea (15), transferred to nitrocellulose paper, and reacted with various fractions of rabbit immune sera in a Western blot analysis. (iii) Quantitation of LPS antibodies against P. multocida. The LPS antibody concentrations in various immune serum fractions were determined by a quantitative precipitin analysis (18). In brief, increasing amounts of purified LPS were added to a series of tubes each containing the same volume of antiserum, and the mixtures were incubated for 1 h at 37°C and 7 days at 4°C. The precipitated antibodies were centrifuged, washed twice with cold saline, and quantitated by a modified Lowry procedure (2). Preimmunized rabbit sera were treated in the same manner as the immune rabbit sera and were used as a control.
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TABLE 1. Protection of mice against homologous challenge by rabbit immune sera directed to P. multocida outer membranesa Expt
Serum fractionb
1
Unadsorbed immune serum Normal serum
2 and 3
Purified LPS antibody Normal serum
4
Effluent of LPS column Normal serum
5
Supernatant from LPS and outer membrane vesicle adsorption Normal serum Eluted antibody from effluent-P. multocida complex Normal serum
6
antibody (jug/mouse)"
LPS
Mean log CFU of P. multocida in ± SEM (no. of mice)d
lungs
5.1 0
3.102 ± 0.482 (10) 6.623 ± 0.167 (10)
0.50
3.268 ± 0.426 (8) 5.935 ± 0.184 (10)
0.50
0.34 0
0
5.305 ± 0.206 (10)
1.683 ± 0.345 (10) 0
0.50), indicating that specific LPS antibodies were noninhibitory against P. multocida proliferation in mice. (iii) Effluents from the LPS column inhibited P. multocida proliferation. Since purified LPS antibodies were nonprotective in mice, we examined the inhibitory qualities of other antibodies. Mice inoculated i.n. with effluents from the LPS column and challenged had a 465-fold reduction in the numbers of P. multocida in the lungs (P < 0.001) (Table 1, experiment 4). This result suggested that antibodies found in the LPS column effluents were important in the inhibition of P. multocida proliferation. (iv) Loss of inhibitory activity by removal of antibodies against OMP. Antibodies against P. multocida OMP were removed by repeated adsorption of effluents (from the LPS column) with membrane vesicles of homologous P. multocida. Mice inoculated with rabbit immune serum which contained no antibodies against OMP had numbers of P. multocida in the lungs very similar to those in mice inoculated with normal rabbit serum (P > 0.50) (Table 1, experiment 5), indicating that antibodies against OMP were important in the inhibition of P. multocida proliferation. (v) Inhibition mediated by antibodies against OMP. Antibodies against OMP were purified by elution of antigenantibody complexes (antibody-intact P. multocida cell com-
PROTECTION OF MICE BY ANTIBODIES TO P. MULTOCIDA OMP
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A
B
C
D
E
A
F
66.2 -
B
C
D
E
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F
45.031.0-
45.021.5-
31.021.5-
144 FIG. 2. Western blot analysis of rabbit antibodies against P. multocida OMP. Outer membrane vesicles of P. multocida extracted by lithium chloride were used as antigens. Lanes: A, unadsorbed outer membrane immune serum; B; outer membrane immune serum after LPS adsorption; C, outer membrane immune serum after LPS and protein adsorption; D, eluted OMP antibodies; E, affinity-purified LPS antibodies; F, negative control (normal rabbit sera prior to immunization). Molecular weight markers (103) are on the left.
plexes). Mice inoculated i.n. with purified antibodies against OMP had a 201-fold reduction in the numbers of P. multocida in the lungs (P < 0.001) as compared with mice inoculated with normal rabbit sera (Table 1, experiment 6). This result indicated that antibodies against OMP inhibited P. multocida proliferation in mice. Characterization of protective antibodies in rabbit immune sera. (i) P. multocida OMP antibodies detected by Western blot analysis. Antibodies against OMP of P. multocida were analyzed by Western blotting with outer membrane vesicles as antigens. Unconcentrated immune sera obtained from each separation procedure were used for passive transfer studies and Western blot analysis. Unadsorbed rabbit immune serum contained various antibodies against OMP with molecular masses ranging from 27 to 66 kDa (Fig. 2, lane A). Adsorption of the immune serum with purified LPS did not remove any antibodies against OMP (Fig. 2, lane B). Antibodies against OMP were removed completely by adsorption of effluents (from the LPS column) with outer membrane vesicles (Fig. 2, lane C). Antibodies against major OMP were eluted from antibody-P. multocida cell complexes, although antibodies against some OMP were diminished in amount (Fig. 2, lane D). As expected, purified LPS antibodies contained very few antibodies against OMP (Fig. 2, lane E). Pooled sera which were obtained from three rabbits prior to immunization and which contained no antibodies against P. multocida OMP (Fig. 2, lane F) were used as controls. (ii) P. multocida LPS antibodies detected by Western blot analysis. Antibodies against purified LPS were demonstrated in unadsorbed immune serum (Fig. 3). The antibodies were reactive to LPS with molecular masses slightly lower than 14.4 kDa and to LPS with molecular masses slightly higher than 14.4 kDa (Fig. 3, lane A). Adsorption of the immune serum with an LPS-Sepharose column removed most of the antibodies against LPS (Fig. 3, lane B). Adsorption of effluents (post LPS column) with outer membrane vesicles
14.4FIG. 3. Western blot analysis of rabbit antibodies against P. multocida LPS. Purified LPS from P. multocida were used as antigens. Lanes: A, unadsorbed outer membrane immune serum; B, outer membrane immune serum after LPS adsorption; C, outer membrane immune serum after LPS and protein adsorption; D, eluted OMP antibodies; E, affinity-purified LPS antibodies; F, negative control (normal rabbit sera prior to immunization). Molecular weight markers (103) are on the left.
totally removed antibodies against LPS (Fig. 3, lane C). Antibodies against LPS were not eluted from antibody-P. multocida cell complexes (Fig. 3, lane D). As expected, abundant amounts of LPS antibodies were demonstrated in purified LPS antibodies (Fig. 3, lane E). Pooled sera which were obtained from three rabbits prior to immunization and which contained no antibodies against P. multocida LPS (Fig. 3, lane F) were used as controls. (iii) LPS antibodies determined by quantitative precipitin analysis. The amounts of LPS antibodies were determined and used to calculate the amounts of LPS antibodies received by each experimental mouse. Each mouse in experiments 1, 2 and 3, and 4 received 5.1, 17.7, and 0.34 p.g of LPS antibodies, respectively (Table 1). Mice in experiment 4, receiving 52-fold fewer LPS antibodies than mice in experiments 2 and 3, were protected against P. multocida proliferation, whereas mice in experiments 2 and 3 were not protected. Similarly, mice in experiment 4, receiving 15-fold fewer LPS antibodies than mice in experiment 1, were protected against P. multocida proliferation to a similar degree as were mice in experiment 1. These results showed that LPS antibodies were not important in the inhibition of P. multocida proliferation. DISCUSSION This study demonstrated that the inhibitory ability of rabbit immune sera against P. multocida proliferation in mice was due to antibodies against P. multocida OMP but not LPS. The inhibitory activity conferred by OMP antibodies was shown basically in two experiments. In the first experiment, LPS antibodies were adsorbed with an LPSSepharose affinity column, and the effluent sera from the column were used in the passive transfer experiments. In the second experiment, eluted OMP antibodies were used to demonstrate inhibition against P. multocida proliferation. In both experiments, a significant difference was shown between experimental and control groups (Table 1). Other
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LU ET AL.
studies (10, 26) also supported the view that P. multocida proteins are involved in protection. Those studies showed that partially purified proteinaceous immunogens of avian strains of P. multocida produced homologous protection in chickens, although the origin of these proteinaceous immunogens was not determined. In this study, we found that OMP antibodies were inhibitory, inferring that purified OMP contain a virulence factor(s) that controls P. multocida proliferation in mice. Our recent monoclonal antibody study further showed that the 37.5-kDa OMP of P. multocida is the target of MAB1608, which protects mice and rabbits against a homologous challenge (12). In the present study, we noted differences in CFU among experimental mice inoculated with normal serum and challenged. The differences in CFU among experimental mice inoculated with normal rabbit serum and challenged with P. multocida may reflect differences in batches of animals used, even though the mice were inbred and supplied by the same vendor. Because of the expected variations, a control group of mice was included in each experiment. The role of LPS antibodies in protection remains unclear. One study showed that rabbit immune sera against purified LPS of P. multocida type E provided protection in mice against a homologous challenge (20). Our study, however, showed that LPS antibodies against P. multocida 3:A did not protect mice against pulmonary infection. In agreement with our results, other studies (17, 21) showed that purified LPS of P. multocida produced no active immune protection in mice. Why LPS antibodies against P. multocida 3:A do not inhibit the pulmonary proliferation of P. multocida in mice remains unknown. Studies with Haemophilus influenzae type b showed that phenotypic variations in vivo reduce the binding of LPS antibodies to the cell surface, a phenomenon that may affect the protective potential of LPS antibodies (9). Although this phenomenon has not been studied in P. multocida, it is well known that P. multocida organisms undergo colony morphology changes when inoculated into animals for in vivo passage (5) or are transferred several times in artificial medium (4). Alternatively, the lack of inhibition of P. multocida proliferation by LPS antibodies may be related to the antibody titers. Since relative titers were not measured, it is possible that the OMP antibody titer was much higher than the LPS antibody titer and resulted in the ineffectiveness of the LPS antibodies. The role of capsular antibodies in resistance is difficult to assess because there is no complete method for the detection of antibodies against the capsule of type A P. multocida. Two approaches were taken to address the issue of P. multocida capsular antibodies. First, we produced rabbit immune sera containing antibodies against OMP and LPS but not capsules by immunizing rabbits with sucrose gradient-purified outer membranes which contained small amounts of capsules. Hyaluronidase treatment of P. multocida organisms resulted in the removal of capsules, since capsules were not demonstrated in enzyme-treated P. multocida organisms by India ink staining. In addition, rabbit immune sera against P. multocida outer membranes contained no antibodies against capsular antigens, as demonstrated by the passive hemagglutination method (23). Second, we adsorbed the immune sera with purified LPS and outer membrane vesicles which contained minute amounts of capsular material and tested the resistance efficacy of the remaining portion of the immune sera. The effluent from the LPS-Sepharose column was as inhibitory against P. multocida proliferation in mice as was the unadsorbed rabbit immune serum, and the purified LPS antibodies were non-
INFECT. IMMUN.
inhibitory against P. multocida proliferation in mice (Table 1). These results showed that antibodies against OMP were responsible for the inhibitory activity observed in the effluent. The supernatant obtained after adsorption of the effluent with outer membrane vesicles, however, was noninhibitory. We concluded that antibodies against P. multocida OMP but not capsules are the major components involved in the inhibition of P. multocida proliferation in mice. In this study, we used a mouse model to determine the role of rabbit immune sera against P. multocida OMP and LPS in
protecting rabbits against pasteurellosis. However, the results obtained in mice may not be the same as those obtained in rabbits. To clarify this issue, we conducted an experiment with rabbit immune sera against a KSCN extract of P. multocida for passive immunity transfer in mice. The selection of the antisera was based on the facts that the KSCN extract had protein and LPS profiles similar to those of the P. multocida outer membranes (14) and the rabbit immune sera against the KSCN extract protected rabbits against an i.n. homologous P. multocida challenge (13). The geometric mean concentration (CFU per gram) of P. multocida in mouse lungs was reduced from 2.05 x 105 (normal rabbit sera) to 3.40 x 102 (rabbit immune sera against a P. multocida KSCN extract); i.e., passive immunization of mice with rabbit immune sera and challenge resulted in a 604-fold reduction (P < 0.001) in the numbers of P. multocida in the lungs. These results clearly showed that rabbit immune sera against the KSCN extract significantly inhibited P. multocida proliferation in mice, indicating that the results in mice were applicable to rabbits. In this study, we observed that rabbit antibodies against P. multocida OMP but not LPS inhibited the pulmonary proliferation of P. multocida in mice. These results led us to believe that antibodies against P. multocida OMP but not LPS most likely protect rabbits against pasteurellosis. Studies in mice certainly provided very useful information for future studies in rabbits. In our view of the development of an effective P. multocida Vaccine, efforts should be focused on P. multocida OMP instead of LPS. In conclusion, we demonstrated that the OMP antibodies but not the LPS antibodies of P. multocida 3:A are inhibitory against P. multocida proliferation in mouse lungs and are presumably protective against a homologous challenge in rabbits. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants RRO1311 and RR00890 from the National Institutes of Health. We thank S. Atashband, Steven Afendis, and L. Watkins for excellent technical assistance. REFERENCES 1. Ashfaq, M. K., and S. G. Campbell. 1986. The influence of opsonins on the bactericidal effect of bovine alveolar macrophages against Pasteurella multocida. Cornell Vet. 76:213-221. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Carter, G. R. 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Adv. Vet. Sci. Comp. Med. 11:321379. 4. Carter, G. R. 1984. Genus I. Pasteurella Trevisan 1887, 94, AL Nom. cons. Opin. 13, Jud. Comm. 1954, 153, p. 552-558. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 5. Carter, G. R., and E. Annau. 1953. Isolation of capsular
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polysaccharides from colonial variants of Pasteurella multocida. Am. J. Vet. Res. 14:475-478. 6. Collins, F. M., C. J. Niederbuhl, and S. G. Campbell. 1983. Bactericidal activity of alveolar and peritoneal macrophages exposed in vitro to three strains of Pasteurella multocida. Infect. Immun. 39:779-784. 7. Collins, F. M., and J. B. Woolcock. 1976. Immune responses to Pasteurella multocida in the mouse. J. Reticuloendothel. Soc. 19:311-321. 8. Flatt, R. E., S. H. Weisbroth, and A. L. Kraus. 1974. Bacterial diseases, p. 193-205. In S. H. Weisbroth, R. E. Flatt, and A. L. Kraus (ed.), The biology of the laboratory rabbit. Academic Press, Inc., New York. 9. Inzana, T. J., and P. Anderson. 1985. Serum factor-dependent resistance of Haemophilus influenzae type b to antibody to lipopolysaccharide. J. Infect. Dis. 151:869-877. 10. Kajikawa, O., and M. Matsumoto. 1984. A protective antigen for turkeys purified from a type 1 strain of Pasteurella multocida. Vet. Microbiol. 10:43-55. 11. Lu, Y.-S., S. Afendis, and S. P. Pakes. 1988. Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits. Infect. Immun. 56:1532-1537. 12. Lu, Y.-S., W. C. Lai, S. P. Pakes, and L. C. Nie. 1991. A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis. Infect. Immun. 59:172-180. 12a.Lu, Y.-S., and S. P. Pakes. Abstr. Annu. Meet. Am. Assoc. Lab. Anim. Sci. 1987, abstr. no. 84, p. 532. 13. Lu, Y.-S., S. P. Pakes, and L. Massey. 1987. Hyperimmune serum from rabbits immunized with potassium thiocyanate extract of Pasteurella multocida protects against homologous challenge. J. Clin. Microbiol. 25:2173-2180. 14. Lu, Y.-S., S. P. Pakes, L. Massey, and C. Stefanu. 1987. A potassium thiocyanate extract vaccine prepared from Pasteurella multocida 3:A protects rabbits against homologous challenge. Infect. Immun. 55:2967-2976. 15. Manning, P. J., M. A. Naasz, D. Delong, and S. L. Leary. 1986. Pasteurellosis in laboratory rabbits: characterization of lipopolysaccharides of Pasteurella multocida by polyacrylamide gel electrophoresis, immunoblot techniques, and enzyme immunoassay. Infect. Immun. 53:460-463. 16. Mukkur, T. K. S. 1979. Immunogenicity of a chaotropically
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extracted protective antigen(s) of Pasteurella multocida type A against experimental pasteurellosis in mice. J. Gen. Microbiol. 113:37-43. Mukkur, T. K. S., N. A. Pyliotis, and A. Banes. 1982. Possible immunological synergism among the protective antigens of Pasteurella multocida type A. J. Comp. Pathol. 92:249-260. Nowotny, A. 1969. Basic exercises in immunochemistry. A laboratory manual, p. 158-160. Springer-Verlag, New York. Okerman, L., L. Spanoghe, and R. M. DeBruycker. 1979. Experimental infections of mice with Pasteurella multocida strains isolated from rabbits. J. Comp. Pathol. 89:51-55. Perreau, P., and J. P. Petit. 1963. Antigene lipopolyosidique de Pasteurella multocida type E. Rev. Elev. Med. Vet. Pays Trop. 16:5-18. Rebers, P. A., and K. L. Heddleston. 1974. Immunologic comparison of Westphal type lipopolysaccharide and free endotoxin from an encapsulated and a nonencapsulated avian strain of Pasteurella multocida. Am. J. Vet. Res. 35:555-560. Rebers, P. A., M. Phillips, R. A. Boykins, and K. R. Rhoades. 1980. Immunizing properties of Westphal lipopolysaccharide from an avian strain of Pasteurella multocida. Am. J. Vet. Res. 41:1650-1654. Sawada, T., R. B. Rimler, and K. R. Rhoades. 1982. Indirect hemagglutination test that uses glutaraldehyde-fixed sheep erythrocytes sensitized with extract antigens for detection of Pasteurella antibody. J. Clin. Microbiol. 15:752-756. Shenep, J. L., R. S. Munson, Jr., S. J. Barenkamp, and D. M. Granoff. 1983. Further studies of the role of noncapsular antibody in protection against experimental Haemophilus influenzae type b bacteremia. Infect. Immun. 42:257-263. Shenep, J. L., R. S. Munson, Jr., and D. M. Granoff. 1982. Human antibody responses to lipopolysaccharide after meningitis due to Haemophilus influenzae type b. J. Infect. Dis. 145:181-190. Syuto, B., and M. Matsumato. 1982. Purification of a protective antigen from a 2.5% saline extract of Pasteurella multocida. Infect. Immun. 37:1218-1226. Woolcock, J. B., and F. M. Collins. 1976. Studies of the immune mechanism in Pasteurella multocida-infected mice. Infect. Immun. 13:949-958. Zar, J. H. 1984. Biostatistical analysis, 2nd ed., p. 185-205. Prentice-Hall, Inc., Englewood Cliffs, N.J.
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Antibodies to Outer Membrane Proteins but Not to Lipopolysaccharide Inhibit Pulmonary Proliferation of Pasteurella multocida in Mice YUE-SHOUNG LU,* H. N. AGUILA, WAYNE C. LAI, AND S. P. PAKES Division of Comparative Medicine, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 Received 4 September 1990/Accepted 21 December 1990
The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 106 CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P < 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P < 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P > 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs.
Pasteurella multocida causes a common and widespread infection in laboratory rabbits. Disease manifestations range from fatal septicemia, severe pleuritis, and pneumonia to less severe sequelae, such as multiple abscesses, chronic rhinitis, and otitis media. Some rabbits may harbor P. multocida without showing clinical signs of pasteurellosis (8). Unsuspecting investigators may have their research compromised when such rabbits are stressed by an experimental procedure and subsequently show clinical signs and lesions. One of our goals is to develop a vaccine that protects rabbits against P. multocida colonization and disease. Recently, we demonstrated that a KSCN extract of P. multocida and rabbit immune sera against a KSCN extract of P. multocida actively and passively protect rabbits against a homologous challenge, respectively (13, 14). It is also known that KSCN extracts of P. multocida contain outer membrane proteins (OMP), lipopolysaccharides (LPS), capsule, and nucleic acids (14) and that rabbits immunized with KSCN extracts produce antibodies against OMP and LPS of homologous P. multocida (13). We also reported that a vaccine prepared from purified outer membranes of P. multocida protected rabbits against a homologous challenge (12a) and that the resulting immune sera contained antibodies against OMP and LPS. Rabbits immunized with purified P. multocida outer membranes were protected against death and colonization in the lungs and had reduced prevalence and severity of pneumonia, numbers of P. multocida in nasal cavities, and prevalence of P. multocida colonization in nonrespiratory organs, such as the liver, spleen, uterus, and tympanic bullae. The goal of this study was to determine whether antibodies against OMP or LPS or both are respon*
sible for resistance. Studies on the relative roles of LPS antibodies and OMP antibodies in resistance to disease may facilitate the development of an effective vaccine to control rabbit pasteurellosis. In this study, we demonstrated that antibodies against P. multocida OMP but not LPS inhibited pulmonary proliferation of P. multocida in mice. MATERIALS AND METHODS Bacterial strains and culture media. P. multocida (strain UT-1; serotype 3:A) isolated from a rabbit with suppurative rhinitis was used in these experiments. This isolate does not produce P. multocida exotoxin (14). The organisms were grown on blood agar plates and cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) at 37°C. All P. multocida organisms used for experiments were in the log phase of growth. Preparation of P. multocida outer membranes. The outer membranes were prepared as described previously (11). The capsules of the organisms were removed enzymatically with hyaluronidase (11), since type A P. multocida is known to contain hyaluronic acid (3). The cells were disrupted and subjected to sucrose gradient centrifugation. Selected fractions from the gradient were pooled and dialyzed extensively against phosphate-buffered saline (PBS). The amount of hyaluronic acid in the final preparation was determined with purified hyaluronic acid (Sigma Chemical Co., St. Louis, Mo.) as a standard (14). Preparation of rabbit immune sera against P. multocida outer membranes. Pasteurella-free New Zealand White female rabbits weighing 2.7 to 3.6 kg were purchased from Hazleton-Dutchland Laboratories, Denver, Pa. The nasal cavities of all rabbits were determined to be free of P. multocida by culturing and to be negative for serum immunoglobulin G antibodies against P. multocida by an enzyme-
Corresponding author. 1470
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1471
Rabbit Immune Serum
LPS-Sepharose Column Effluent
Adsorption with Outer Membrane Vesicles
LPS antibody-LPS-Sepharose | Elution
Adsorption with intact P.mulitcdacells
LPS antibody
Elution luted antibody
Supematant FIG. 1. Fractionation of rabbit immune sera against P. multocida outer membranes.
linked immunosorbent assay (13). Three rabbits were inoculated intranasally (i.n.) with purified P. multocida outer membranes (1 mg of protein containing 121 ,ug of hyaluronic acid per rabbit per inoculation) in PBS at days 0, 7, 14, and 35. Four weeks later, rabbits were anesthetized intravenously with pentobarbitol sodium to collect sera. Rabbits immunized by this protocol confer resistance to a homologous challenge. Pooled rabbit immune sera were used in the experiments. Experimental design. First, we precipitated the rabbit immune sera with ammonium sulfate and separated the antibodies into OMP antibodies and LPS antibodies. Second, we characterized the various antibodies by Western immunoblot analyses with outer membrane vesicles or purified LPS as antigens and determined the LPS antibody concentrations in various antibody fractions by quantitative precipitin analysis. Third, we tested the efficacy of each fraction of antibody in the inhibition of P. multocida proliferation in mice by passive transfer studies. Separation of rabbit immune sera. Pooled immune sera and preimmunized rabbit sera were precipitated with ammonium sulfate and separated as outlined in Fig. 1. (i) Preparation of P. multocida LPS. Purified LPS was prepared from P. multocida (3:A) by the hot phenol water method (22). (ii) Purification of LPS antibodies by affinity chromatography. LPS'antibodies in the outer membrane immune sera were purified by affinity chromatography (24, 25), which consisted of coupling purified LPS to cyanogen bromideactivated Sepharose 4B (Pharmacia, Piscataway, N.J.) and eluting antibodies from the column. In brief, 4 mg of LPS was suspended in coupling buffer and reacted overnight at 4°C with 1 g of CNBr-activated Sepharose 4B. Residual reactive groups were blocked with Tris-HCl buffer (pH 8.0) for 2 h. The matrix was washed extensively with coupling buffer to remove noncovalently bound LPS before equilibration with PBS containing 0.5% bovine serum albumin. Immune sera were adsorbed in this matrix, and LPS antibodies were eluted with 3.5 M MgCl2. The eluted fraction was dialyzed extensively against PBS at 4°C, checked for sterility, and used for experiments. (iii) Removal of antibodies against OMP of P. multocida. The effluent immune sera (sera from adsorption through the LPS-Sepharose column) were incubated with an equal volume of outer membrane 'Vesicles (14) of homologous P.
multocida (1 mg/ml) for 3 h at 37°C and centrifuged at 106,000 x g for 60 min. The supernatants were saved. This procedure was repeated four times. Final supernatants were tested for sterility and used for experiments. (iv) Purification of antibodies against P. multocida OMP. For determination of whether antibodies against OMP were the protective components of the immune sera, the effluent immune sera were adsorbed with intact P. multocida cells and the antibodies were eluted from the cell-antibody complex (11). In brief, freshly cultured homologous P. multocida cells (108 CFU in 0.5 ml of PBS) were mixed with an equal volume of serum and incubated for 1 h at 4°C with gentle agitation. The mixture was centrifuged, and the pellet was saved. An equal volume of fresh effluent immune serum was added to the pellet, and the procedure was repeated six times. The final pellet was washed with PBS and suspended in 1 ml of 0.2 M glycine-HCl buffer (pH 2.8) to dissociate the antibodies and P. multocida cells. The suspension was centrifuged, and the supernatant containing the antibodies was collected and adjusted to pH 7.0 with Tris buffer. The eluted antibodies, containing 0.1% bovine serum albumin, were stored at -20°C until used. Preimmunized rabbit sera, used as a'control in the passive transfer experiments, were treated in the same manner as the rabbit immune sera. Characterization of separated rabbit antibodies. (i) Identification of antibodies against P. multocida OMP. The various fractions of rabbit immune sera were analyzed by Western blotting (11, 13) with P. multocida outer membrane vesicles (14) as antigens. (ii) Identification of antibodies against P. multocida LPS. Purified P. multocida LPS were separated in a 15% separating gel containing 4 M urea (15), transferred to nitrocellulose paper, and reacted with various fractions of rabbit immune sera in a Western blot analysis. (iii) Quantitation of LPS antibodies against P. multocida. The LPS antibody concentrations in various immune serum fractions were determined by a quantitative precipitin analysis (18). In brief, increasing amounts of purified LPS were added to a series of tubes each containing the same volume of antiserum, and the mixtures were incubated for 1 h at 37°C and 7 days at 4°C. The precipitated antibodies were centrifuged, washed twice with cold saline, and quantitated by a modified Lowry procedure (2). Preimmunized rabbit sera were treated in the same manner as the immune rabbit sera and were used as a control.
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TABLE 1. Protection of mice against homologous challenge by rabbit immune sera directed to P. multocida outer membranesa Expt
Serum fractionb
1
Unadsorbed immune serum Normal serum
2 and 3
Purified LPS antibody Normal serum
4
Effluent of LPS column Normal serum
5
Supernatant from LPS and outer membrane vesicle adsorption Normal serum Eluted antibody from effluent-P. multocida complex Normal serum
6
antibody (jug/mouse)"
LPS
Mean log CFU of P. multocida in ± SEM (no. of mice)d
lungs
5.1 0
3.102 ± 0.482 (10) 6.623 ± 0.167 (10)
0.50
3.268 ± 0.426 (8) 5.935 ± 0.184 (10)
0.50
0.34 0
0
5.305 ± 0.206 (10)
1.683 ± 0.345 (10) 0
0.50), indicating that specific LPS antibodies were noninhibitory against P. multocida proliferation in mice. (iii) Effluents from the LPS column inhibited P. multocida proliferation. Since purified LPS antibodies were nonprotective in mice, we examined the inhibitory qualities of other antibodies. Mice inoculated i.n. with effluents from the LPS column and challenged had a 465-fold reduction in the numbers of P. multocida in the lungs (P < 0.001) (Table 1, experiment 4). This result suggested that antibodies found in the LPS column effluents were important in the inhibition of P. multocida proliferation. (iv) Loss of inhibitory activity by removal of antibodies against OMP. Antibodies against P. multocida OMP were removed by repeated adsorption of effluents (from the LPS column) with membrane vesicles of homologous P. multocida. Mice inoculated with rabbit immune serum which contained no antibodies against OMP had numbers of P. multocida in the lungs very similar to those in mice inoculated with normal rabbit serum (P > 0.50) (Table 1, experiment 5), indicating that antibodies against OMP were important in the inhibition of P. multocida proliferation. (v) Inhibition mediated by antibodies against OMP. Antibodies against OMP were purified by elution of antigenantibody complexes (antibody-intact P. multocida cell com-
PROTECTION OF MICE BY ANTIBODIES TO P. MULTOCIDA OMP
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A
B
C
D
E
A
F
66.2 -
B
C
D
E
1473
F
45.031.0-
45.021.5-
31.021.5-
144 FIG. 2. Western blot analysis of rabbit antibodies against P. multocida OMP. Outer membrane vesicles of P. multocida extracted by lithium chloride were used as antigens. Lanes: A, unadsorbed outer membrane immune serum; B; outer membrane immune serum after LPS adsorption; C, outer membrane immune serum after LPS and protein adsorption; D, eluted OMP antibodies; E, affinity-purified LPS antibodies; F, negative control (normal rabbit sera prior to immunization). Molecular weight markers (103) are on the left.
plexes). Mice inoculated i.n. with purified antibodies against OMP had a 201-fold reduction in the numbers of P. multocida in the lungs (P < 0.001) as compared with mice inoculated with normal rabbit sera (Table 1, experiment 6). This result indicated that antibodies against OMP inhibited P. multocida proliferation in mice. Characterization of protective antibodies in rabbit immune sera. (i) P. multocida OMP antibodies detected by Western blot analysis. Antibodies against OMP of P. multocida were analyzed by Western blotting with outer membrane vesicles as antigens. Unconcentrated immune sera obtained from each separation procedure were used for passive transfer studies and Western blot analysis. Unadsorbed rabbit immune serum contained various antibodies against OMP with molecular masses ranging from 27 to 66 kDa (Fig. 2, lane A). Adsorption of the immune serum with purified LPS did not remove any antibodies against OMP (Fig. 2, lane B). Antibodies against OMP were removed completely by adsorption of effluents (from the LPS column) with outer membrane vesicles (Fig. 2, lane C). Antibodies against major OMP were eluted from antibody-P. multocida cell complexes, although antibodies against some OMP were diminished in amount (Fig. 2, lane D). As expected, purified LPS antibodies contained very few antibodies against OMP (Fig. 2, lane E). Pooled sera which were obtained from three rabbits prior to immunization and which contained no antibodies against P. multocida OMP (Fig. 2, lane F) were used as controls. (ii) P. multocida LPS antibodies detected by Western blot analysis. Antibodies against purified LPS were demonstrated in unadsorbed immune serum (Fig. 3). The antibodies were reactive to LPS with molecular masses slightly lower than 14.4 kDa and to LPS with molecular masses slightly higher than 14.4 kDa (Fig. 3, lane A). Adsorption of the immune serum with an LPS-Sepharose column removed most of the antibodies against LPS (Fig. 3, lane B). Adsorption of effluents (post LPS column) with outer membrane vesicles
14.4FIG. 3. Western blot analysis of rabbit antibodies against P. multocida LPS. Purified LPS from P. multocida were used as antigens. Lanes: A, unadsorbed outer membrane immune serum; B, outer membrane immune serum after LPS adsorption; C, outer membrane immune serum after LPS and protein adsorption; D, eluted OMP antibodies; E, affinity-purified LPS antibodies; F, negative control (normal rabbit sera prior to immunization). Molecular weight markers (103) are on the left.
totally removed antibodies against LPS (Fig. 3, lane C). Antibodies against LPS were not eluted from antibody-P. multocida cell complexes (Fig. 3, lane D). As expected, abundant amounts of LPS antibodies were demonstrated in purified LPS antibodies (Fig. 3, lane E). Pooled sera which were obtained from three rabbits prior to immunization and which contained no antibodies against P. multocida LPS (Fig. 3, lane F) were used as controls. (iii) LPS antibodies determined by quantitative precipitin analysis. The amounts of LPS antibodies were determined and used to calculate the amounts of LPS antibodies received by each experimental mouse. Each mouse in experiments 1, 2 and 3, and 4 received 5.1, 17.7, and 0.34 p.g of LPS antibodies, respectively (Table 1). Mice in experiment 4, receiving 52-fold fewer LPS antibodies than mice in experiments 2 and 3, were protected against P. multocida proliferation, whereas mice in experiments 2 and 3 were not protected. Similarly, mice in experiment 4, receiving 15-fold fewer LPS antibodies than mice in experiment 1, were protected against P. multocida proliferation to a similar degree as were mice in experiment 1. These results showed that LPS antibodies were not important in the inhibition of P. multocida proliferation. DISCUSSION This study demonstrated that the inhibitory ability of rabbit immune sera against P. multocida proliferation in mice was due to antibodies against P. multocida OMP but not LPS. The inhibitory activity conferred by OMP antibodies was shown basically in two experiments. In the first experiment, LPS antibodies were adsorbed with an LPSSepharose affinity column, and the effluent sera from the column were used in the passive transfer experiments. In the second experiment, eluted OMP antibodies were used to demonstrate inhibition against P. multocida proliferation. In both experiments, a significant difference was shown between experimental and control groups (Table 1). Other
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LU ET AL.
studies (10, 26) also supported the view that P. multocida proteins are involved in protection. Those studies showed that partially purified proteinaceous immunogens of avian strains of P. multocida produced homologous protection in chickens, although the origin of these proteinaceous immunogens was not determined. In this study, we found that OMP antibodies were inhibitory, inferring that purified OMP contain a virulence factor(s) that controls P. multocida proliferation in mice. Our recent monoclonal antibody study further showed that the 37.5-kDa OMP of P. multocida is the target of MAB1608, which protects mice and rabbits against a homologous challenge (12). In the present study, we noted differences in CFU among experimental mice inoculated with normal serum and challenged. The differences in CFU among experimental mice inoculated with normal rabbit serum and challenged with P. multocida may reflect differences in batches of animals used, even though the mice were inbred and supplied by the same vendor. Because of the expected variations, a control group of mice was included in each experiment. The role of LPS antibodies in protection remains unclear. One study showed that rabbit immune sera against purified LPS of P. multocida type E provided protection in mice against a homologous challenge (20). Our study, however, showed that LPS antibodies against P. multocida 3:A did not protect mice against pulmonary infection. In agreement with our results, other studies (17, 21) showed that purified LPS of P. multocida produced no active immune protection in mice. Why LPS antibodies against P. multocida 3:A do not inhibit the pulmonary proliferation of P. multocida in mice remains unknown. Studies with Haemophilus influenzae type b showed that phenotypic variations in vivo reduce the binding of LPS antibodies to the cell surface, a phenomenon that may affect the protective potential of LPS antibodies (9). Although this phenomenon has not been studied in P. multocida, it is well known that P. multocida organisms undergo colony morphology changes when inoculated into animals for in vivo passage (5) or are transferred several times in artificial medium (4). Alternatively, the lack of inhibition of P. multocida proliferation by LPS antibodies may be related to the antibody titers. Since relative titers were not measured, it is possible that the OMP antibody titer was much higher than the LPS antibody titer and resulted in the ineffectiveness of the LPS antibodies. The role of capsular antibodies in resistance is difficult to assess because there is no complete method for the detection of antibodies against the capsule of type A P. multocida. Two approaches were taken to address the issue of P. multocida capsular antibodies. First, we produced rabbit immune sera containing antibodies against OMP and LPS but not capsules by immunizing rabbits with sucrose gradient-purified outer membranes which contained small amounts of capsules. Hyaluronidase treatment of P. multocida organisms resulted in the removal of capsules, since capsules were not demonstrated in enzyme-treated P. multocida organisms by India ink staining. In addition, rabbit immune sera against P. multocida outer membranes contained no antibodies against capsular antigens, as demonstrated by the passive hemagglutination method (23). Second, we adsorbed the immune sera with purified LPS and outer membrane vesicles which contained minute amounts of capsular material and tested the resistance efficacy of the remaining portion of the immune sera. The effluent from the LPS-Sepharose column was as inhibitory against P. multocida proliferation in mice as was the unadsorbed rabbit immune serum, and the purified LPS antibodies were non-
INFECT. IMMUN.
inhibitory against P. multocida proliferation in mice (Table 1). These results showed that antibodies against OMP were responsible for the inhibitory activity observed in the effluent. The supernatant obtained after adsorption of the effluent with outer membrane vesicles, however, was noninhibitory. We concluded that antibodies against P. multocida OMP but not capsules are the major components involved in the inhibition of P. multocida proliferation in mice. In this study, we used a mouse model to determine the role of rabbit immune sera against P. multocida OMP and LPS in
protecting rabbits against pasteurellosis. However, the results obtained in mice may not be the same as those obtained in rabbits. To clarify this issue, we conducted an experiment with rabbit immune sera against a KSCN extract of P. multocida for passive immunity transfer in mice. The selection of the antisera was based on the facts that the KSCN extract had protein and LPS profiles similar to those of the P. multocida outer membranes (14) and the rabbit immune sera against the KSCN extract protected rabbits against an i.n. homologous P. multocida challenge (13). The geometric mean concentration (CFU per gram) of P. multocida in mouse lungs was reduced from 2.05 x 105 (normal rabbit sera) to 3.40 x 102 (rabbit immune sera against a P. multocida KSCN extract); i.e., passive immunization of mice with rabbit immune sera and challenge resulted in a 604-fold reduction (P < 0.001) in the numbers of P. multocida in the lungs. These results clearly showed that rabbit immune sera against the KSCN extract significantly inhibited P. multocida proliferation in mice, indicating that the results in mice were applicable to rabbits. In this study, we observed that rabbit antibodies against P. multocida OMP but not LPS inhibited the pulmonary proliferation of P. multocida in mice. These results led us to believe that antibodies against P. multocida OMP but not LPS most likely protect rabbits against pasteurellosis. Studies in mice certainly provided very useful information for future studies in rabbits. In our view of the development of an effective P. multocida Vaccine, efforts should be focused on P. multocida OMP instead of LPS. In conclusion, we demonstrated that the OMP antibodies but not the LPS antibodies of P. multocida 3:A are inhibitory against P. multocida proliferation in mouse lungs and are presumably protective against a homologous challenge in rabbits. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants RRO1311 and RR00890 from the National Institutes of Health. We thank S. Atashband, Steven Afendis, and L. Watkins for excellent technical assistance. REFERENCES 1. Ashfaq, M. K., and S. G. Campbell. 1986. The influence of opsonins on the bactericidal effect of bovine alveolar macrophages against Pasteurella multocida. Cornell Vet. 76:213-221. 2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Carter, G. R. 1967. Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Adv. Vet. Sci. Comp. Med. 11:321379. 4. Carter, G. R. 1984. Genus I. Pasteurella Trevisan 1887, 94, AL Nom. cons. Opin. 13, Jud. Comm. 1954, 153, p. 552-558. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 5. Carter, G. R., and E. Annau. 1953. Isolation of capsular
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polysaccharides from colonial variants of Pasteurella multocida. Am. J. Vet. Res. 14:475-478. 6. Collins, F. M., C. J. Niederbuhl, and S. G. Campbell. 1983. Bactericidal activity of alveolar and peritoneal macrophages exposed in vitro to three strains of Pasteurella multocida. Infect. Immun. 39:779-784. 7. Collins, F. M., and J. B. Woolcock. 1976. Immune responses to Pasteurella multocida in the mouse. J. Reticuloendothel. Soc. 19:311-321. 8. Flatt, R. E., S. H. Weisbroth, and A. L. Kraus. 1974. Bacterial diseases, p. 193-205. In S. H. Weisbroth, R. E. Flatt, and A. L. Kraus (ed.), The biology of the laboratory rabbit. Academic Press, Inc., New York. 9. Inzana, T. J., and P. Anderson. 1985. Serum factor-dependent resistance of Haemophilus influenzae type b to antibody to lipopolysaccharide. J. Infect. Dis. 151:869-877. 10. Kajikawa, O., and M. Matsumoto. 1984. A protective antigen for turkeys purified from a type 1 strain of Pasteurella multocida. Vet. Microbiol. 10:43-55. 11. Lu, Y.-S., S. Afendis, and S. P. Pakes. 1988. Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits. Infect. Immun. 56:1532-1537. 12. Lu, Y.-S., W. C. Lai, S. P. Pakes, and L. C. Nie. 1991. A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis. Infect. Immun. 59:172-180. 12a.Lu, Y.-S., and S. P. Pakes. Abstr. Annu. Meet. Am. Assoc. Lab. Anim. Sci. 1987, abstr. no. 84, p. 532. 13. Lu, Y.-S., S. P. Pakes, and L. Massey. 1987. Hyperimmune serum from rabbits immunized with potassium thiocyanate extract of Pasteurella multocida protects against homologous challenge. J. Clin. Microbiol. 25:2173-2180. 14. Lu, Y.-S., S. P. Pakes, L. Massey, and C. Stefanu. 1987. A potassium thiocyanate extract vaccine prepared from Pasteurella multocida 3:A protects rabbits against homologous challenge. Infect. Immun. 55:2967-2976. 15. Manning, P. J., M. A. Naasz, D. Delong, and S. L. Leary. 1986. Pasteurellosis in laboratory rabbits: characterization of lipopolysaccharides of Pasteurella multocida by polyacrylamide gel electrophoresis, immunoblot techniques, and enzyme immunoassay. Infect. Immun. 53:460-463. 16. Mukkur, T. K. S. 1979. Immunogenicity of a chaotropically
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