Immunization with bacterial antigens: pasteurellosis.

Pasteurella piscicida is the aetiological agent of pasteurellosis or pseudotuberculosis, one of the most threatening diseases of wild and cultured marine fish. This bacterium has been reported from many geographical areas including USA, Japan, and the Mediterranean countries. In this review, the biochemical, serological, and molecular characteristics of the pathogen are described. In addition, its main virulence mechanisms, such as the presence of capsule, the iron uptake system, and the phospholipase activity, as well as their putative role in the pathogenicity of P. piscicida are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, with a special emphasis on the vaccination programmes and the most effective protective antigens to be included in the vaccine formulations.

Influence of the capsular layer on the virulence of Pasteurella piscicida for fish.

The influence of the capsule of Pasteurella piscicida on the cell surface properties of this microorganism as well as on the virulence and the capacity of the strains to grow in fish sera was examined. Although all the P. piscicida strains synthetized an additional exostructure in glucose-enriched-medium, only virulent strains constitutively synthetized capsule. The cell surface of all the P. piscicida isolates showed a low hydrophobic nature. No strains pelleted in broth culture (SP-) and all of them were stable after boiling (PAB). All isolates attached to the fish cell line CHSE-214 with values of adherence ranging from 2 to 5% of the initial bacterial inoculum. The presence of induced capsular material caused changes in some cell surface characteristics such as hydrophobicity and stability after boiling. A decrease in the adherence capacity of all the P. piscicida strains was also observed. However, the capsule increased the degree of virulence for fish of the nonpathogenic strains (LD50 was reduced in about 4 log) and conferred to all the isolates resistance to serum killing. Therefore, these results indicate that the presence of capsule can play an important role in the pathogenesis of P. piscicida.

Hemagglutination by Pasteurellaceae isolated from rodents.

Pasteurellaceae notably P. pneumotropica, have been associated with severe outbreaks of respiratory disease in several species of rodents. Host-specific parasitism of Pasteurellaceae in rodents has hardly been studied. Since host tropism in many bacteria involves adhesive mechanisms, we examined the hemagglutinating (HA) properties of 44 isolates from different rodent species (mouse (15) rat (8), hamster (9), gerbil (10) and Mastomys (2)). Only 13 mouse isolates and the 2 Mastomys isolates hemagglutinated human (type O Rh+) and canine red blood cells (RBCs). No HA was found using RBCs from 10 other animal species. HA was not inhibited by simple sugars and glycoconjugates, but was completely inhibited by heating of bacterial cells for 10 min at 80 or 100 degrees C, partially inhibited by glutaraldehyde and inhibited in a dose-dependent mode by NaIO4, suggesting the involvement of bacterial polysaccharide structures in the HA process. Enrichment procedures did not reveal the presence of HA- subpopulations in HA+ isolates or the presence of HA+ subpopulations in HA- isolates. Electron microscopy revealed the presence of fimbriae both in HA+ and HA- isolates. A regularly structured (RS) layer was detected on cells of part of the HA+ isolates only. Our results suggest that Pasteurellaceae of mice and Mastomys may be related and differ from isolates isolated from other rodent species.

Evidence of phenotypic dichotomy within an individual Pasteurella multocida type strain and among some haemorrhagic septicaemia-causing field isolates.

Haemorrhagic septicaemia-causing strains of Pasteurella multocida were identified by a disease-specific ELISA. Some strains, however, were of the same serotype as those which cause haemorrhagic septicaemia (HS) but were negative when tested in the disease specific ELISA. The suspect false negative isolates were passaged in mice and retested in the HS ELISA with the same result. Immunoelectron microscopy was used to examine further these suspect HS-causing strains. Monoclonal antibodies and protein A-gold showed that the suspect negative organisms were a mixture of phenotypes with less than 10 per cent, and usually less than 2 per cent, of the population expressing HS-associated epitopes. The degree of staining on the organisms expressing the HS-epitopes was of the same intensity as the positive control organism. Expression of the HS-associated epitopes is presumably too low to allow detection in the current HS ELISA.

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain.

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Adherence of Pasteurella multocida isolated from pigs and relationship with capsular type and dermonecrotic toxin production.

Pasteurella multocida can often be isolated from pneumonic lungs in pigs. There is little information about the pathogenesis of this infection. Attachment of microorganisms to eucaryotic cells is considered to be a prerequisite for colonization of the host in the pathogenesis of bacterial infections. Forty-seven P multocida strains isolated from pigs in France, and belonging to capsular type A or D were tested for their ability to agglutinate human erythrocytes, and to adhere to tracheal and lung cells. Each isolate was tested for dermonecrotic toxin production. Adherent strains were further observed by electron microscopy to look for attachment structure. Only type A strains agglutinated human O erythrocytes, but no relationship was observed between hemagglutination and dermonecrotic toxin production. The results of the adherence tests showed a greater affinity (P less than 0.05) of type A strains for lung cells (50% were adherent, whereas only 20% of type D strains were adherent) but did not reveal any correlation between adherence and the presence of dermonecrotic toxin. Microscope observations showed that these P multocida strains did not possess any pili-like structures. In conclusion, by means of the adherence test we were able to demonstrate a stronger adherence of type A strains and this adherence did not seem to be related to pili-like structures.

[Pasteurella multocida (type D and A) and atrophic rhinitis of swine--hemagglutination, fimbriae and adhesion in vitro in the nasal mucosa of swine fetuses]. / Pasteurella multocida (Typ D und A) und Rhinitis atrophicans des Schweines--Hämagglutination, Fimbrien sowie Adhäsion in vitro an Nasenschleimhaut von Schweinefeten.

79 strains of P. multocida were investigated, mostly isolated from porcine nasal cavities, and a mannose-resistant hemagglutination with guinea pig and human group 0, but not with porcine erythrocytes was found. Fimbriae as adhesins were demonstrated only on 2 strains. A correlation between capsular type, hemagglutination, fimbriation and toxigenicity on the examined P. multocida strains was not observed. Three strains were investigated for the adherence to the nasal mucosa of neonatal pigs; aggregates shown by scanning electron microscopy indicate colonization; a correlation of adherence with surface proteins and slime production of P. multocida is discussed.

[Electron microscopic studies of mucosal extirpates of rabbits after after in vitro contact with Pasteurella multocida subsp. multocida strains]. / Elektronenmikroskopische Untersuchungen an Schleimhautexstirpaten von Kaninchen nach in vitro-Kontakt mit Pasteurella multocida subsp. multocida-Stämmen.

In vitro experiments of adhesion and colonisation of mucosal surface fragments (nose, trachea) from freshly killed rabbits with four strains of Pasteurella multocida subsp. multocida with different types of capsule antigen (A, D, without capsule) showed the following results: Above all the capsule serovar A strain, producing a capsule of hyaluronic acid and fimbria, were able to adhere and to form microcolonies on the mucosal surface. Microvilli of epithelial cells and mucus producing cells were recognized as the place of adhesion. The formation of microcolonies occurred with a destruction of the kinocilia, which was caused by a bacteria free culture filtrate as well.

Bacterial adhesion to mucosal surfaces with special reference to Pasteurella multocida isolates from atrophic rhinitis.

Adhesion to mucosal cells is an important virulence attribute of bacterial pathogens colonizing these sites. Bacteria of the upper respiratory system, such as members of the genus Bordetella, have well-defined adhesins. The main adhesin of B. pertussis is the filamentous hemagglutinin which can be used by other bacteria for attachment. The main adhesin of B. bronchiseptica is the bovine erythrocyte hemagglutinin. In both Bordetella species the presence of fimbriae does not appear critical to adhesion. In contrast, atrophic rhinitis (AR)-producing strains of Pasteurella multocida colonize poorly the pig's nasal mucosa. We performed an in vitro trial using newborn pigs' turbinate explants and showed that two toxigenic strains (serotype D fimbria + and serotype A fimbria -) were adherent when observed by scanning electron microscopy (SEM). Intranasal inoculation of both six week old and newborn SPF pigs with various strains of P. multocida also resulted in colonization. Adhesion was best achieved by toxigenic strains, regardless of possession of fimbria, hemagglutinin or capsular serotype. Colonization was more abundant and constant in tonsils. Nasal colonization was sporadic and sparse. Colonization of trachea and lung was only observed with serotype A strains. The results showed that toxigenic P. multocida can colonize the upper respiratory tract, especially the tonsils, of pigs.

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059.

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.

Presence of bacterial glycocalyx and fimbriae on Pasteurella haemolytica in feedlot cattle with pneumonic pasteurellosis.

This investigation was conducted to determine if Pasteurella haemolytica within feedlot cattle affected by pneumonic pasteurellosis express fimbriae (pili) and bacterial glycocalyx. Bacteriological culture of pulmonary tissue from three calves with fibrinous pneumonia resulted in heavy growth of P. haemolytica. Transmission electron microscopy of the lungs showed numerous microcolonies of gram-negative bacteria with morphology typical of Pasteurella haemolytica. The cells within these microcolonies possessed bacterial glycocalyces which stained with ruthenium red. Glycocalyx-encased microcolonies were also present in specimens examined by scanning electron microscopy. Typical P. haemolytica cells were evident in a tracheal specimen and these bacteria had radial glycocalyces consistent with polysaccharide and proteinaceous material condensed on linear structures suggestive of fimbriae. The pathogenetic importance of the bacterial glycocalyx and fimbriae in shipping fever pneumonia has yet to be established but their presence in clinical cases of Pasteurella pneumonia in feedlot cattle further supports a possible role in the initiation and progression of this disease as well as bacterial resistance to antimicrobial agents.

Distribution of a monoclonal antibody-recognized protective protein immunogen on the outer membranes of Pasteurella multocida rabbit isolates.

The distribution of a monoclonal antibody (MAb)-recognized protective protein immunogen on the outer membrane of 153 Pasteurella multocida rabbit isolates was determined by dot blot (DB) analysis. MAb 1608 reacted with 36 (24%) of the 153 clinical isolates. The DB-positive clinical isolates expressed capsular antigens A, D, and nontypable and somatic antigens 2, 3, 10, 12, 15, and nontypable. Western blot (immunoblot) analysis with adsorbed and eluted MAb 1608 confirmed that the antigenic determinant identified was located on the cell surface. With MAb 1608 as a probe for antibody-accessible radioimmunoassay, 31 of 36 DB-positive P. multocida rabbit isolates were shown to have surface-exposed and antibody-accessible antigenic determinants, while 44 of 44 DB-negative isolates were negative by antibody-accessible radioimmunoassay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed DB-negative P. multocida isolates both with (6 of 13, 46%) and without (7 of 13, 54%) the 37.5-kilodalton protein. This study establishes that the protective antigenic determinant of the 37.5-kilodalton outer membrane protein is present in 24% of rabbit clinical isolates tested and is detectable in P. multocida strains distributed among the major somatic types (3, 10, 12, and 15) and the capsular types (A and D) commonly isolated from rabbits in North America.

Electron microscopic examination of cells of Pasteurella haemolytica-A1 in experimentally infected cattle.

Several modern electron microscopy techniques were used to examine Pasteurella haemolytica (biotype A, serotype 1) (strain B122) recovered from experimentally infected cattle and in situ within the lung tissue of experimentally infected cattle. Glycocalyx four to five times thicker than that seen on P. haemolytica grown in vitro was evident on bacterial cells recovered from live infected calves by pulmonary lavage. Fimbriae were also present on cells recovered by lavage. A thick glycocalyx was also seen on P. haemolytica-A1 within the lungs of experimentally infected cattle at necropsy. In summary, cells of P. haemolytica-A1 in experimentally infected cattle have fimbriae and glycocalyx on their cell surfaces and these structures appear to be important in bacterial colonization of the bovine respiratory tract and pathogenesis of shipping fever (Pasteurella) pneumonia.

Expression of pili and capsule by the avian strain P-1059 of Pasteurella multocida.

The avian strain P-1059 of Pasteurella multocida was grown on blood agar (BA), on dextrose-starch agar (DSA), or in Heddleston's hydrogen sulfide test broth. Cells were examined for the presence of pili using electron microscopy after staining with phosphotungstic acid, and they were examined for capsule after ruthenium red staining. Pili were found on the capsulated iridescent type, P-1059I, and on two non-capsulated variants, the blue, P-1059B, and the gray, P-1059G. Many cells grown on BA were heavily piliated. In contrast, fewer cells grown on DSA had pili, and piliation was only slight to moderate. The P-1059I, P-1059B, and P-1059G produced pellicles when grown on broth medium. Pili were found on the circumference of the cells grown on either agar or broth medium. Occasionally a pilus connecting two cells was seen on cells cultured in broth. Cultivation of the P-1059I on DSA containing the iron-chelating agent alpha,alpha'-bipyridyl produced a non-capsulated blue variant. The non-capsulated variant reverted to P-1059I when grown on BA but did not revert when grown on DSA.

Purification of fimbriae from Pasteurella haemolytica A-1.

Pasteurella haemolytica A-1 produces large, rigid fimbriae with a diameter of 12 nm and a denisty of 1.32. Their subunit molecular weight was 35,000 as determined by SDS-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies raised against native fimbriae. The isoelectric point of the purified fimbriae was 4.8. While Pasteurella haemolytica whole cells plus a crude shear fraction were capable of agglutinating bovine erythrocytes, purified fimbriae exhibited no such activity.

Adherence of Pasteurella multocida or Bordetella bronchiseptica to the swine nasal epithelial cell in vitro.

The interaction of Bordetella bronchiseptica or Pasteurella multocida with swine nasal epithelial cells was studied in vitro. The mean number of B. bronchiseptica organisms adhered per cell was about three times as high as that of P. multocida (P less than 0.01), and the adherence was specifically inhibited by the homologous antiserum prepared with the whole-cell antigen of each bacterium. The poor affinity of P. multocida to the swine nasal mucosa as compared with that of B. bronchiseptica was also demonstrated in the cultured fragments of the nasal mucosa. When observed with a scanning electron microscope, B. bronchiseptica organisms colonized the fragments, whereas few P. multocida organisms adhered. Morphologically, the P. multocida-infected fragments had an essentially normal structure, whereas marked degeneration and marked desquamation of the epithelial cells and severe inflammatory reactions were observed in many areas of the B. bronchiseptica-infected fragments. These morphological observations were consistent with those for the nasal mucosa of P. multocida- or B. bronchiseptica-infected neonatal pigs (T. Nakai, K. Kume, H. Yoshikawa, T. Oyamada, and T. Yoshikawa, Jpn. J. Vet. Sci. 48:693-701, 1986; T. Oyamada, T. Yoshikawa, H. Yoshikawa, M. Shimizu, T. Nakai, and K. Kume, Jpn. J. Vet. Sci. 48:377-387, 1986). Cultured swine nasal fragments, however, were equally injured when they were incubated in a medium containing purified dermonecrotic toxin (DNT) preparations of B. bronchiseptica or P. multocida. Therefore, these DNT preparations can induce morphological damage closely resembling that induced in vivo. Hence, colonization of B. bronchiseptica and production of its DNT on the swine nasal mucosa appear to result in the production of mucosal damage. On the other hand, P. multocida seems to lack the ability to colonize normal swine nasal mucosa, thus resulting in no production or the slight production of DNT to such an extent as to produce mucosal damage. The present data support our previous hypothesis (Nakai et al.; Oyamada et al.) that B. bronchiseptica induces swine atrophic rhinitis, whereas P. multocida does not.

Electron microscopic visualization of capsular material of Pasteurella multocida types A and D labeled with polycationic ferritin.

The presence of capsular material on cells of Pasteurella multocida types A and D was determined by transmission electron microscopy after polycationic ferritin labeling. The capsule of agar-grown isolates of P. multocida type A was thick (70 to 90 nm) and regular, whereas that of type D isolates was thinner (20 to 30 nm) and irregular. Such layers were seen on cells from 4- to 6-h broth cultures, but cells from older cultures (12 to 18 h) had very little cell-associated capsular material. Our data indicate that the capsular material of P. multocida types A and D is morphologically different and that capsule production in broth culture is maximal during early log phase.

Ingestion and killing of Pasteurella haemolytica A1 by bovine neutrophils in vitro.

In this study, various parameters affecting the ability of bovine neutrophils to ingest and kill a virulent strain of Pasteurella haemolytica A1 in vitro were examined. Ingestion of P. haemolytica was serum dependent (optimal serum concentration 10%) and was mediated principally by heat-stable opsonins, presumably antibodies, that could be removed by absorption with formalin-killed P. haemolytica. Ingested P. haemolytica were killed by neutrophils within 1-4 h incubation; the magnitude of killing being directly dependent on the number of neutrophils present. The number of viable P. haemolytica was reduced by approximately 1.5 log at bacterial concentrations of 0.01-100 P. haemolytica per neutrophil; a concomitant reduction in neutrophil viability was observed at the highest bacterial concentration (100:1). Bovine neutrophils underwent a vigorous luminol-enhanced chemiluminescence response after ingesting opsonized P. haemolytica, thus indicating that reactive oxygen intermediates were being formed that could have contributed to the intracellular killing of P. haemolytica.

Variability of cell surface hydrophobicity among Pasteurella multocida somatic serotype and Actinobacillus lignieresii strains.

Pasteurella multocida possesses a characteristically gram-negative ultrastructure, yet its inability to grow in the presence of hydrophobic compounds and the general penicillin susceptibility of genera making up the family Pasteurellaceae suggest a cell envelope having atypical permeability properties. The cell surface hydrophobicity properties of strains representing 15 of the 16 somatic serotypes of P. multocida and three strains of Actinobacillus lignieresii were assessed with hydrocarbon adherence and hydrophobic interaction chromatographic assays. These methods revealed surface hydrophobicity to vary dramatically among strains in both species. No direct correlation was observed with species, growth rate, or susceptibility to the antibiotics oxytetracycline (polar), polymyxin B (amphiphilic), or novobiocin (nonpolar) as measured with MIC determinations. All strains were susceptible to the antibiotics, although A. lignieresii was significantly less susceptible than P. multocida to novobiocin. These data suggest that cell surface hydrophobicity in P. multocida may be influenced by the type of lipopolysaccharide present but is not directly related to permeability of the antibiotics examined. The wide diversity of hydrophobic properties exhibited by strains of both P. multocida and A. lignieresii precludes the use of this parameter as a taxonomic acid.

Electron microscopic description of glycocalyx and fimbriae on the surface of Pasteurella haemolytica-A1.

Several electron microscopic techniques were used to examine the surface of cells of Pasteurella haemolytica (biotype A, serotype 1) grown in vitro. All methods showed the presence of a very extensive glycocalyx on logarithmic phase (6 h) cells grown in liquid media. The anionic glycocalyx of these cells stained well with ruthenium red, but collapsed during dehydration for electron microscopy unless stabilized with specific antibodies. When the same techniques were used to examine cells in the stationary phase (18 h) the glycocalyx was much reduced. Large numbers of fimbriae were seen on both 6 h and 18 h cells grown in fluid media without shaking. In summary, logarithmic phase cells of P. haemolytica have both fimbriae and extensive anionic glycocalyx at their surface and we suggest that either or both of these structures may be important in the colonization of the bovine respiratory tract and the subsequent pathogenesis of Pasteurella pneumonia.