Abstract
Background/Aim
Pasteurella multocida is a significant cause of morbidity and mortality in rabbits, as well as other species. Some isolates elaborate a heat-labile toxin (PMT) that has been shown to be an important virulence factor. Though previous studies have demonstrated protective immunity can be conferred via immunization of rabbits with heat-inactivated PMT (IPMT), we investigated the ability of immunization to impact colonization of P. multocida.
Materials and Methods
Rabbits were immunized at days 0, 7 and 14 with either phosphate buffered saline (PBS), the mucosal adjuvant cholera toxin (CT), IMPT or IPMT + CT. Male New Zealand white rabbits were used and confirmed to be free of P. multocida prior to experimentation.
Results
Serum IgG and nasal lavage fluid IgA responses directed against PMT were found in rabbits immunized with IPMT, with or without CT, but not in those immunized with only PBS or CT; and the addition of CT to IPMT enhanced the response. Significantly more P. multocida CFUs (p≤0.05) were cultured from the lungs of rabbits immunized with IPMT, with or without CT, compared to those administered only PBS or CT, although no differences were observed in nasal lavage fluid samples. Further, immunization IPMT, with or without CT, conferred protection against pleuritis and pneumonia.
Conclusion
PMT, in addition to its role as a virulence factor, may serve as a colonization factor for P. multocida in the lungs of rabbits.
Keywords: Pasteurella multocida, rabbit, toxin, colonization factor
Pasteurella multocida is a common bacterial pathogen of domestic rabbits. In addition, clinical disease associated with P. multocida infection may be subclinical or manifested as disease characterized by rhinitis, pneumonia, abscessation of viscera and subcutaneous sites, metritis, orchitis, septicemia, and otitis media (1).
Several virulence factors have been suggested for P. multocida, including lipopolysaccharide (2-4), adhesion factors (5,6), lipase (7), and heat-labile toxin (8-15). P. multocida heat-labile toxin (PMT) is a cytoplasmic protein (16) with a molecular weight of 150,000 (11,17,18). Elaboration of PMT is common among disease-producing isolates of P. multocida from swine in which it plays a central role in the induction of atrophic rhinitis (12,14,19). In swine, turkeys, and rabbits, PMT is synthesized principally by capsular type D isolates of P. multocida (20-22), although toxigenic type A isolates from humans and swine have also been described (23-25). When administered intranasally to rabbits, PMT induces pneumonia, pleuritis, hepatic necrosis, splenic lymphoid atrophy, and nasal turbinate atrophy (13).
Swine immunized with formaldehyde-inactivated, affinity-purified PMT were protected against development of atrophic rhinitis (26-28). However, swine with spontaneous atrophic rhinitis rarely had detectable anti-PMT serum antibody (29,30). Rodents immunized with inactivated PMT developed a serum anti-PMT antibody response that was dose-dependent and were protected against hepatic necrosis and death following challenge with native PMT (26,31). Immunization of rabbits with heat-inactivated PMT (IPMT) stimulated protective immunity to challenge with PMT, and this effect was enhanced by coadministration with cholera toxin, a potent adjuvant for the mucosal immune system (32).
In swine, administration of antisera from pigs immunized with formaldehyde-inactivated PMT protected against colonization by toxigenic P. multocida (33). Furthermore, immunization of swine resulted in decreased colonization by P. multocida in the nasal cavity (34).
In vitro studies with rabbit tissue explants showed that PMT enhanced the adherence of P. multocida to the trachea and aorta (35). The study described here extends that finding and was undertaken to examine the ability of immunization with IPMT to diminish P. multocida colonization in the nasopharynx, lungs, inner ear, and liver in rabbits. In addition, the effect of immunization, with and without cholera toxin, on development of disease in rabbits challenged with toxigenic P. multocida was examined.
Materials and Methods
Animals. Pasteurella-free New Zealand white male rabbits weighing 1.8 to 2.4 Kg (Inotiv, Inc., Indianapolis, IN, USA) were used for this study. The Pasteurella-free status of the animals was confirmed by culture of deep nasal swabs prior to experimentation. Use of rabbits for this study was approved by the Institutional Animal Care and Use Committee. Rabbits were housed in stainless steel cages and provided a daily ration of 110 g of commercial rabbit feed (Lab Rabbit Chow HF 5326; Purina Mills, Inc., Richmond, IN, USA). The room in which the rabbits were housed was maintained at 15 to 16.9˚C and was illuminated on a 12-h light/dark cycle.
Immunogen. Purified PMT used for toxoid preparation was prepared by sequential ammonium sulfate precipitation, ion-exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis as described (13). The toxoid (IPMT) was prepared by heating PMT at 70˚C for 30 min. Cholera toxin (CT) was obtained from a commercial source (Sigma Chemical, St. Louis, MO, USA).
Enzyme-linked immunosorbent assay (ELISA). A modification of a previously described (33) ELISA was used to detect anti-PMT IgA and IgG. Briefly, each well of polystyrene microtitration plates was coated with 1 μg of purified PMT in 100 μl of 50 mM carbonate/bicarbonate buffer (pH 9.6). After incubation at 4˚C for 12 h, plates were thoroughly washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and coated for 24 h with 3% bovine serum albumin (Sigma Chemical Co.). Plates were again washed and dilutions of serum (1:10) and nasal lavage fluid (1:2) in 0.03% Tween 20 in PBS (pH 7.4) were added to the wells and incubated. After washing, 1:500 dilutions of horseradish peroxidase conjugated to goat anti-rabbit IgG (Sigma Chemical Co.) or goat anti-rabbit IgA (Accurate Chemical and Scientific, Inc., Westbury, NY, USA) were added to wells in 1:500 concentrations. After incubation and washing, substrate (o-phenylaminediamine, Sigma Chemical Co.) was added, and optical density at 490 nm was measured 30 min later using a Vmax microplate reader (Molecular Devices Corp., Menlo Park, CA, USA).
Experimental design. Groups of five rabbits were immunized intranasally (IN) on days 0, 7, and 14 with either PBS (pH 7.4) with or without 100 μg of CT or 5 μg of IMPT with or without 100 μg of CT. Immunogens were administered in 1.0 ml of PBS, equally divided between the nares. The immunization regimen was chosen based upon earlier studies which demonstrated this to be an effective method to stimulate anti-PMT antibody responses in both serum and nasal lavage fluid (32). Blood was obtained from the auricular artery before initial immunization (day 0) and on days 7, 10, and 14 after initial immunization. Nasal lavage samples were obtained at these same timepoints by means of a previously described procedure (32). Briefly, 1.0 ml of sterile PBS was instilled into one nostril with the head directed slightly downward toward the contralateral nostril. Fluid that was sneezed out of ran out the contralateral nostril was collected in a sterile petri dish. This process was repeated for both nostrils and the samples from each rabbit were pooled for each timepoint.
To examine the ability of IPMT immunization to protect rabbits against colonization with homologous P. multocida, immunized rabbits were challenged IN 16 days after initial immunization with 1.0×106 colony forming units (CFUs) of a toxigenic P. multocida strain, serotype 3, 12, 15:D which had originally been isolated from the bone marrow of a rabbit. Ten days after challenge, rabbits were euthanized with an intravenous overdose of sodium pentobarbital and necropsied.
Bacterial load and pathologic evaluation. At necropsy, swab specimens of the lungs and nasopharynx were cultured on tryptic soy agar containing 5% sheep blood and were examined for growth of P. multocida after incubation at 37˚C for 24 h. For quantitative comparison, culture of lungs was performed by macerating a 1 g sample from the apical portion of the left cardiac lobe and culturing 100 μl of 1:10-1:10,000 ten-fold dilutions of this suspension. The nasopharynx was vigorously swabbed, the swab vortexed at high speed for 15 sec in 2 ml of sterile saline, and 100 μl of 1:1-1:10,000 ten-fold dilutions of this suspension cultured on tryptic soy agar with 5% sheep blood. P. multocida CFUs were enumerated from lung and nasopharyngeal cultures after 24 h of incubation.
After fixation in 10% neutral-buffered formalin, lung specimens were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Nasal turbinate specimens were decalcified in RDC decalcifying solution (Creative Biolabs, Inc., Shirley, NY, USA) after formalin fixation, then embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined microscopically. The degree of histologic change, usually seen as inflammation and necrosis for the lungs and as atrophy of the nasal turbinates, was graded on a numerical scale as previously described (13). Briefly, a score of 1 to 5 was assigned (0=no change, 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe) by an evaluator blinded to experimental group to each tissue on the basis of extent and degree of change. For the lungs, each of the lobes was given a score and an average was calculated for the entire lung.
Statistical analysis. Numbers of P. multocida CFUs from the lung and nasopharynx of rabbits and ELISA values were compared between experimental groups with the Wilcoxon rank sum test. Statistical significance was reached when p≤0.05.
Results
Serum anti-PMT antibody. Immunization with IPMT, with or without CT, stimulated a marked increase in serum anti-PMT IgG beginning at 10 days and continuing through 14 days after initial immunization (Figure 1). Serum anti-PMT IgG was significantly (p≤0.05) greater in rabbits immunized with only IPMT at 14 after initial immunization compared to those immunized with PBS or CT only. The response in rabbits immunized with IPMT plus CT was significantly greater than that in rabbits administered only PBS or CT by 10 days after initial immunization. Serum anti-PMT IgG activity in IPMT-immunized rabbits continued to increase through day 14. The responses in rabbits immunized with IPMT plus CT were significantly greater than those in rabbits immunized with IPMT alone only at 14 days after initial immunization (p≤0.05). There was no detectable serum anti-PMT IgA in rabbits from any of the treatment groups (data not shown).
Figure 1.
Serum anti-PMT IgG expressed as intensity of optical density at 90 nm. Serum anti-PMT IgG was significantly (p≤0.05) greater in rabbits immunized with only IPMT at 14 days after initial immunization compared to those immunized with PBS or CT only. The responses in rabbits immunized with IPMT plus CT were significantly greater than those in rabbits administered only PBS or CT by 10 days after initial immunization and continuing through 14 days. The responses in rabbits immunized with IPMT plus CT were significantly greater than those in rabbits immunized with IPMT alone only at 14 days after initial immunization (*p≤0.05). PMT: Pasteurella multocida heat-labile toxin; IPMT: heat-inactivated Pasteurella multocida heat-labile toxin; PBS: phosphate buffered saline; CT: cholera toxin.
Nasal lavage fluid anti-PMT antibody. Immunization with IPMT, with or without CT, stimulated a marked increase in anti-PMT IgA in nasal lavage samples beginning at day 10 following initial immunization and continuing through day 14 (Figure 2). Rabbits immunized with IPMT only demonstrated significantly greater anti-PMT IgA at day 14 in nasal lavage samples than those immunized with PBS or CT only. At days 10 and 14 the responses in rabbits immunized with IPMT plus CT were significantly greater than those in rabbits administered only PBS or CT (p≤0.05); and at day 14 values were significantly greater than those in rabbits immunized with only IPMT. There was no detectable anti-PMT IgG in nasal lavage samples from rabbits in any of the experimental groups (data not shown).
Figure 2.
Nasal lavage fluid anti-PMT IgA expressed as intensity of optical density at 90 nm. Rabbits immunized with IPMT only demonstrated significantly greater anti-PMT IgA at day 14 in nasal lavage samples than those immunized with PBS or CT only. At days 10 and 14 the responses in rabbits immunized with IPMT plus CT were significantly greater than those in rabbits administered only PBS or CT (p≤0.05); and at day 14 values were significantly greater than those in rabbits immunized with only IPMT (*p≤0.05 and **p≤0.02). PMT: Pasteurella multocida heat-labile toxin; IPMT: heat-inactivated Pasteurella multocida heat-labile toxin; PBS: phosphate buffered saline; CT: cholera toxin.
Culture of tissues for P. multocida. Results of nasopharyngeal cultures are shown in Figure 3. No significant differences in numbers of P. multocida CFUs were found between any of the treatment groups. In contrast, significantly fewer (p≤0.02) P. multocida CFUs were cultured from the lungs of rabbits immunized with IPMT, with or without CT, than from the lungs of rabbits administered only PBS or CT (Figure 4). There was no significant difference in numbers of CFUs cultured from the lungs of rabbits immunized with only IPMT compared to cultures of lungs of rabbits immunized with IPMT plus CT (Figure 4).
Figure 3.
Bacterial load of P. multocida in nasal lavage samples expressed as mean colony forming units (CFUs). No significant differences between treatment groups were found. PMT: Pasteurella multocida heat-labile toxin; IPMT: heat-inactivated Pasteurella multocida heat-labile toxin; PBS: phosphate buffered saline; CT: cholera toxin.
Figure 4.
Bacterial load of P. multocida in lung samples expressed as mean colony forming units (CFUs). Significantly fewer (p≤0.02) P. multocida CFUs were cultured from the lungs of rabbits immunized with IPMT, with or without CT, than from the lungs of rabbits administered only PBS or CT. There were no significant differences in numbers of CFUs cultured from the lungs of rabbits immunized with only IPMT compared to cultures of lungs of rabbits immunized with IPMT plus CT. PMT: Pasteurella multocida heat-labile toxin; IPMT: heat-inactivated Pasteurella multocida heat-labile toxin; PBS: phosphate buffered saline; CT: cholera toxin.
Histopathology. Results of histopathological evaluations are summarized in Table I. Rabbits from all groups demonstrated some degree of pleuritis and pneumonia. Pleuritis was characterized by varying degrees of fibrin deposition on the pulmonary pleural surface and influx of neutrophils into the pleura. Pneumonia was characterized by varying degrees of multifocal, perivascular necrotizing pneumonia, pulmonary hemorrhage, and edema. Pneumonia was observed in 4 out of 5 rabbits in the group immunized with IPMT plus CT and in all rabbits in the other treatment groups. Scores for severity of pleuritis and pneumonia were significantly less (p≤0.05) in rabbits immunized with IPMT, with or without CT, compared to rabbit administered only PBS or CT. All rabbits developed atrophy of the nasal turbinates. The differences in scores for severity of nasal turbinate atrophy were not significantly different between treatment groups.
Table I. Scores for histopathologic change were assigned by a pathologist blinded to treatment group.
Scores of 1 to 5 were assigned (0=no change, 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe) based on degree of change. *±Standard deviation. PMT: Pasteurella multocida heat-labile toxin; IPMT: heat-inactivated Pasteurella multocida heat-labile toxin; PBS: phosphate buffered saline; CT: cholera toxin.
Discussion
P. multocida possesses several virulence factors, some of which likely influence colonization of mucosal surfaces. For example, capsule type A isolates from rabbits produce fimbriae which mediate adhesion to pharyngeal cells (5). In swine, colonization of the nasal cavity with toxigenic P. multocida was enhanced in challenged pigs which were not immunized against PMT, suggesting that PMT is a colonization factor (34).
A variety of biologic activities have been demonstrated for PMT. In swine, the toxin produces atrophy of the nasal turbinate bones, likely from osteolytic activity (15,18,36-38) although a role for impaired osteoblastic activity has also been postulated (14,15,39). In rats, administration of PMT induces severe hepatic damage and testicular atrophy (31,40,41). Further, PMT is a powerful mitogen for Swiss 3T3 cells and Rat-1 fibroblasts at picomolar concentrations (42,43). The specific mechanism of PMT action is undefined, however several lines of evidence suggest a role for protein kinase C activation and subsequent hydrolysis of polyphosphoinositides (44-47).
In the present study, we investigated the possible role of PMT as a colonization factor for P. multocida in the rabbit nasopharynx and lungs. Immunization of rabbits with IPMT stimulated partial protective immunity against infection. Significantly fewer CFUs of P. multocida were cultured from the lungs of IPMT-immunized rabbits than from the lungs of saline- or CT-treated control rabbits. In contrast, no difference was found between treatment groups in the number of P. multocida CFUs cultured from nasopharyngeal swab samples. Further, immunization with IPMT, with or without CT, conferred protection against pneumonia and pleuritis. Studies conducted in vitro have demonstrated the ability of PMT to enhance adhesion of P. multocida to lung tissue but not tracheal tissue in rabbits (35). Our results extend that finding to in vivo conditions.
Intranasal inoculation of rabbits with purified PMT results in necrotizing perivascular pneumonia, hepatic necrosis and splenic atrophy (13). These systemic effects suggest that the toxin can be readily absorbed into the blood stream of rabbits. The heat-labile toxin of enterotoxigenic Escherichia coli and the streptolysin S toxin of Streptococcus pyogenes are neutralized more effectively when produced by nonadherent bacteria, while toxin produced by bacteria adherent to cells appears to be relatively inaccessible to neutralizing toxin inhibitors (48). Possibly, in a similar fashion adherent P. multocida organisms in the nasopharynx are unaffected, or less affected, by neutralizing anti-PMT antibody, while bloodborne PMT that might prime the lung for P. multocida adherence is inactivated by circulating and local antibody.
PMT is an immunogenic protein, and immunization of rabbits with IPMT stimulates protective immunity to pneumonia, pleuritis, hepatic necrosis and fibrosis, splenic atrophy and testicular atrophy following challenge with PMT (32). A significant increase in serum IgG directed against PMT was associated with a reduction in the number of CFUs cultured from the lungs and in severity of pleuritis and pneumonia. This association is consistent with the suggestion that circulating PMT influences adherence of P. multocida in the lungs.
Conclusion
Immunization of rabbits with inactivated heat-labile toxin of Pasteurella multocida conferred protection against challenge with PMT-producing organisms. Importantly, fewer P. multocida CFUs were found in the lungs of immunized rabbits compared to controls. In contrast, there was no difference in the number of CFUs found in the nasopharynx between groups of rabbits. These results suggest that PMT influences adherence of P. multocida to the lungs of rabbits and may, through an undetermined mechanism of action, represent a colonization factor.
Conflicts of Interest
The Author has no conflicts of interest with respect to the contents of this manuscript.
Funding
The work described herein was internally funded by the institution.
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