INFECTION AND IMMUNITY, Dec. 1991, p. 4517-4523 0019-9567/91/124517-07$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 59, No. 12
The Outer Membrane of Pasteurella multocida 3:A Protects Rabbits against Homologous Challenge YUE-SHOUNG LU,l* WAYNE C. LAI,1 STEVEN P. PAKES,1 AND C. STEFANU2 Department of Pathology, Division of Comparative Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235,1 and Department of Preventive Medicine, State University of New York Health Science Center, Syracuse, New York 132102 Received 14 May 1991/Accepted 10 September 1991
The protective efficacy of a vaccine purified from the Pasteurella multocida 3:A outer membrane (OM) was evaluated in rabbits by homologous challenge. Twenty-seven rabbits were divided into four groups: 1, vaccinated with OM and challenged; 2, nonvaccinated and challenged; 3, vaccinated with OM only; and 4, nonvaccinated and not challenged. Rabbits were immunized intranasally with 1 mg of OM protein on days 0, 7, 14, and 35, challenged intranasally on day 49, and killed on day 63. Mortality rates were 0, 67, 0, and 0% for groups 1 through 4, respectively. The prevalence of pneumonia was reduced from 73 (group 2) to 20% (group 1). The severity of pneumonia was reduced from 0.62 (group 2) to 0.07 (group 1), as measured by the group lesion index. The number of P. multocida in nasal cavities was reduced from 3.89 x 105 (group 2) to 6.19 x l92 (group 1). The geometric mean number of P. multocida in lungs was 8,360,000-fold less in group 1 than in group 2. Similarly, the prevalence of P. multocida colonization in nonrespiratory organs was reduced from 47 (group 2) to 4% (group 1). Furthermore, group 1 and 3 rabbits developed significantly elevated immunoglobulin A antibodies in nasal secretions and lung lavages and significantly elevated immunoglobulin G antibodies in lung lavages and sera. In addition, rabbit immune sera contained antibodies against P. multocida OM proteins and lipopolysaccharides and inhibited P. multocida proliferation in mouse lungs. These results indicate that a vaccine prepared from the OM of P. multocida provides a significant protection in rabbits against homologous challenge.
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 (6). Infected rabbits are unsuitable for research involving respiratory, genital, and sensory systems because of colonization of the organisms and lesions in these systems. One of our goals is to develop a vaccine that protects rabbits against P. multocida disease. Because the current experimental vaccines do not provide a complete protection in rabbits (17, 28), there is interest in defining more specific and accurate responses of rabbits to P. multocida potential protective immunogens. Recent studies have demonstrated that potassium thiocyanate (KSCN) extract of P. multocida and rabbit immune sera against KSCN extract of P. multocida actively and passively protect rabbits against homologous challenge (16, 17, 28). KSCN extract of P. multocida contains outer membrane proteins (OMPs), lipopolysaccharides (LPS), capsular material, and nucleic acids (17). Rabbits immunized with KSCN extract produce antibodies against OMPs and LPS of homologous P. multocida (16, 28). To further identify the protective components of the KSCN extract of P. multocida, we vaccinated rabbits with the purified OM, which contains OMPs and LPS. In this study, we immunized rabbits intranasally (i.n.) with the purified OMs and challenged them i.n. with homologous P. multocida. The results showed that the OM of P. multocida elicited excellent protection in rabbits, as was evident by the reduction of numbers of P. multocida in nasal cavities and *
Corresponding author. 4517
lungs, the reduction in prevalence of P. multocida colonization in nonrespiratory organs, and the reduction in prevalence and severity of pneumonia. MATERIALS AND METHODS Animals. Pasteurella-free New Zealand White female rabbits, weighing 2.7 to 3.6 kg, were purchased from HazletonDutchland Laboratories, Denver, Pa. Before vaccination, nasal cavities of each rabbit were cultured three times for P. multocida and sera were tested for P. multocida immunoglobulin G (IgG) antibodies by an enzyme-linked immunosorbent assay (ELISA). Rabbits free of P. multocida and P. multocida IgG antibodies were used in the experiments. Source and serotype of P. multocida. Wild-type P. multocida UT-1 was isolated from a rabbit with suppurative rhinitis. The strain belongs to serotype 3:A and was maintained by lyophilization. Preparation and characterization of P. multocida OMs. The OMs were prepared as described previously (13). Briefly, overnight-cultured P. multocida organisms were washed twice in phosphate-buffered saline (PBS), suspended in 0.1 M PBS (pH 6.0), and incubated with hyaluronidase to remove the capsule which contains hyaluronic acid (3). Removal of the capsule was monitored chemically (5, 13) by using purified hyaluronic acid as a standard. The treated organisms were then disrupted and subjected to sucrose gradient centrifugations. Selected fractions from the gradient were pooled, dialyzed extensively against PBS, and assayed for total protein by a modification of the Lowry method (1). The buoyant density of the purified OMs, and membrane contents revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to characterize the OMs (12, 13).
LU ET AL.
Experimental design. Twenty-seven rabbits were divided into four groups and treated as follows. Group 1 (nine rabbits) was inoculated i.n. with purified P. multocida OMs (1 mg of protein containing 121 ,ug of hyaluronic acid per rabbit per inoculation) in PBS on days 0, 7, 14, and 35; challenged i.n. on day 49; and killed on day 63 by an overdose of pentobarbital sodium IV. Group 2 (12 rabbits) was not vaccinated and was challenged in the same manner as the group 1 rabbits. Group 3 (three rabbits) was treated the same as group 1 except that the rabbits were not challenged. Group 4 (three rabbits) was not vaccinated and not challenged. This immunization protocol was chosen because, in KSCN extract experiments, it elicited an excellent protective immunity in rabbits (17). The criteria for determining protection include mortality, pathologic changes (prevalence and severity of pneumonia, rhinitis, and tympanitis), microbiologic parameters (prevalence and number of P. multocida in nasal cavities and lungs and prevalence of P. multocida in nonrespiratory organs), immunologic responses (IgA antibody in nasal secretions and lung lavages and IgG antibody in lung lavages and sera), and inhibitory activity of rabbit immune sera against P. multocida proliferation in mice. Preparation of challenge organisms. Wild-type P. multocida UT-1 organisms maintained in a lyophilized stock were grown on dextrose starch agar plates (Difco Laboratories, Detroit, Mich.). Organisms cultured overnight were suspended in 0.01 M PBS (pH 7.2), centrifuged, washed twice in PBS, diluted, and inoculated into rabbits. The inoculum contained 2.6 x 107 organisms per ml as determined by CFU in tryptic soy agar with 0.3% yeast extract. Rabbits in groups 1 and 2 were inoculated i.n. with the challenge organism (1 ml per rabbit) by using Pasteur pipettes. Microbiological studies. The prevalence and numbers of P. multocida in nasal cavities and lungs and the prevalence of P. multocida in nonrespiratory organs were used as criteria of protection. Bilateral nasal swabs collected from group 1 and 2 rabbits on days 2, 6, 8, 10, and 14 postchallenge were inoculated into blood agar plates to determine the numbers of P. multocida in nasal cavities (15, 16). At necropsy, bilateral nasal swabs; left and right tympanic bulla swabs; and the liver, spleen, uterus, and lungs were collected sterilely from each rabbit. The nasal swabs were vortexed in 1 ml of PBS to make a suspension. The suspension was then serially diluted 10-fold and inoculated onto blood agar plates in triplicate at each dilution to determine the number of CFU per nasal swab. Parts of the lungs from each rabbit were homogenized in PBS to make a 10% (wt/vol) suspension. The lung suspensions were diluted 10-fold and plated on blood agar to determine the number of CFU per gram of lungs. A small piece of liver, spleen, and uterus and left and right tympanic bulla swabs were inoculated onto blood agar plates to determine the presence or absence of P. multocida. Immunological studies. (i) Determination of antibody levels by ELISA. Nasal secretions, lung lavages, and sera were obtained from rabbits as previously described (16) and stored at -20°C until assayed. Nasal secretions and lung lavages were collected at necropsy, and sera were collected at days 24 and 63 after the first i.n. immunization. Serum IgG and lung lavage IgG were measured by an ELISA, with P. multocida OM vesicles (13, 19) as the antigen and alkaline phophatase-conjugated goat anti-rabbit IgG (heavy chain specific; Miles Laboratories, Elkhart, Ind.) as the secondary antibody (16). IgA antibody levels in nasal secretions and lung lavages were measured by a similar assay, except that alkaline phosphatase-conjugated goat anti-rabbit secretory
IgA (cx chain and secretory component specific; Cappel Laboratories, Cochranville, Pa.) was the secondary antibody (16). Plates contained appropriate controls in duplicate, including sera known to be positive; sera known to be negative; and wells without antigen, antibody, or conjugate. The optical density (OD) reading of the uncoated wells was subtracted from those of all other sample readings. The OD readings of samples known to be positive were averaged and normalized to a predetermined value. All sample readings were then normalized and extrapolated to 100 min. The OD readings are presented as geometric means (16, 28). These were determined by using the log1o of each value for each rabbit to calculate the mean and standard error of the mean (SEM). Antilogs of the mean log of the OD, 1 SEM above and 1 SEM below the mean log OD, represent the geometric mean, with an upper and lower limit of variance about that mean. (ii) Immunoblot analysis. Pooled immune sera, collected at day 63 from rabbits immunized with OMs without challenge (group 3), were analyzed by Western blot (immunoblot). Homologous P. multocida OM vesicles (13, 19) or purified LPS were used as antigens (27). SDS-PAGE, electrophoretic transfer of P. multocida proteins and LPS to nitrocellulose membranes, and immunoblotting assays were performed as previously described (13, 18, 33). Nonspecific binding sites on nitrocellulose membranes were blocked in PBS solution containing 0.05% Tween 20 and 1% bovine serum albumin (pH 7.2). Serum specimens were diluted 1:500 in PBS-Tween 20 (0.05%). Antibody binding was localized by using horseradish peroxidase-conjugated goat anti-rabbit IgG (heavy and light chain specific; Bio-Rad Laboratories). A substrate solution containing 4-chloro-1-naphthol and hydrogen peroxide was used for color development. Pathology. Rabbits that died spontaneously or were euthanatized at the end of experiments (day 63) were necropsied. The nasal turbinates, lung, and tympanic bullae were fixed in 10% neutral, buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined for microscopic lesions. Microscopic lesions of nasal turbinates (three sections), lungs (five sections, with one section from each lobe), and tympanic bullae (two sections) from each rabbit were scored as 0 (normal), 1 (mild), 2 (moderate), or 3 (severe) (16, 17). For histopathologic grading, the slides were coded and evaluated as unknowns, and the evaluations were repeated twice. The slide codes were then revealed, and the results were analyzed. To compare a score, the sum of the lesion scores for each organ from an individual rabbit within a group was divided by the sum of the maximum possible score to determine the group lesion index (GLI). A GLI of 1.0 indicates the most severe lesion possible for each organ of a group (16, 17). Passive transfer studies. Passive transfer experiments in mice were conducted to determine whether rabbit immune sera against OMs contain antibodies that inhibit P. multocida proliferation. Mice were divided into two groups of 10 animals per group, anesthetized with methoxyflurane, inoculated i.n. with 50 ,ul of unconcentrated immune (group 3) and normal (group 4) rabbit sera, respectively, recovered from anesthesia for 2 h, challenged i.n. with live P. multocida (2 x 106 CFU), and euthanatized by a methoxyflurane overdose at 48 h postchallenge. Lungs were homogenized to make a 10% (wt/vol) suspension and assayed for numbers of P. multocida organisms (14, 15). Typical P. multocida colonies were counted, and selected colonies were confirmed by standard biochemical characteristics. I.n. administration was used because we wanted to concentrate the antibody in
P. MULTOCIDA OUTER MEMBRANE PROTECTION OF RABBITS
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TABLE 1. Reduction of lesions and mortality in rabbits immunized with P. multocida OMs and challenged with virulent P. multocida organisms Rabbit group
No. of rabbits
Vaccinated, challenged Not vaccinated, challenged Vaccinated, not challenged Not vaccinated, not challenged
2 3 4
Mean GLI ± SEM
Mean GLI ± SEM
0.07 ± 0.02
0.62 ± 0.05
0.09 ± 0.03
0/9 (0) 8/12 (67)
a Each rabbit in groups 1 and 3 was immunized i.n. with 1 mg of OM on days 0, 7, 14, and 35. Group 1 and 2 rabbits were challenged i.n. with 2.6 x 107 cells of virulent homologous P. multocida UT-1 on day 49 and killed on day 63. b Prevalence indicates the number of positive lobes (sections) per total number of lobes (sections) examined (five lung lobes and three nasal sections from each rabbit were examined). Mean GLI indicates the severity of the group lesions, calculated as described in the text. A GLI of 1.0 is the most severe change possible for a group. Prevalence and mean GLI of pneumonia were significantly lower (P < 0.001) for group 1 than group 2. c Mortality is measured as the number of dead rabbits per total number of rabbits in the group. The mortality rate for group 1 was significantly (P < 0.001) lower than that for group 2.
the lungs, which is the major target organ of P. multocida. Lungs were collected 48 h postchallenge because antibodies against P. multocida provide maximum protection in mice at 48 h post-i.n. challenge (15). Statistical analysis. The analysis of variance was applied to determine significant differences in numbers of P. multocida in nasal cavities and lungs, lesion indices, and immune responses among the groups of rabbits. The chi-square test was used to determine significant differences in mortality, the prevalence of lesions, and the prevalence of P. multocida colonization in lungs and nonrespiratory organs among groups (34). RESULTS Characterization of the OM. The buoyant density of the OM was 1.22 g/cm3, which is identical to the buoyant density of P. multocida OM reported previously (13). The OM content, as determined by SDS-PAGE analysis, is composed of proteins measuring 24.6, 27, 30.4, 37.5, 49.5, and 64.5 kDa. These OMP profiles were identical to results reported recently (13). Clinical observations. Eight rabbits from group 2 (nonvaccinated and challenged) died 3 to 10 days postchallenge. They were depressed, anoretic, and dyspneic before death. Rabbits in groups 1, 3, and 4 did not develop any clinical signs of P. multocida infection. Mortality rates were 0, 67, 0, and 0% for groups 1 (vaccinated-challenged), 2 (nonvaccinated-challenged), 3 (vaccinated-nonchallenged), and 4 (nonvaccinated-nonchallenged), respectively. A significant difference (P < 0.001) was observed between groups 1 and 2, indicating that P. multocida OM protects rabbits against lethal challenge with P. multocida. Pathology. The prevalence and severity of lesions in lungs, nasal cavities, and tympanic bullae were compared among groups. (i) Pneumonia. The prevalences of pneumonia in lung lobes were 20% (9 of 45), 73% (44 of 60), 0% (0 of 15), and 0% (0 of 15) for groups 1 through 4, respectively, and the difference between groups 1 and 2 was significant (P < 0.001). As shown in Table 1, the GLI, which indicates the lesion severity of the group, was reduced from 0.62 (group 2) to 0.07 (group 1), a significant ninefold reduction (P < 0.001). Nine (from eight rabbits) of the 45 lobes of the group 1
rabbits had mild pneumonia, and 36 lobes were normal. In contrast, 29 (from 11 rabbits) of the 60 lobes of group 2 had severe pneumonia, 10 (from 6 rabbits) had moderate pneumonia, and 5 (from 4 rabbits) had mild pneumonia; the remaining 16 lobes were normal. All 15 lobes in groups 3 and 4 were normal. (ii) Rhinitis. The prevalence of rhinitis was reduced from 25% (9 of 36) in group 2 to 0% (0 of 27) in group 1, a significant reduction (P < 0.001). However, there was no significant difference in the GLI between group 1 and 2 rabbits. Among the 36 nasal sections of the group 2 rabbits, 8 (from five rabbits) had mild and 1 (from one rabbit) had moderate rhinitis; the remaining 27 sections were normal. Nasal sections from groups 1, 3, and 4 were normal. Only 1 of the 12 rabbits in group 2 had a mild tympanitis, and none of the rabbits in groups 1 and 3 developed tympanitis (data not shown); thus, the efficacy of vaccination in preventing tympanitis could not be evaluated. None of the group 4 rabbits developed tympanitis. No gross lesions in other tissues of any of the experimental rabbits were observed. Microbiology. (i) Reduction of nasal colonization by immunization. The effect of immunization with OMs on the prevalence and numbers of P. multocida colonies in nasal cavities was evaluated. The number of rabbits cultured at various days ranged from 3 to 12, as shown in Fig. 1. The geometric mean CFU for an individual rabbit was calculated and logarithmically converted. The mean and SEM of the log CFU of the group at various days were then calculated and compared among the groups. The challenge organisms colonized the nasal cavities of all rabbits in group 1 and 2 beginning on day 2 after challenge and persisting until the rabbits died or were euthanatized at 14 days postchallenge. A quantitative analysis of the numbers of P. multocida colonies in nasal cavities showed the immunized-challenged rabbits (group 1) had lower numbers of P. multocida colonies than those of nonimmunized-challenged rabbits (group 2). Immunization resulted in 15-, 107-, 18-, 6-, and 6-fold reductions at days 2, 6, 8, 10, and 14 postchallenge, respectively. The reductions were significant at days 2 (P < 0.005), 6 (P < 0.001), and 8 (P < 0.05) postchallenge. P. multocida organisms were not isolated from nasal cavities of group 3 and 4 rabbits.
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DAYS AFTER CHALLENGE FIG. 1. Numbers of P. multocida in nasal cavities of rabbits inoculated with purified P. multocida OMs and challenged (0; group 1), without purified OMs and challenged (A; group 2), and with purified OMs but not challenged (0; group 3). Nasal swab samples (two per rabbit) collected at days 2, 6, 8, 10, and 14 postchallenge were cultured on blood agar plates. Data represent the log of the geometric mean CFU and SEM; numbers in parentheses indicate numbers of rabbits tested; the geometric mean CFU per rabbit of the group was calculated first and converted to the logarithmic number, and then the mean of the log of the geometric mean of the group was calculated. The log of geometric mean CFU of group 1 rabbits was significantly less than that of the corresponding group 2 rabbits at days 2 (P < 0.005), 6 (P < 0.001), and 8 (P < 0.05).
(ii) Isolation of P. multocida from various tissues at necP. multocida is known to infect the respiratory tract (nasal cavity and lungs) and nonrespiratory organs such as the tympanic bullae, liver, spleen, and uterus of rabbits. The
effect of immunization with OMs was evaluated further by determining the numbers of P. multocida in the respiratory tract and the prevalence of P. multocida colonization in the nonrespiratory organs at necropsy (Table 2). Immunization of rabbits with OMs reduced the number of P. multocida organisms in nasal cavities from 3.89 x 105 (group 2) to 6.19 x 102 (group 1), a significant 628-fold reduction (P < 0.001). Similarly, immunization of rabbits dramatically reduced the number of P. multocida organisms in lungs from 8.36 x 106 in group 2 to 0 in group 1 rabbits; i.e., immunization of rabbits with OMs resulted in an 8,360,000-fold reduction (P < 0.001) in the number of P. multocida organisms in lungs (Table 2). As shown in Table 2, 47% (28 of 60) of the nonrespiratory tissue samples in group 2 rabbits were positive for P. multocida compared with 4% (2 of 45) of the group 1 rabbits. Immunization resulted in a significant 43% reduction (P < 0.001) of P. multocida colonization in nonrespiratory organs. P. multocida organisms were isolated from the liver (7 of 12), spleen (5 of 12), uterus (2 of 12), and left (9 of 12) and right (5 of 12) tympanic bullae of group 2 rabbits, whereas the same P. multocida organisms were isolated only from the left (1 of 9) and right (1 of 9) tympanic bullae of group 1 rabbits. P. multocida organisms were not isolated from the respiratory and nonrespiratory organs of group 3 and 4 rabbits. Immune responses. (i) Nasal, pulmonary, and serum antibody responses measured by ELISA. As shown in Table 3, rabbits (group 3) immunized i.n. with purified P. multocida OM developed significantly (P < 0.05) higher nasal IgA, pulmonary IgA, pulmonary IgG, and serum IgG (days 24 and 63) antibodies against P. multocida than did the control rabbits (group 4). Group 2 rabbits, which were nonimmunized but challenged, did not produce significant amounts of antibodies at day 14 postchallenge, except for pulmonary IgG and serum IgG. Rabbits which were immunized and challenged (group 1) developed significantly higher (P < 0.001) amounts of antibodies, including IgA (nasal and pulmonary) and IgG (pulmonary and serum) than did the controls. Rabbits which were subjected to immunization and challenge (group 1) also had significant (P < 0.01) elevations
TABLE 2. Isolation of P. multocida from nasal cavities, lungs, and nonrespiratory organs of rabbits which were immunized with OMs and challenged with virulent P. multocida P. multocida in nasal Rabbit
Vaccinated, challenged Not vaccinated, challenged Vaccinated, not challenged Not vaccinated, not
P. multocida in lungs
Rabbis N fPrevalence
Mean + SEM
Mean ± SEMd
oe ±0 9/9 (100) 2.792e ± 0.374 6.19 x 102 0/9 (O)f 0 12/12 (100) 5.590 ± 0.378 3.89 x 105 11/12 (92) 6.920 ± 0.348 8.36 x 106
posit(e/total 2/45 (4)B 28/60 (47)h
0 ± 0
aTreatments for each group of rabbits were described in Table 1. At necropsy, nasal swabs (two per rabbit) and portion of lungs were collected and cultured for P. multocida as described in the text. b Prevalence indicates the number of positive samples per total number sampled; values in parenthesis are percentages. Mean (- SEM) indicates the mean of the log of the geometric mean CFU per rabbit. The geometric mean CFU per rabbit was calculated and converted to the logarithmic number; then the mean of the log of the geometric mean of the group was calculated. c A small piece of liver, spleen, and uterus and swabs collected from left and right tympanic bullae were inoculated into blood agar plates for P. multocida isolation. d Mean of the log of the geometric mean CFU per gram of lung tissue. The geometric mean CFU per gram of lung in each rabbit was calculated and converted to the logarithmic number. Then the mean of the log of the geometric mean of the group was calculated. e Mean CFU significantly less (P < 0.001) than that of group 2. f Prevalence of P. multocida in lungs significantly less (P < 0.001) than that of group 2. g Prevalence significantly less (P < 0.001) than that of group 2. P. multocida organisms were isolated from tympanic bullae only. h P. multocida organisms were isolated from the liver, spleen, uterus, and tympanic bullae.
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TABLE 3. ELISA antibody responses of rabbits immunized with OMs and challenged with virulent P. multocidaa Geometric mean (range) ELISA [no. of samples]b Rabbit group
1 2 3 4
1.640 0.177 0.800 0.091
(1.780-1.520)c  (0.227-0.138)d  (1.070-0.597)  (0.123-0.068) 
2.755 0.254 0.655 0.106
(2.865-2.648)c  (0.354-0.182)d  (2.162-0.198)  (0.141-0.079) 
0.552 0.539 0.599 0.061
(0.640-0.477) (0.762-0.381) (1.010-0.346) (0.083-0.041)
0.586 0.231 0.844 0.201
(0.644-0.533) (0.458-0.116) (1.026-0.693) (0.253-0.159)
2.461 0.605 2.454 0.247
(2.704-2.240)  (0.676-0.542)d  (2.655-2.270)  (0.381-0.161) 
a Treatments for each group of rabbits are described in Table 1. b Values shown are the geometric mean readings for the group. The numbers in parenthesis represent the geometric mean ± 1 SEM. Nasal and lung lavage samples were obtained at necropsy; serum samples were collected at days 24 and 63 after the first vaccination of group 1 and 3 rabbits and at days 7 and 14 postchallenge of group 2 rabbits. IgA levels were determined from undiluted nasal and lung lavage samples, whereas IgG levels were determined from 1:10 dilutions of lung lavage samples and 1:20 dilutions of serum samples. Geometric mean readings of group 1 and 3 rabbits were significantly higher than those of group 4 rabbits at P