The Disease

Pathological Changes

The dominant pathological lesion of atrophic rhinitis is an atrophy of the nasal turbinate bones as assessed by transverse section of the nasal cavity at the level of the first/second upper premolar teeth where the dorsal and ventral conchae are maximally developed in the normal pig (Fig. 1). The changes in the snout usually appear between 6 and 12 weeks of age of the pigs (181). In mild to moderate cases the ventral scrolls of the turbinates are the most commonly affected area; they vary from slightly shrunken to complete atrophy. In more severe cases, atrophy of the dorsal scrolls of the ventral turbinate and the dorsal and ethmoidal turbinates occurs. In the most severe form, all turbinate structures are completely absent. Lateral deviation of the nasal septum also is often observed. The very evident clinical signs of nasal atrophy have focused attention on this area. However, it is possible that pathology is also induced in other parts of the body, but has not been observed because it has not been looked for. For example, lesions have been found in bladder epithelium during experimental infections (95).

Figure 1

Cross-sections of snouts of 6-week-old pigs. (a) Uninfected pig showing normal anatomy of nasal structures. (b) Pig after infection with B. bronchiseptica and toxigenic P. multocida. The left dorsal turbinate is absent, and only a band of connective tissue (more...)

Virulence Determinants Expressed by B. bronchiseptica

Bacterial species of the genus Bordetella are respiratory pathogens. B. bronchiseptica is widely distributed in nature. Besides turbinate atrophy and bronchopneumonia in swine, it causes respiratory disorders in a variety of other mammals, including dogs and rabbits (66), and an increasing number of cases in humans have also been reported (234). B. pertussis is the etiologic agent of whooping cough, a respiratory disorder in humans, and is a close relative of B. bronchiseptica. The two bacteria have even been classified as a subspecies based on 23S RNA sequence comparison (150). Bordetellas express a variety of virulence factors (adhesins and toxins) that affect their interaction with the eukaryotic host and potentially with other bacteria. Intensive research has been conducted on the virulence determinants of B. pertussis and less on the other species. However, B. bronchiseptica shares most of its potential virulence factors with B. pertussis with the exception of pertussis toxin that is not expressed by B. bronchiseptica, so that research on B. pertussis has greatly helped us to understand B. bronchiseptica. For that reason some of what is described in this section refers to the properties of B. pertussis, but where possible we will describe the specific B. bronchiseptica virulence determinants.

Toxins

Bordetellas make several novel toxins. These include the tracheal cytotoxin and three protein toxins: adenylate cyclase toxin, dermonecrotic toxin (DNT), and, in the case of B. pertussis, pertussis toxin (PT).

Adenylate Cyclase

The adenylate cyclase was discovered almost by chance in a B. pertussis vaccine preparation (232). Its appearance in the culture medium of growing bacteria marked it out from other bacterial adenylate cyclases and suggested that it could be a virulence determinant (83). Subsequent work showed that a substantial amount of the adenylate cyclase activity was located extracytoplasmically in the bacteria (85). A further, initially surprising finding was that the Bordetella adenylate cyclase was stimulated by the eukaryotic calcium-binding protein calmodulin (CaM) (233). The enzyme was also found in B. bronchiseptica (48), and later it was shown to be calmodulin dependent (114).

Transposon inactivation of the adenylate cyclase gene showed that it was essential for virulence in the mouse model (228), where it appeared to play a key role in colonization (67, 107). The adenylate cyclase can enter some cell types to raise cyclic AMP levels, which was might lead to "phagocyte impotence" as suggested (31). The invasive adenylate cyclase was shown to be a large molecule (78), although various other values for its molecular mass were published. This confusion was resolved when the cya gene was cloned, sequenced (62), and subsequently analyzed (63). The N terminus of the protein was enzymatically active and bound CaM. (This was the part found in the smaller adenylate cyclases that had been isolated.) The C-terminal part had striking homology to Escherichia coli hemolysin, a member of the RTX family of bacterial protein toxins. RTX toxins are pore-forming toxins that characteristically have flanking genes involved in the modification of the hemolytic protein and its transport from the bacteria. Homologous accessory genes were found in Bordetella (63, 72). The domain of the adenylate cyclase that is homologous to RTX toxins provides the Bordetella hemolytic activity, but its main purpose appears to be to effect entry of the adenylate cyclase into host cells. The gene from B. bronchiseptica shows 98% identity to the B. pertussis gene (9).

The Bordetella adenylate cyclase is very active (233), and this probably explains its activation by CaM, which is a eukaryotic protein not found in bordetellas (233). The presence of such an active adenylate cyclase inside bordetellas might be detrimental to the regulation of gene function, although there appears to be no literature on Bordetella cyclic AMP-regulated genes, or might deplete ATP and be highly energy inefficient. The reliance on CaM ensures that the enzyme is only active where needed, within a eukaryotic cell. This calmodulin sensitivity is similar to the other known toxic adenylate cyclase, that of Bacillus anthracis (124). Cyclic AMP is an important signaling molecule in eukaryotes (146, 214) which is produced in response to extracellular signals arriving at the cell membrane. These stimulate receptors coupled to the heterotrimeric G proteins G_i and G_s, which regulate the activity of membrane-bound adenylate cyclase. Cyclic AMP acts by binding to regulatory subunits of protein kinase A to stimulate release of the active enzyme, which has multiple effects within the cell (Fig. 2).

Figure 2

Diagram of some of the signal transduction pathways affected by the dermonecrotic (DNT) and adenylate cyclase (Ad cyclase) toxins from B. bronchiseptica and by the P. multocida toxin (PMT). Targets modified by the toxins are shown by open arrows. Direct (more...)

The Bordetella adenylate cyclase has caused macrophage apoptosis both in vitro (105) and in vivo (68), and also affects monocytes by modulating the tumor necrosis factor α and superoxide response (157). The toxin blocks phagocytosis by neutrophils (225). Very recently, the adenylate cyclase induced apoptosis in dendritic cells (B. P. Mahon, personal communication). In addition it has been shown that the adenylate cyclase affects platelet function by inhibiting their ability to aggregate, and this has had a direct effect on bleeding time in vivo (98). Similar experiments have been conducted with the B. bronchiseptica adenylate cyclase (80). Mutants with a nonpolar deletion in cyaA were as efficient as wild-type bacteria in killing macrophages, and this was attributed to the existence of type III delivered virulence factors. When this system was also mutated, the B. bronchiseptica adenylate cyclase was found to have a significant effect on killing. There was a minimal effect on bacterial colonization, but a significant effect on neutrophil infiltration and pathological damage.

Antibodies to the adenylate cyclase are protective, and it has been suggested that adenylate cyclase could be a valuable vaccine component (69, 84).

Dermonecrotic Toxin

DNT, or heat-labile toxin, was the first virulence factor to be identified in Bordetella (12). Its dermonecrotic nature was determined following subcutaneous injection (127). However, B. pertussis strains with a mutated DNT had 50% lethal doses (LD₅₀s) for mice that were identical to those of wild-type strains (226), suggesting that DNT is not essential for virulence. Similar observations were made with a naturally acquired DNT-negative B. bronchiseptica strain of porcine origin in both intravenously (131) and intracerebrally (123) inoculated mice. Nevertheless, DNT is found in all the Bordetella species where it is highly conserved (222), and it is regulated along with most of the other virulence genes by the bvg system (156, 227). Recently, there has been renewed interest in the toxin, as its mechanism of action has been identified. In addition, it has been clear for some time that DNT from B. bronchiseptica has a role to play in atrophic rhinitis, as discussed later in this section.

DNT is a large intracellularly acting toxin that is taken up by target cells in a pH-dependent process (112). DNT acts on proteins of the Rho family (88, 92). These are small GTP-binding proteins that serve as molecular switches that interact with effector proteins when bound to GTP, but which contain an intrinsic GTPase and are inactive after hydrolysis of the GTP to GDP (174). They regulate many aspects of cellular function, including cytoskeletal organization and signaling pathways linked to gene expression and cell cycle progression. DNT appears to modify all members of the Rho family: Rho that is involved in stress fiber formation, Rac that regulates the appearance of membrane ruffling, and cdc42 that controls filopodia formation. DNT catalyzes either the deamidation (88) or transglutamination (137, 191) of glutamine 63 to inhibit the GTPase activity of the G protein. This results in its permanent activation, leading to the tyrosine phosphorylation and activation of the focal adhesion kinase (112), another key regulator of the cytoskeleton and intracellular signaling pathways. The equivalent modification induced by the E. coli cytotoxic necrotizing factor also leads via Rho to activation of the regulator of prostaglandin synthesis, COX-2 (216), and activation of the JNK via Rac or cdc42 (125), and it is likely that these effects will also occur with DNT. Since the Rho proteins have various effectors it is likely that cellular intoxication by DNT will lead to several sequelae (Fig. 2).

Cells treated with DNT display several morphological effects. Distinct membrane ruffling is observed, and the appearance of actin stress fibers and focal adhesions (92), linked to phosphorylation of the focal adhesion kinase (112). Quiescent resting cells are stimulated to enter the cell cycle and undergo DNA synthesis, but cytokinesis is blocked (89). All such effects can be attributed to activation of Rho proteins, since no evidence of activation of other signaling pathways exists (112).

Purification of DNT has proved to be difficult (238), and the field has been advanced most rapidly by the application of molecular biology. The gene sequence for DNT was first identified in B. pertussis (222), although expression of a recombinant DNT was first produced from B. bronchiseptica (170). The two proteins are practically identical. The start of the DNT sequence does not have a signal sequence, and there are no known accessory proteins that might aid secretion. Although it is possible that a novel mechanism for secretion exists, all available evidence suggests that DNT is only released on bacterial cell lysis (38, 153), and that DNT is released by dying bacterial cells to aid the remaining bacteria in an infection.

Intracellularly acting toxins often have two or three separate domains that mediate binding to the target cell, translocation of the enzymatically active part of the toxin across the membrane, and a catalytic domain (145). The only sequence homology to other proteins is in the C-terminal region of DNT to a similar region of cytotoxic necrotizing factor (222). This region is the catalytic site of both toxins, while the N-terminal regions of the proteins encode cell-binding and uptake functions (103, 122).

The role of DNT in bordetellosis remains elusive, in part, because its presence does not affect the LD₅₀ of bordetellas for mice (226). However, the mouse is an unsatisfactory model for whooping cough (106, 159). In addition, DNT is produced by all Bordetella species and its expression is regulated like other virulence determinants (227). It also has a potent and specific activity. It is likely that DNT plays a subtle role, perhaps affecting bacterial clearance or immunity both to Bordetella and other pathogens. The ability of DNT to suppress immune function might be expected for a toxin that interferes with the normal functions of Rho proteins. Such effects would facilitate the secondary infection that is usually found in Bordetella disease. In addition DNT has a direct effect on bone formation as is discussed later. Thus, most observers now believe that DNT has an important but undefined role in pathogenesis.

The importance of DNT in the pathogenesis of turbinate atrophy in pigs is more clearly established. This property of DNT was first suspected when Hanada et al. (76) showed that repeated intranasal inoculation of piglets with cell-free sonicated extracts from virulent B. bronchiseptica that contained high levels of DNT could produce nasal lesions similar to those seen in naturally occurring atrophic rhinitis. This view was supported by the comparison of a naturally acquired DNT− strain of porcine origin to a DNT+ strain also of porcine origin (131, 132). The mutant showed a similar production of adenylate cyclase-hemolysin and at least one adhesin (132). It exerted identical virulence in mice and colonized the nasal cavity of gnotobiotic pigs in a number comparable with the DNT+ strain, but only pigs infected with the latter strain developed turbinate atrophy. The slight possibility that other unknown differences between the strains could have been responsible for turbinate atrophy has been addressed by the use of genetically constructed isogenic DNT− strains. The parent and the mutant strains of B. bronchiseptica each possessed similar virulence for mice (134), but turbinate atrophy was only observed in pigs infected with the DNT+ strain, and not in those infected with the DNT− strain (S. L. Brockmeier, K. B. Register, T. Magyar, A. J. Lax, G. D. Pullinger, and R. A. Kunkle, submitted for publication). These data strongly suggest that DNT is required for the induction of turbinate atrophy in pigs that is linked to B. bronchiseptica infection.

Tracheal Cytotoxin

The production of tracheal cytotoxin is a common characteristic of Bordetella species (32, 64). Tracheal cytotoxin is a disaccharide-tetrapeptide derivative of the peptidoglycan layer of the cell wall that both structurally and functionally falls into the muramyl peptide family. Unlike most other gram-negative bacteria, B. pertussis releases a large amount of this glycopeptide into the culture supernatant, usually during log-phase growth (32). Exposure to purified tracheal cytotoxin specifically damages ciliated epithelial cells, causing ciliostasis and extrusion of these cells (33, 64, 65). It has been demonstrated in hamster trachea epithelial cells that the lactyl tetrapeptide portion of the molecule is responsible for its full toxic activity (130). Tracheal cytotoxin also has a toxic effect on other cells, impairing neutrophil function at low concentrations with toxicity found in larger quantities (40). The toxicity conferred by tracheal cytotoxin is caused by the induction of interleukin-1 in host cells (82), which activates host cell nitric oxide synthase leading to high levels of nitric oxide (NO) radicals (81). NO destroys iron-dependent enzymes, eventually inhibiting mitochondrial function and DNA synthesis in nearby host cells (82). The role of B. bronchiseptica tracheal cytotoxin has not been examined in detail. Dugal et al. (45) reported that a heat-stable substance of low molecular weight produced by B. bronchiseptica, possibly the tracheal cytotoxin, could induce ciliostasis of the tracheal epithelium with a concomitant accumulation of mucus.

The Regulation of Bordetella Virulence Determinants

Early experimentation with bordetellas showed that the main virulence characteristics were regulated in two ways: first, by an apparently irreversible phase shift in which virulence was rapidly lost upon repeated subculture (126), and second, by antigenic modulation whereby these characteristics could be reversibly controlled. The switch between the antigenic modes could be triggered by the addition of different salts or by cultivation at lower temperatures (113), or by nicotinic acid (192). B. bronchiseptica has a similar mechanism (155). The two switches are linked, with the loss of virulence on subculture resulting from in vitro selection of an infrequent mutation (114). This mutation occurs in a global regulator locus (bvgAS) that responds to environmental conditions (143, 206). The BvgAS regulatory system has been comprehensively reviewed (11, 36, 189, 198). A region of six cytosine bases at the promoter is clearly vulnerable to replication error, and insertional frameshift mutations have been identified in this locus (206), which would give the opportunity for reversion to the virulent phase. The bvgAS genes constitute a His-Asp phosphorelay relay system that regulates a wide variety of virulence determinants. Transcription of the bvgAS genes is controlled by four promoters, three of which are bvg-regulated. BvgS is a membrane-located histidine kinase. It is not known what signals BvgS responds to in vivo, although temperature is one possibility. BvgA is aspartic acid phosphorylated by BvgS and is a transcriptional activator that binds to the promoter elements of the regulated genes (177). It is interesting that the FHA gene is activated several hours prior to activation of toxin genes due to the differing affinities of phosphorylated BvgA for the respective promoter elements (188), implying that adhesion takes place before aggressins are expressed.

The BvgAS system regulates all elements of adhesion (FHA, pertactin, and fimbriae) and the dermonecrotic and adenylate cyclase toxins as well as the type III secretion system (Fig. 3); tracheal cytotoxin is not regulated by Bvg. In addition, several genes are repressed in the Bvg+ mode, the so-called vrg or bvg-repressed genes (109). These include motility (3) and the urease gene in B. bronchiseptica (140). The in vivo role of the vrg genes is unclear, as indeed is the role of bvg modulation. The various possibilities are discussed in some depth by Bock and Gross (11), who point out that B. bronchiseptica and B. pertussis express quite different vrg genes, and also that these genes, and bvgmediated modulation, may either have a role in environmental growth or in some as-yet not-understood aspect of pathogenesis. B. bronchiseptica, among the Bordetella species, is able to survive and even grow, among nutrient-poor circumstances, and at a temperature as low as 10°C (167, 168). Cotter and Miller (35) demonstrated that the Bvg− phase is advantageous for persistence under nutrient-limiting conditions compared with the Bvg+ phase. Furthermore, modulated B. bronchiseptica showed enhanced adhesion to ciliated porcine nasal epithelial cells in vitro (172) and gave increased colonization of the rat nasal cavity (18). B. bronchiseptica may have an environmental phase where the bvg-regulated virulence factors are redundant and not produced. At the same time, the increased adhesive ability coupled to reduced expression of virulence at reduced temperature might be beneficial for establishment in a host species and evasion of host defenses. In particular, this could lead to the establishment of a chronic infection. However, identification and characterization of the presumed adhesin and a better understanding of the potential role of modulated or phase-locked strains in vivo would be required to prove this hypothesis.

Figure 3

Diagram of some of the potential virulence determinants regulated by the bvg genes in Bordetella species. Abbreviations: bvg, Bordetella virulence gene; vrg, vir (bvg)-repressed gene; FHA, filamentous hemagglutinin.

Virulence Determinants Expressed by P. multocida

P. multocida was one of the first bacteria to be linked to disease and then tamed by a vaccine, in Louis Pasteur's seminal work in 1881. Since then surprisingly little has been learned about its virulence. P. multocida causes two serious animal diseases in the developing world: fowl cholera and hemorrhagic septicemia in cattle. In the West, it is a major determinant of atrophic rhinitis in pigs and is the most common cause of infection in wounds inflicted by dogs and cats. Resurgent interest in this bacterium has led to new developments which should greatly aid further analysis of virulence, namely the publication of the complete genome sequence of an avian strain of P. multocida (139) and the application of signature-tagged mutagenesis to identify in vivo expressed genes that are likely to be important in pathogenesis (56). Nevertheless, the best understood virulence determinant in P. multocida is still the dermonecrotic toxin. This virulence factor is only found in a limited set of strains, mainly of porcine origin. Since this is of crucial importance in the disease of atrophic rhinitis, we discuss it in some detail.

Atrophic Rhinitis and Bone Metabolism

Bone is a complex tissue that is subject to constant remodeling throughout life. The two major cell types that comprise bone are osteoblasts, which form bone, and multinucleated osteoclasts, which are responsible for bone resorption. These cells are derived from different lineages: osteoblasts from mesenchymal stem cells, and osteoclasts from the monocyte/macrophage lineage (209). Various markers can be used to identify these cells and assess the stage of differentiation. Bone formation and resorption are known to be tightly coupled, and it appears that most systemic signals that regulate this process (e.g., hormones, growth factors, and cytokines) target osteoblasts, which in turn release signaling molecules that regulate control osteoclast differentiation and activity (209).

The Interaction between B. bronchiseptica and P. multocida

There are three crucial stages in the pathogenesis of most infectious diseases: the pathogens must overcome the specific and nonspecific defense mechanisms of the host; they have to establish and multiply in or on the target tissue; and finally they should produce toxic or other harmful factors which are responsible, directly or indirectly, for the clinical signs of disease. As shown in this section, B. bronchiseptica and P. multocida, the two etiological agents of atrophic rhinitis, may share the tasks of fulfilling these stages.

Harris and Switzer (79) first reported that a type-D isolate of P. multocida failed to colonize the nasal cavity of pigs, but established and persisted if the animals had been previously infected with B. bronchiseptica. Since then it has been clearly established that toxigenic P. multocida needs some predisposing factor to colonize the nasal cavity in sufficient numbers to cause irreversible atrophy of the turbinate bones (161, 183). Gnotobiotic pigs infected with toxigenic P. multocida alone were colonized poorly, and only slight turbinate damage was seen at slaughter, whereas in mixed infections with B. bronchiseptica and toxigenic P. multocida colonization by large numbers of P. multocida occurred and severe disease was produced (183). Other predisposing agents have also been discovered. Intranasal pretreatment with dilute acetic acid was found to successfully enhance colonization by toxigenic P. multocida in experimentally infected pigs (160). Histological changes were not detected in the nasal cavity 6 days after acetic acid treatment, and it was reported that acetic acid caused only transient modification of the nasal respiratory epithelium resulting in stagnation of the nasal mucus, which was presumed to make the nasal environment favorable to colonization by P. multocida (57). Aerial pollutants (e.g., dust and gaseous ammonia) can also contribute to the severity of lesions associated with atrophic rhinitis by facilitating colonization of the pig's upper respiratory tract by P. multocida (73–75). On the other hand, others did not find significant differences in the extent or frequency of conchal atrophy between ammonia-exposed pigs and controls, or in the frequency of isolation of toxigenic P. multocida from conchae and tonsils (5). In conclusion, B. bronchiseptica, which is highly prevalent in the pig population, still seems to be the most important predisposing factor, although other factors like chemical irritants and dust may also exacerbate the disease.

The ability of B. bronchiseptica to encourage P. multocida establishment and growth could result from a direct effect on colonization, by promoting direct bacterium-bacterium interaction to aid attachment, or by the induction of host damage that could lead to either enhanced binding or improved nutrient acquisition. Alternatively B. bronchiseptica infection could impair host response mechanisms, leading to better P. multocida colonization. There is currently insufficient information available to establish how many of these putative mechanisms operate, but there is evidence that several are involved.

Although the exact role for the array of putative adhesins expressed by B. bronchiseptica has not yet been clarified, it is clearly established that B. bronchiseptica has an outstanding capacity to attach firmly to nasal epithelial cells (235) and generate profound ultrastructural changes in the nasal mucosa (46). Tracheal rings either infected with B. bronchiseptica or treated with a cell-free B. bronchiseptica supernatant were reported to enhance P. multocida adherence, with the suggestion that tracheal cytotoxin was the important factor (45). Similarly, filtered supernatants of B. bronchiseptica sonicates enhanced P. multocida colonization in vivo (2). A further possible attachment mechanism is the so-called piracy of adhesins where pretreatment of ciliated cells or pathogenic bacteria with FHA can promote adherence to cilia in vitro and in vivo (219). Since P. multocida attaches poorly to nasal epithelial cells, utilization of extracellular FHA produced by B. bronchiseptica could contribute to its establishment in the nasal cavity, although this hypothesis needs further investigation. In summary, these observations suggest that B. bronchiseptica directly enhances P. multocida attachment.

The role of the B. bronchiseptica DNT in P. multocida colonization has been analyzed in some detail. Nasal colonization of pigs by P. multocida was substantially increased (by a factor of >10⁴ CFU) by prior treatment with B. bronchiseptica (183) and resulted in an infection that persisted. A naturally occurring porcine isolate of B. bronchiseptica which did not produce DNT was compared with a wild-type strain for its ability to colonize and also to aid P. multocida colonization (26, 132). Later experiments used isogenic dnt mutant strains of B. bronchiseptica strains (134; Brockmeier et al., submitted). In all infection experiments using DNT-negative strains P. multocida colonized to lower levels (between 10- and 100-fold less) and declined after several days. An avirulent (Bvg⁻) variant of B. bronchiseptica was poorer at promoting P. multocida colonization than the dnt mutant strains (132). These results strongly suggest that DNT is the key factor that creates conditions in the nasal cavity most favorable for persistent colonization by large numbers of toxigenic P. multocida. Furthermore, these findings indicate that other virulence determinants produced by virulent strains of B. bronchiseptica may assist the growth of P. multocida in the nasal cavity but to a lesser extent than DNT.

It has not been shown whether DNT enhances P. multocida colonization by causing local tissue damage or by affecting immune function, though both mechanisms are likely to play a role. Lesions in the nasal cavity induced by monoinfection of pigs with DNT producing B. bronchiseptica, namely, severe hyperplasia and dysplasia of the epithelium, squamous cell metaplasia, loss of cilia from epithelial cells, marked fibrosis of the lamina propria, and a mild to moderate resorption of bone, are not seen in infections with the DNT-negative strain (132). It is unknown which, if any, of these histopathological changes might promote colonization by toxigenic P. multocida. In addition the specific cellular effect of DNT on Rho function has been shown to paralyze immune cells (87, 159; Mahon, personal communication) and this is likely to be important in the disease. The Bordetella factors responsible for the enhancement of P. multocida growth in the absence of DNT have not been investigated. However one distinct possibility is that released adenylate cyclase could enhance P. multocida colonization given its well characterized effect on immune function. Tracheal cytotoxin and type III secretion products might also assist in the establishment of P. multocida through impairing ciliary activity and the immune reaction of the host, respectively.

The enhancement of colonization occurs in both directions, since it is also clear that P. multocida colonization can boost colonization by B. bronchiseptica (180). It is known that PMT treatment can decrease the antibody response to bystander antigens (221). Indeed it appears that the combined action of B. bronchiseptica DNT and PMT creates conditions favorable to the growth of both bacteria, which are not reproduced by the action of either toxin alone. Three key observations support this hypothesis. First, in P. multocida infected pigs the wild-type strain of B. bronchiseptica colonized in greater numbers than the DNT-negative strain, although they colonized in similar numbers when given as single infections (132). Furthermore, there was no difference in colonization by the DNT-negative strain of B. bronchiseptica in single infections or in combined infections with toxigenic P. multocida. This suggests that the enhanced colonization of B. bronchiseptica in pigs infected with P. multocida depended on the presence of the B. bronchiseptica DNT. Second, infection with toxigenic but not with nontoxigenic P. multocida enhanced the growth of B. bronchiseptica (180). Conversely, colonization by a nontoxigenic strain of P. multocida was increased only marginally in B. bronchiseptica infected pigs, whereas the growth of a toxigenic strain of P. multocida was greatly enhanced (183). This suggests that it is PMT which increased the growth of B. bronchiseptica and that the enhanced growth of P. multocida in B. bronchiseptica-infected pigs depends on the presence of PMT. Finally, in pigs pretreated intranasally with dilute acetic acid, toxigenic P. multocida colonized briefly in numbers as great as those seen in combined infections with B. bronchiseptica before declining abruptly, although the importance of PMT in promoting P. multocida colonization was emphasized by the observation that colonization was less if PMT was blocked by antibody (24). The damage in the turbinates of such pigs was as great as in combined infections, yet clearly the induced pathology did not permit continued colonization by P. multocida in large numbers. In summary, damage from PMT does not by itself support the growth of large numbers of P. multocida but does enhance colonization by B. bronchiseptica so long as DNT is also present. Conversely, colonization by P. multocida is increased in B. bronchiseptica infected pigs as long as the strain of P. multocida is toxigenic.

Finally, do the two bacteria synergize to produce turbinate damage and the other pathology that is seen in this disease? The degree of turbinate bone loss in mixed infection experiments seemed to correlate with the numbers of P. multocida present and by inference with the likely quantity of PMT released (26), and it is known that PMT alone can reproduce the severe loss of turbinate bone that characterizes the disease (44, 96, 116, 182). It remains unclear whether Bordetella DNT synergizes with PMT to lead to greater bone destruction, or other pathological changes. While DNT directly targets and activates proteins of the Rho family by chemical modification, PMT is likely to activate Rho more indirectly, and thus perhaps more transiently. In addition PMT affects other cell-signaling pathways. The analysis of potential synergistic reactions could best be tackled in vitro using purified toxins, and this has yet to be performed.

Conclusion

The question remains: what is atrophic rhinitis? The generally accepted most important signs of the disease are severe and persistent turbinate atrophy, snout deformation, and, perhaps more controversially, reduced growth rate. At the present stage of our knowledge, PMT seems to play a dominant role in developing these characteristic lesions, and so toxigenic P. multocida is considered to be the primary etiological agent of atrophic rhinitis. On the other hand, a substantial body of evidence shows that P. multocida is unable to fulfill its role without predisposing circumstances, a set of imprecisely determined changes to the nasal mucosa that creates a niche suitable for the establishment of this pathogen. From this point of view, atrophic rhinitis could be defined as a multifactorial disease because several factors seem able to assist colonization of the host by sufficient numbers of P. multocida. It could even be classed as an opportunistic pathogen. However, the specific synergistic interactions between B. bronchiseptica and P. multocida support the notion that it is a genuine polymicrobial disease, which in addition may serve as a useful model system for more complex mixed infections. Nevertheless, even for this relatively simple polymicrobial disease, further research is clearly necessary to achieve a deeper understanding of the molecular interactions involved, which, in turn, may lead to novel therapeutic approaches.

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Footnotes

We dedicate this chapter to the late Richard B. Rimler, who made many contributions to the study of atrophic rhinitis.