Serum sensitivity and lipopolysaccharide characteristics in Bordetella bronchiseptica, B. pertussis and B. parapertussis

The viability of four strains of Bordetella bronchiseptica, two strains of B. pertussis and one strain of B. parapertussis exposed to hyperimmune and pre-colostrum porcine serum was examined. Viable cell numbers (cfu/ml) of the B. pertussis strains and a rough strain of B. bronchiseptica (CSU-P-1) decreased by 99% and 99.99%, respectively, after exposure for 1 h to porcine hyperimmune serum. In contrast, smooth B. bronchiseptica strains and the B. parapertussis strain showed no significant decrease in viable cell numbers after the same treatment. B. bronchiseptica strain CSU-P-1 also showed a 99% decrease in viable cell numbers after exposure to pre-colostrum porcine serum for 1 h whereas the other strains tested showed no decrease in viable numbers under the same conditions. Heating the hyperimmune and pre-colostrum serum at 56 degrees C for 30 min resulted in the loss of bactericidal activity suggesting the involvement of complement in both systems. Analysis of silver-stained SDS-PAGE profiles of lipopolysaccharide (LPS) extracted from the bacterial cells indicated that the smooth strains of B. bronchiseptica and the B. parapertussis strain possessed high mol. wt O-side chain-like material, whereas the B. pertussis strains and B. bronchiseptica strain CSU-P-1 did not. Gel filtration of acid-hydrolysed LPS samples indicated two distinct carbohydrate peaks for the strains with high mol. wt O-side chain-like material, whereas the other strains each yielded one distinct peak. Western-blot analysis indicated a positive reaction for anti-B. bronchiseptica antibodies to the high mol. wt O-side chain-like material of all serum-resistant strains used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the dermonecrotic toxin in members of the genus Bordetella.

All members of the genus Bordetella and Pasteurella multocida (a gram-negative bacillus genetically unrelated to Bordetella spp., yet often sharing the same ecological niche) produce a dermonecrotic toxin (DNT). The amount of toxin produced and the time required for appearance of the lesions are identical for Bordetella pertussis, B. parapertussis, and B. bronchiseptica but different for P. multocida and B. avium. DNT has been reported to act by promoting vasoconstriction; however, vasoactive compounds (verapamil, prazosin, hydralazine, tolazoline, or isoxsuprine) are able to reverse the action of the toxin only slightly. Vasoconstrictors (atropine, serotonin, epinephrine, or endothelin) did not produce DNT-like lesions. We have characterized a region of DNA essential for DNT expression. We have determined by Southern analysis that the restriction map of the DNT gene is nearly identical in B. pertussis, B. parapertussis, and B. bronchiseptica, but the sequences are not present in toxigenic B. avium and P. multocida strains. A gentamicin resistance-origin of transfer cassette cloned into a 1.8-kb NotI-BamHI fragment results in constructs which can be mobilized and recombined into the Bordetella chromosome, rendering the resultant B. pertussis, B. parapertussis, and B. bronchiseptica strains negative for DNT. A 5-kb BamHI-ApaI fragment from the B. pertussis chromosome was sequenced and revealed homology to the Escherichia coli CNF1 (cytotoxic necrotizing factor 1) toxin.     

Adherence of Bordetella bronchiseptica and Pasteurella multocida to swine nasal ciliated epithelial cells in vitro

A bacterial adherence assay using swine nasal turbinate fragments was established. Turbinate fragments were incubated with Bordetella bronchiseptica or Pasteurella multocida type D at different concentrations or for different incubation times at 37 degrees C on a shaker at 120 rev/min. B. bronchiseptica phase I strains exhibited strong adherence to swine nasal ciliated epithelial cells. The number of adherent bacteria per cell increased when the bacterial concentration or incubation time increased (0, 15, 30, and 60 min); however, the number of adherent bacteria decreased after 3 or 6 hours' incubation due to the loss of cilia from cells. The optimal bacterial concentration and incubation time were 1 x 10(9) organisms/ml and one hour respectively, which resulted in 7.48 +/- 0.66 (Mean +/- SEM; B. bronchiseptica strain 03) and 9.31 +/- 0.54 (B. bronchiseptica strain 013) adherent bacteria per cell. In contrast to B. bronchiseptica phase I strains, rough phase strains of B. bronchiseptica and all P. multocida strains tested showed no adherence to swine nasal ciliated epithelial cells. All B. bronchiseptica phase I strains could agglutinate calf RBC but rough phase strains could not. Furthermore, pretreatment of B. bronchiseptica phase I organisms with 1 mg/ml or 2 mg/ml of trypsin significantly inhibited the adherence of B. bronchiseptica to ciliated epithelial cells; however, trypsin (2 mg/ml) treatment of bacteria did not decrease their ability to agglutinate calf RBC. From these results we conclude that, in addition to hemagglutinin, other proteinaceous components exist on the surface of virulent B. bronchiseptica that are sensitive to 2 mg/ml trypsin; these are suggested to be the adhesins for the adherence of B. bronchiseptica to swine nasal ciliated epithelial cells.     

Virulence of Bordetella bronchiseptica in the porcine respiratory tract

The virulence of Bordetella bronchiseptica in gnotobiotic piglets was studied by intranasal infection with 11 cultures derived from eight strains isolated from pigs (4), dogs (2), a human subject and a monkey. Six of the cultures contained organisms in phase I and five contained phenotypically different phase-III or -IV organisms. Of the phase-III and -IV cultures, four were derived from strains that had been isolated in phase I. Colonisation of the nasal cavity was investigated by counting bacteria in nasal swabs and washings. The toxigenicity of cell extracts from each strain and variant was determined by tests of lethality in mice or of cytopathogenicity in cell cultures. The results showed that two phase-I cultures from pigs colonised the nasal cavity and respiratory tract of gnotobiotic piglets better than did four phase-I cultures from other species. Phase-I organisms invariably produced capsules, fimbriae and mannose-resistant haemagglutination of guinea-pig erythrocytes. Four of five cultures in phases III and IV consisted of organisms that did not produce capsules, fimbriae or haemagglutination and colonised the nasal cavity poorly. Phase variation from I to III occurred in culture and in vivo, but variation from III to I occurred in vivo only and was accompanied by enhanced colonisation. Gnotobiotic piglets infected with porcine phase-I organisms exhibited atrophy of the nasal turbinate bones after 28 days; these organisms produced significantly more toxin than did bacteria in phase I from other species, or those in phases III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the Dermonecrotic Toxin of Bordetella bronchiseptica in the Pathogenesis of Respiratory Disease in Swine

Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT(+) parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.     

Bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein A

In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg- phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg- strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.     

Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different.     

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.     

Effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of osteoblastic clone MC3T3-e1 cells.

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.     

Stimulation of bone resorption by inflamed nasal mucosa, dermonecrotic toxin-containing conditioned medium from Pasteurella multocida, and purified dermonecrotic toxin from P. multocida.

The effects of inflamed nasal mucosa from pigs with atrophic rhinitis (AR), cell extract from Bordetella bronchiseptica, conditioned medium from Pasteurella multocida, and purified dermonecrotic toxin (DNT) from P. multocida on mouse fetal long bones in organ culture were studied. Inflamed nasal "AR mucosa" stimulated the release of 45Ca from prelabeled cultures, while histologically the formation of calcified matrix was impaired as well. B. bronchiseptica cell extract only transiently increased 45Ca release, but also impaired the formation of matrix. 45Ca release was also stimulated by DNT-containing conditioned medium from P. multocida and by purified DNT. The effect of DNT was biphasic: low doses (1 to 25 ng/ml) slightly stimulated bone resorption, higher doses were inhibitory. The stimulatory action of DNT on 45Ca release was accompanied by an increase in numbers of preosteoclasts and osteoclasts. The significance of these findings for the pathogenesis of AR is discussed.     

Comparison of isolation methods for the recovery of Bordetella bronchiseptica and Pasteurella multocida from the nasal cavities of piglets.

Nasal swabs from 241 piglets from 12 herds with clinical atrophic rhinitis and 283 piglets from 14 herds without clinical atrophic rhinitis were examined for the presence of Bordetella bronchiseptica and/or Pasteurella multocida. For B. bronchiseptica, swabs were streaked on three selective media. Blood agar supplemented with cephalexin was the most satisfactory selective culture medium for the isolation of B. bronchiseptica. For P. multocida, swabs were also streaked on three selective media. Mice were also used for isolation of P. multocida from the nasal cavities of pigs. The mouse inoculation test was not found to be the definitive test for the isolation of P. multocida. A significant number of P. multocida strains were avirulent in the mouse model. The modified Knight medium (without potassium tellurite) was the best single method for isolating P. multocida. However, a combination of mouse passage and direct culture on selective media increased the rate of isolation. There was no marked difference in the prevalence of B. bronchiseptica or P. multocida in swine herds with or without clinical atrophic rhinitis. Both capsular types A and D were present in the nasal cavities of the pigs with or without clinical atrophic rhinitis.     

Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp.

A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation.     

Nucleotide sequence homology to pertussis toxin gene in Bordetella bronchiseptica and Bordetella parapertussis.

Multiple strains of Bordetella parapertussis and B. bronchiseptica were examined for the presence of nucleotide sequences which hybridized with a cloned 4.5-kilobase (kb) fragment of B. pertussis DNA containing the genes responsible for pertussis toxin expression. All six B. parapertussis strains tested had nucleic acid sequences that hybridized with the cloned 4.5-kb fragment in Southern blot analyses. When the B. parapertussis DNA was digested with restriction endonuclease PstI, the pattern of hybridization was identical to that obtained with B. pertussis. Only five of the seven B. bronchiseptica strains tested had sequences that hybridized with the 4.5-kb fragment. Three of these B. bronchiseptica strains had a hybridization pattern identical to B. pertussis upon PstI digestion and Southern blot analyses. Two B. bronchiseptica strains were shown to lack a PstI cleavage site downstream from the region analogous to that coding for the pertussis toxin structural genes. Monoclonal antibody analyses were unable to detect pertussis toxin subunits S1 and S2 in Western blots with cellular material or culture supernatant from several B. bronchiseptica and B. parapertussis strains that possessed the DNA homologies. In addition, preliminary Northern hybridizations with RNA isolated from B. bronchiseptica and B. parapertussis strains suggested that the homologous regions were not transcribed. The data show that the gene coding for the toxic component of B. pertussis is common in other Bordetella species, though the gene probably is not expressed.     

Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella avium.

We examined Bordetella avium for virulence factors common to Bordetella pertussis, including pertussis toxin, filamentous hemagglutinin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin. B. avium produced a dermonecrotic toxin and a tracheal cytotoxin. The dermonecrotic toxin of B. avium is a 155,000-molecular-weight, heat-labile protein which was lethal for mice, guinea pigs, young chickens, and turkey poults and produced dermonecrosis when injected intradermally into guinea pigs, chickens, and turkey poults. High-pressure liquid chromatography of B. avium culture supernatant fluid revealed the presence of a tracheal cytotoxin chemically identical to that produced by B. pertussis. B. avium isolates were negative for B. pertussis-like filamentous hemagglutinin and pertussis toxin when assayed with antibody against B. pertussis filamentous hemagglutinin and pertussis toxin. Furthermore, B. avium failed to induce the clustered CHO cell morphology characteristic of pertussis toxin. Adenylate cyclase assays indicated that B. avium does not produce an extracytoplasmic adenylate cyclase, even after passage through embryonated turkey eggs. Since production of virulence proteins by B. pertussis is regulated by growth in media containing nicotinamide or MgSO4 or by growth at reduced temperatures, we determined the effect of these supplements and growth conditions on production of dermonecrotic toxin by B. avium. Production of dermonecrotic toxin in B. avium was not altered by growth in media containing 100 microM FeSO4 or 500 micrograms of nicotinamide per ml or by growth at 25 or 42 degrees C, but production was significantly decreased by growth in media containing 20 mM MgSO4 and slightly reduced by growth in media containing 500 micrograms of nicotinic acid per ml. These studies revealed that B. avium is similar to B. pertussis in that both species produce a dermonecrotic toxin and a tracheal cytotoxin and production of dermonecrotic toxin is regulated by nicotinamide and MgSO4. The presence of dermonecrotic toxin and tracheal cytotoxin in all Bordetella species indicates that these products may be important virulence factors in bordetellosis.     

Analysis of proteins encoded by the ptx and ptl genes of Bordetella bronchiseptica and Bordetella parapertussis.

Bordetella pertussis is the only bacteria] species which is known to produce pertussis toxin (PT); however, both Bordetella bronchiseptica and Bordetella parapertussis contain regions homologous to the ptx genes of B. pertussis that encode the toxin subunits. After finding that several children with B. parapertussis infections exhibited modest antibody titers to PT, we examined the ptx genes of both B. parapertussis and B. bronchiseptica to determine whether they would encode stable, functional proteins even though their promoters are thought to be inactive under the conditions that have been examined. We inserted a functional promoter directly upstream of the ptx-ptl region of both species and examined culture supernatants of the resulting strains for PT activity. Biologically active PT was found in the culture supernatants of both engineered species. The toxin encoded by the B. parapertussis ptx genes appeared more labile in culture supernatants than did toxin produced by either B. pertussis or the engineered strain of B. bronchiseptica. This lability might be due to the lack of a full-length S2 subunit. We also investigated the ptl genes of these species, which are necessary for the secretion of this toxin, and found that both B. bronchiseptica and B. parapertussis contain at least certain of these genes, including ptlE and ptlF. Moreover, B. bronchiseptica appeared to contain all essential ptl genes since the introduction of a functional promoter directly upstream of the ptx-ptl region resulted in both production and efficient secretion of toxin. These results indicate that despite a number of amino acid changes in the sequences of the toxins, the toxins encoded by B. bronchiseptica and B. parapertussis are active.     

Molecular characterization of two Bordetella bronchiseptica strains isolated from children with coughs.

During a surveillance program associated with the Italian clinical trial for the evaluation of new acellular pertussis vaccines, two bacterial isolates were obtained in cultures of samples from immunocompetent infants who had episodes of cough. Both clinical isolates were identified as Bordetella bronchiseptica by biochemical criteria, although both strains agglutinated with antisera specific for Bordetella parapertussis, suggesting that the strains exhibited some characteristics of both B. bronchiseptica and B. parapertussis. Both children from whom these strains were isolated exhibited an increase in serum antibody titer to pertussis toxin (PT), a protein that is produced by Bordetella pertussis but that is not thought to be produced by B. bronchiseptica. We therefore examined whether the clinical isolates were capable of producing PT. Neither strain produced PT under laboratory conditions, although both strains appeared to contain a portion of the ptx region that encodes the structural subunits of PT. In order to determine whether the ptx genes may encode functional proteins, we inserted an active promoter directly upstream of the ptx region of one of these strains. Biologically active PT was produced, suggesting that this strain contains the genetic information necessary to encode an active PT molecule. Sequence analysis of the ptx promoter region of both strains indicated that, while they shared homology with the B. bronchiseptica ATCC 4617 sequence, they contained certain sequence motifs that are characteristic of B. parapertussis and certain motifs that are characteristic of B. pertussis. Taken together, these findings suggest that variant strains of B. bronchiseptica exist and might be capable of causing significant illness in humans.     

Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids.

Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase.     

Reduced virulence of a Bordetella bronchiseptica siderophore mutant in neonatal swine

One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.     

Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in enzyme-linked immunosorbent assay.

The progenies of specific-pathogen-free sows which had been immunized with Bordetella bronchiseptica vaccines of various origin before parturition were challenged intranasally with B. bronchiseptica within 5 days of birth. Sera of piglets were taken weekly and investigated by enzyme-linked immunosorbent assay against a mixture of B. bronchiseptica cell surface antigens containing curled fibers and fimbriae, lipopolysaccharide, and a mixture of proteins mostly derived from the outer membrane. The serological response to this antigenic mixture was paradoxical; the highest titers were obtained with the least effective vaccines. Antibodies which did relate to protection were oriented against the outer-membrane-derived proteins, one of which, of 68,000 molecular weight, appeared to be particularly important for two reasons. First, its concentration within the antigenic mixture was dependent upon cultural conditions; of all the proteins present in virulent strains, it was the first to disappear upon modulation. Second, it was absent from a strain which was unable to induce atrophic rhinitis in specific-pathogen-free piglets. Although all vaccines tested had some beneficial effect on the various clinical manifestations of the disease, only two vaccines were effective (P less than 0.001) in the prevention of nasal pathological changes. These two vaccines also stimulated the highest titers against the 68,000-molecular-weight protein. A mouse protection test utilizing a lethal intraperitoneal challenge failed to monitor the efficacy of vaccines for protection against atrophic rhinitis.     

Purification and characterization of Bordetella bronchiseptica dermonecrotic toxin

Dermonecrotic toxin produced by Bordetella bronchiseptica was purified by chromatography on DEAE Toyopearl 650M and on Bio-Gel HTP, gel filtration on Sephadex G-200, and subsequent chromatography on Bio-Gel HTP and on SP Toyopearl 650M. The purified toxin was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance liquid chromatography. There was a 90-fold increase in the dermonecrotic titer per mg protein in guinea pigs and the recovery of activity was 17.6% of that of the original cell extract. The purified toxin is a single-chain protein with a molecular weight of 145,000 and an isolelectric point of 6.3-6.7. Its minimal necrotizing dose is approximately 0.4 ng. It was completely inactivated by heating for 20 min at 56 degrees C. It contained no endotoxin, carbohydrates, nucleic acids, or hemagglutinins.