Veterinary Microbiology, 25 (1990) 253-265 Elsevier Science Publishers B.V., Amsterdam
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Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin N. Chanter and J.M. Rutter* AFRC, Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, Great Britain (Accepted 11 April 1990)
ABSTRACT Chanter, N. and Rutter, J.M., 1990. Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin. Vet. Microbiol., 25: 253-265.
Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain ofPasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated ( 1 ) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.
INTRODUCTION
Atrophic rhinitis is a disease of growing pigs which is characterised in its severest form by complete or near complete destruction of the nasal turbinate bones (Rutter, 1985). The severe disease has been reproduced experimentally only by infection with toxigenic isolates of Pasteurella multocida (Pedersen and Barfod, 1981; Rutter and Rojas, 1982), which are normally isolated from herds with the disease (Schoss and Theil, 1983; Rutter et al., 1984; Nielsen et al., 1986), or by inoculation with a protein toxin purified from *Present address: Veterinary Medicines Directorate, Central Veterinary Laboratory, Weybridge, Surrey, Great Britain.
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these bacteria (Chanter et al., 1986a; Dominick and Rimler, 1986). Multiplication of P. multocida to produce enough toxin to cause severe atrophic rhinitis requires either prior infection with Bordetella bronchiseptica (Rutter and Rojas, 1982 ) or pretreatment of the nasal cavity with dilute acetic acid (Pedersen and Elling, 1984). The factors associated with B. bronchiseptica infection or acetic acid treatment that enhance colonisation by toxigenic P. multocida are unknown but are thought to represent an important part of the complex set of events in natural cases that ends in the elaboration of sufficient toxin to damage the nasal turbinate bones. The purified toxoid has been shown to be immunogenic (Nakai et al., 1984; Chanter et al., 1986b) even though antitoxin can be detected in few experimental (Rutter, 1983 ) or natural (Rutter et al., 1984) cases of disease. Vaccines containing purified toxoid have been used to protect against natural or experimental disease (Pedersen and Barfod, 1982; Baars et al., 1986; Kobisch and Pennings, 1986; Nagy et al., 1986; Voets, 1988; Foged et al., 1989) and claims have been made of the ability of a toxoid vaccine to eliminate infection (Baars et al., 1986 ). Most isolates oftoxigenic P. multocida belong to capsule type D but toxigenic isolates occur which produce capsule type A. The antigenic relationship between toxins from isolates with different capsule types is not known nor has the cross-protection provided by different toxoids been compared. B. bronchiseptica is widely prevalent in pig herds, experimentally it causes a mild turbinate atrophy and predisposes pigs to colonisation by toxigenic P. multocida (Rutter, 1985 ). This has meant that commercial vaccines for atrophic rhinitis include a bordetella bacterin but the effects of this on an antibody response to the toxin ofP. multocida have not been reported. The objectives of this study were to (1) assess the protective and cross protective properties of antibody to toxin from different capsule types compared with the protective capacity of antisera to crude lysate or bacterin which contained little antitoxin; and (2) examine the role of toxin as a colonisation factor. The results showed that antibody to toxin proved to be protective and because of the use ofbordetella bacterins in commercial atrophic rhinitis vaccines, a preliminary investigation was made to see if a bordetella bacterin enhanced or reduced an antibody response to the pasteurella toxin. A key element of this study was the use of gnotobiotic or specific pathogen free pigs and the acetic acid model of challenge with P. multocida alone; this protocol excluded interference from superinfection with B. bronchiseptica or other P. multocida and maternal or actively aquired antibodies to these bacteria. MATERIALS AND METHODS
Bacteria P. multocida strain LFB3 was a capsule type D toxigenic isolate from a British pig with clinical atrophic rhinitis (Rutter, 1983 ) and is known to pro-
PASTEURELLA MULTOCIDA IN ATROPHIC RHINITIS OF PIGS
2 55
duce disease. P. multocida strain 4661 was a toxigenic capsule type A isolate from Denmark (provided by K.B. Pedersen, Serum Laboratorium, Copenhagen). Nontoxigenic P. multocida strain P37 was isolated from a pig in a herd without a history of atrophic rhinitis; strains LFB3, 4661 and P37 were grown on 5% horse blood agar at 37 ° C overnight. B. bronchiseptica strain B58 was isolated in Hungary from a herd with clinical atrophic rhinitis (Magyar et al., 1988); B58 was grown on Bordet-Gengou agar containing 20% (v/v) sheep blood at 37°C for 72 h. Numbers of P. multocida recovered from the experimental infections (see below) were counted by spread plate inoculation of 0.1 ml of serial dilutions of nasal washings in phosphate buffered saline (PBS) at pH 7.0 on each of three 5% horse blood agar plates/dilution (0.1 ml/plate). Inoculated plates were incubated for 24 h at 37 °C and numbers ofP. multocida in nasal washes were calculated from the dilution used to inoculate the plates on which discrete colonies grew (within the range 30-300) and from the number of colonies counted.
Purified toxin, crude toxin, bacterin and inactivation Crude lysates ofP. multocida strains LFB3 and 4661 were prepared by the lysis method of Rimler and Brogden (1986). Crude lysates, treated with RNase, DNase, benzamidine and phenylmethylsulphonylfluoride were sequentially fractionated by DEAE Sephacel chromatography and preparative polyacrylamide gel electrophoresis (Chanter et al., 1986a). In the final step purified toxin was electroeluted from the polyacrylamide gel. Purity of preparations was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels (SDS-PAGE), (Laemmli, 1970) stained with silver (Chanter et al., 1986a). Toxins purified from LFB3 or 4661 had indistinguishable molecular weights and were at least 99% pure as judged by silver strains of serial dilutions of toxins separated by SDS-PAGE. Toxoids were prepared by treatment of purified toxins or crude lysate from LFB3 with 1% formaldehyde at 37 °C for 1 h. A bacterin of LFB3 or B58 was prepared by incubation of bacteria suspended to approximately 109/ml in 1% formaldehyde at 37 °C for 1 h. Immunoassays In an ELISA, microtitre plates (Falcon - Becton Dickinson) were coated overnight with 100/tl of 1 #g/ml of purified toxin or 10 mg/ml of crude lysate of LFB3 in 50 mM carbonate/bicarbonate buffer, pH 9.6. Plates were washed in PBS containing 0.03% Tween 20 (PBS/Tween) and incubated at 37 °C for 1 h with serial dilutions of pig antiserum in PBS/Tween with 1% dehydrated skimmed milk (Marvel, Cadbury, Ltd. ). Plates were washed again three times in PBS/Tween and 100/d of 1:5000 rabbit anti-swine Ig conjugated to horseradish peroxidase (Nordic Immunological Laboratories) in PBS/Tween/
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skimmed milk were added to each well and incubated at 37 °C for 3 h. Plates were washed again and developed with 100 #1 of 0.04% O-phenylenediamine in 0.015% hydrogen peroxide buffered with 0.15 M citrate/hydrogen orthophosphate, pH 5.0 for 15 min when the reaction was stopped with 10 #1 of 2 N sulphuric acid. Control cells were treated similarly, excluding either the antigen or the pig antiserum. Optical densities (492 n m ) of the wells were measured with a Titretec Microplate Reader; the titre of a serum was taken as the highest dilution which gave an optical density of at least twice that for both controls. Proteins separated by SDS-PAGE were immunoblotted by the method of Towbin et al. ( 1979 ) using a Transblot apparatus and the recommended protocols of the manufacturer (Biorad), biotinylated anti-pig immunoglobulin and ~25I-streptavidin (Amersham).
e/gs Gnotobiotic pigs were reared in plastic isolators in pairs and remained free from accidental contamination with other bacteria or fungi. Culture of regular faecal swabs onto blood agar prior to experimental infection did not yield growth and after infection bacteria or fungi, other than the inoculum, did not grow on nasal samples inoculated onto horse blood agar. Specific pathogen free pigs were derived from gnotobiotic pigs by oral inoculation of a preparation of a 'standard flora'. This was produced by serial passage of a cows faeces through two gnotobiotic calves which remained healthy (J.H. Morgan, personal communication). Faeces from the last calf was then serially passaged through two gnotobiotic pigs without signs of disease or infection with P. multocida or B. bronchiseptica. Faeces from the last pig were mixed and stored at - 7 0 ° C diluted to 20% (w/v) in Robertsons cooked meat m e d i u m containing 20% glycerol. Pigs 4 days of age were given 0.2 ml of stored faeces in 5 ml of canned condensed milk. At 4 weeks of age pigs were transferred to the high security building at IAH where extraneous infection with P. multocida or B. bronchiseptica could be prevented.
Gnotobiotic pig ant&era Pig (a) was inoculated twice subcutaneously with 36 #g of purified toxoid from LFB3 in 2.5 ml of Freunds incomplete adjuvant (FIA) with 35 days between injections, and finally 35 days later with 114 #g of toxoid in 3 ml of saline. Pig (b) was inoculated subcutaneously with 44 #g of purified toxoid from 4661 in 2 ml of FIA and with 56 #g in 4 ml of saline 38 days later. Differences in the amounts of toxoid used to immunise pigs (a) and (b) were attributable to the m a x i m u m amounts of toxin available at the time. Pig (c) was inoculated subcutaneously with 2.7 mg of crude lysate from LFB3 in 2 ml of FIA and again with 5.2 mg in 2 ml of saline 38 days later. Pig (d) was inoculated subcutaneously with LFB3 bacterin, corresponding to approxi-
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mately 10 9 cfu before inactivation, in 2 ml of FIA and again 38 days later with twice the dose of antigen in saline. All pigs were bled out at slaughter 10 days after the last injection. Blood was allowed to clot and the serum fraction collected. Serum was inactivated at 56 °C for 30 min, sterilised using an 0.22/tm cellulose filter and stored at - 20 ° C. Pigs (a) or (b) had titres of 500 against crude lysate of LFB3 but titres of 12 500 and 62 500, respectively, when purified toxin from LFB3 was used as antigen. Pigs (c) or (d) had titres of greater than 625 000 when crude lysate of LFB3 was used as antigen in an ELISA; antibody was not detected when purified toxin was used as antigen. Turbinate bones of all pigs were undamaged at slaughter.
Passive protection of gnotobiotic pigs by antisera and the role of toxin in colonisation Thirty two gnotobiotic pigs derived from five litters were divided into eight groups and kept as pairs without mixing groups. Group A (seven pigs, at least one from each litter) served as an uninfected and unprotected control for the calculation of % turbinate atrophy of other pigs at slaughter. Group B (four pigs from two litters ), given a total of 100 ml of PBS each, were infected but not given antiserum. Group C (four pigs from three litters) were each given 100 ml of antiserum, as described below, to the purified toxoid of LFB3. Each m e m b e r of group D (four pigs from two litters) was given 12.5, 25, 50 or 100 ml of antiserum to the purified toxoid of 4661; where pigs were given less than 100 ml the injection was made up to 100 ml by dilution in PBS (see below). Group E (four pigs from two litters) were given 100 ml of antiserum to the crude lysate (two pigs) or to the bacterin (two pigs). Group F (two pigs from the same litter) were given 50 ml of antiserum to the crude lysate plus 50 ml of antiserum to the bacterin. Pigs in groups B to F were infected with toxigenic P. multocida LFB3 using the acetic acid model of infection ( Pedersen and Elling, 1984 ). Group G (four pigs from two litters ) and group H (three pigs from two litters) were infected with nontoxigenic P. multocida P37 but only group G was pretreated with acetic acid. Pigs were injected intraperitoneally, where appropriate, with a total of four doses of 25 ml ( 100 ml in total for each pig) of serum, serum diluted in PBS or PBS, each dose given separately at 8, 10, 12 or 14 days of age. They were instilled intranasally, where applicable, with 0.5 ml of 1% acetic acid in PBS at 9 and 10 days of age in each nostril and infected with approximately 108 cfu ofP. multocida at 11 days of age. A nasal wash was collected 2 days before and 2 days after infection, and thereafter weekly until slaughter at 10 weeks of age. Nasal washes were made by instillation of 10 ml of PBS into one nostril and collection of sample from the other nostril. After weighing and slaughter of pigs a visual estimate was made of % turbinate atrophy by examination of snouts sectioned longitudinally with the
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septum removed. Damage was also measured by removal and weighing of the ventral turbinates and calculation of % turbinate atrophy from turbinate to body weight ratio compared with that of the mean for the uninfected group of controls (group A).
Effect of a bordetella bacterin on an antitoxoid response Ten specific pathogen free pigs derived from one litter were divided into five groups (two pigs per group) and injected subcutaneously at 29 and 56 days of age with vaccines made with an adjuvant. Pigs in group 1 were given 2 ml of either 25% alhydrogel (Superfos a / s ) in Tris/HC1, pH 7.4, (Tris buffer) or FIA emulsified in Tris buffer. Group 2 were given 25 #g of toxoid from LFB3 in FIA/Tris buffer and group 3 were given the same preparation also containing bordetella bacterin equivalent to 109 cfu. Groups 4 and 5 were treated as groups 2 and 3, respectively, but FIA was replaced with alhydrogel. All pigs were re-immunised with the same antigens at 84 days of age and bled at slaughter 7 days later. Alhydrogel vaccines were prepared by overnight incubation with constant mixing at room temperature of a combination of one part toxoid plus two parts Tris buffer, or where appropriate bacterin in Tris buffer, and one part alhydrogel. Statistical analyses An analysis of variance model was fitted to the numbers of bacteria recovered in nasal washings from each pig throughout the experiment to identify any differences between groups. An analysis of variance was also used, where applicable, to test the significance of differences in turbinate damage between groups. RESULTS
Passive protection of gnotobiotic pigs by antisera All control uninfected pigs (group A) had undamaged turbinate bones at slaughter. In contrast, in control infected pigs given PBS (group B ) turbinate bones were estimated to be reduced by 80-90% at slaughter; this was confirmed when the reduction was calculated (Table 1 ). Protection was seen in pigs in group C given antiserum to the purified toxoid from LFB3 when damage to turbinate bones was not visible at slaughter. However, the calculated turbinate to body weight ratio for one pig indicated 30% turbinate atrophy; the same calculation based solely on its uninfected littermate control gave a result of 12%. This resulted in the small calculated turbinate atrophy for group C (Table 1 ). Cross-protection was seen in group D given antiserum to the purified toxoid of 4661 (group D ) when damage to the turbinate bones was not visible at slaughter in three pigs. Damage was seen in one pig of group D but this was
PASTEURELLA MULTOCIDA IN ATROPHIC RH1NITIS OF PIGS
259
TABLE 1
Percentage turbinate damage in pigs infected with toxigenic P. multocida LFB3 calculated from the turbinate to body weight ratio compared with the mean value for the uninfected control group A Treatment and values for individual pigs
Mean turbinate wt/body wt X 10 - 4 (range)
% turbinate atrophy
Group A ( n = 7 ) , uninfected control Group B ( n = 4 ) , given PBS Group C ( n = 4 ) , given antiserum to LFB3 toxoid Group D ( n = 4 ) , given antiserum to 4661 toxoid Group E ( n = 4 ) , given anti-crude lysate or anti-bacterin Group F ( n = 2 ) , given mixture of antisera to crude lysate or bacterin
2.71 (2.46-3.7)
0
0.44 (0.082-0.9)
83
2.59 (1.92-2.98)
4
2.37 (1.67-2.8)
13
0.34 (0.19-0.43)
87
1.11 (0.93-1.29)
59
confined to the left turbinates only (estimated to be reduced by 40%); this pig was given 50 ml of antiserum. Otherwise there was no correlation between the amount of antiserum given or protection; pigs given 25 or 12.5 ml of antitoxin were as equally protected as the pig given 100 ml. Protection was not seen in pigs given antiserum to the crude lysate or bacterin of LFB3 (group E) because turbinate damage seen at slaughter was severe (turbinate bones estimated to be reduced by 90%); this was confirmed when the reduction was calculated (Table 1 ). However, some protection was seen in pigs given a mixture of antisera to crude lysate and bacterin from LFB3 (group F) when damage was significantly less than that seen in pigs in group E given antiserum to crude lysate or to bacterin (P was less than 0.01 ). An immunoblot of antisera to crude lysate or bacterin, using crude lysate of LFB3 separated by SDS-PAGE as antigen, showed that a combination of these sera would give a product which contained antibodies to more antigens of P. multocida than each serum alone (Fig. 1 ); both antisera to the purified toxoids in immunoblots with crude lysate reacted with a single polypeptide of the same molecular weight as the toxin.
The role of toxin in colonisation The numbers of P. multocida which colonised the nasal cavities of groups B to F were compared with those recovered from pigs given acetic acid and infected with nontoxigenic P. multocida P37 (group G) or infected with P37
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N. C H A N T E R AND J.M. R U T T E R
A
13
Fig. 1. Immunoblot of crude lysate ofP. multocida LFB3 separated by SDS-PAGE and probed with antiserum to crude lysate (lane A), antiserum to bacterin (lane B). Markers indicate polypeptides uniquely detected by one or other antiserum. without acetic acid treatment (group H). Statistical comparison of each group with the others showed that colonisation by toxigenic or nontoxigenic P. multocida took the form o f one of two patterns into which all groups fell (Fig. 2 ). There was no significant difference in colonisation by toxigenic P. multocida in pigs given PBS, antiserum to crude lysate or bacterin or a mixture of anticrude lysate and anti-bacterin (groups B, E and F). Also, there was no significant difference in colonisation by toxigenic or nontoxigenic P. multocida in pigs given antiserum to a purified toxoid or given nontoxigenic strain P37, with or without prior acetic acid treatments (groups C, D, G and H ) . However, colonisation by P. multocida in groups C, D, G and H was by significantly fewer bacteria than in groups B, E and F (Fig. 2); the value of P for differences between group F and groups D or G was < 0.05 and the value of P for differences between the remaining groups was < 0.01.
PASTEURELLA MULTOCIDA IN A T R O P H I C R H I N I T I S O F PIGS
8
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Weeks after infection
Weeks after infection
Fig. 2. P. multocida in nasal washings of pigs. Graph (A): ( I ) , group B pigs given PBS and infected with toxigenic P. multocida LFB3; ( O ) , group E pigs given antiserum to the crude lysate or bacterin and infected with LFB3; ( • ) , group F pigs given a mixture ofantisera to crude lysate and bacterin and infected with LFB3. Graph (B): ( • ), group C pigs given antiserum to LFB3 toxoid and infected with LFB3; ( • ) , group D pigs given antiserum to 4661 toxoid and infected with LFB3; ( 0 ) , group G pigs treated with acetic acid and infected with nontoxigenic P. multocida P37; ( I ) , group H pigs infected with P37. Inset graph: ( ~ ) , mean of values for graph A (groups B, E, and F); (El), mean of values for graph B (groups C, D, G and H). Colonisation by each group in graph B was significantly different from each group in graph A (P at least less than 0.05 for each group in one line tested against each group in the other line).
Effect o f a bordetella bacterin on an antitoxoid response
Antitoxin was produced by pigs immunised with toxoid in alhydrogel without bordetella bacterin (group 4; log~o titres of 2 and 5), or with toxoid in FIA with bordetella bacterin (group 3; log~o titres of 4 and 4) or without bacterin (group 2; log~o titres of 3 and 3). However, pigs immunised with adjuvant alone (group 1 ) or with toxoid and bordetella bacterin in alhydrogel (group 5 ) did not produce antitoxin. DISCUSSION
The importance of antitoxin production after vaccination was illustrated when seven out of eight pigs given antitoxin were completely protected against turbinate damage. Pigs given antiserum to the crude lysate or bacterin were not protected even though these sera contained large amounts of antibody to bacteria detected by ELISA using crude lysate ofP. multocida as antigen. The slight mean turbinate atrophy detected from turbinate to body weight ratios in pigs given antitoxoid in groups C and D may be due to variation inherent
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N. CHANTERANDJ.M. RUTTER
in this method (see range of values for control group A). Alternatively, incomplete protection may have been detected in some pigs which was not detectable by visual examination. Nonetheless, visual examination did not suggest turbinate damage except in the one pig in group D. This pig given 50 ml of antiserum to the toxin from the type A strain was not fully protected because one of the ventral turbinates was reduced by 40%. Why this was so when the other turbinate was normal, and pigs given either 25 or 12.5 ml of the same serum where protected even though colonised by indistinguishable numbers of toxigenic bacteria, is unclear. The general protection against turbinate atrophy caused by infection with a toxigenic strain of capsule type D, in pigs given antibody to the toxoid from a capsule type A isolate, suggested a very close similarity or identity between toxins from these bacteria. The intermediate damage in pigs passively immunised with a mixture of antisera to crude lysate and bacterin suggested a degree of protection. One possible explanation may come from the observation that these sera when combined contained antibodies to more antigens than each serum alone. The partial protection could not be explained by the presence of undetectable amounts of antibody to toxin because if either antisera had contained antitoxin, then protection would have been seen in pigs given either antiserum to bacterin or to crude lysate (group E). Consequently, there may be a mechanism of protection based on a combination of antibodies to different antigens. If this occurred it was apparently unrelated to a reduction in colonisation; perhaps a different mechanism of protection may either alter colonisation by P. multocida away from a precise site where toxin can cause damage, or reduce the expression of toxin. Toxigenic P. multocida colonised pigs, treated with acetic acid and given antiserum to purified toxoid, to a degree indistinguishable from that seen with pigs infected with nontoxigenic P. multocida, with or without prior acetic acid treatment. Colonisation in these animals was also significantly less than that seen with infections of toxigenic P. multocida in pigs unprotected by PBS or antiserum to crude lysate or bacterin. These observations suggested that the mechanism by which acetic acid enhanced colonisation by P. multocida was dependent on the action of the toxin in an additive or synergistic fashion. This possibility was supported because colonisation by nontoxigenic P. multocida was not enhanced by treatment of the nasal cavity with acetic acid. However, this line of evidence is dependent on the assumption that nontoxigenic P. multocida possess the same colonisation factors as toxigenic P. rnultocida, other than the toxin itself. The cytotoxin of B. bronchiseptica has been shown to enhance colonisation by toxigenic P. multocida (Chanter et al., 1989) but the mechanism for this, like the action of acetic acid treatment, is unknown. There was, however, evidence that the stimulatory effect of the bordetella cytotoxin was dependent on, or synergistically enhanced, by the action of the pasteurella toxin. It has
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been speculated that adhesion to turbinate mucosal cells may be a necessary step in the pathogenesis of atrophic rhinitis. However, several authors (reviewed by Chanter and Rutter, 1989) using in vitro tests have not consistently detected adhesion to these or other cells by toxigenic P. multocida. The toxin does not, therefore, presumably act as an adhesin although attempts to detect adhesion in vivo or compare turbinate mucosa colonised by toxigenic or nontoxigenic P. multocida have not been published. There remains speculation that the toxin enhances colonisation by causing damage and the release of nutrients more favourable for growth or that it interferes with nonspecific or specific immunological defences to reduce clearance of bacteria. However, in the experiment described here, even when the action of the toxin was not eliminated by antitoxin, numbers of toxigenic P. multocida still declined at a time when damage to the mucosa would have been at its most severe. Consequently, colonisation by toxigenic P. multocida seems to be a complex and dynamic process with much still to be learnt. The reduction in colonisation caused by antitoxin nonetheless did not reduce the mean numbers of bacteria to much below 103 c f u / m l of nasal wash. This reduction was much less than the complete elimination of infection observed by others (Baars et al., 1986). Nontoxigenic P. multocida do not seem to require toxin to establish colonisation so it would seem unlikely that antibody to toxin alone could eliminate colonisation by toxigenic bacteria from pig herds. Failure by others to recover toxigenic P. multocida from pigs containing antitoxin may have been due to low sensitivity of isolation techniques because of the background flora in conventional animals. Alternatively it is still possible that these bacteria were not present because of competition from the normal flora. The failure of pigs to respond to the toxoid in alhydrogel with a bacterin of B. bronchiseptica was unexpected, in view of the well-recognised adjuvant properties of bacterins made from the closely related organism B. pertussis. The mechanism explaining these results is not known but it is particularly relevant to vaccination against atrophic rhinitis. The wide spread prevalence ofB. bronchiseptica in pig herds and experimental evidence that these bacteria can both cause mild turbinate damage themselves and also predispose pigs to colonisation by toxigenic P. multocida, will ensure that atrophic rhinitis vaccines are likely to contain a bordetella component. Colonisation by B. bronchiseptica requires an adhesin (Collings and Rutter, 1985; Semjen and Magyar, 1985 ) and the cytotoxin of B. bronchiseptica is a key factor which enhances colonisation by toxigenic P. multocida (Chanter et al., 1989). Currently, atrophic rhinitis vaccines contain a bordetella bacterin rather than important subunit components. A P. multocida toxoid vaccine containing important subunit components ofB. bronchiseptica may not only provide better protection against bordetella and pasteurella infections but may also reduce the likelihood of failed responses to the P. multocida toxoid. This would be
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true so long as any of the isolated bordetella components did not interfere with an antitoxin response. ACKNOWLEDGEMENTS
The authors would like to thank Mr. M. Dennis and his staff for the production and care of pigs, Miss K. Walker, Miss H. Williamson and Miss C. Thomson for technical assistance and Mrs. K. Bunch for statistical analyses. REFERENCES Baars, J.C., Pennings, A. and Storm, P.K., 1986. Challenge and field experiments with an experimental atrophic rhinitis vaccine, containing Pasteurella multocida DNT-toxoid and Bordetella bronchiseptica. Proc. ninth Congress of the International Pig Veterinary Society, p. 247. Chanter, N. and Rutter, J.M., 1989. Pasteurellosis in pigs and the determinants of virulence of toxigenic Pasteurella multocida. In: C. Adlam and J.M. Rutter (Editors), Academic Press, Harcourt Brace Iovanovich, London, pp. 161-196. Chanter, N., Rutter, J.M. and Mackenzie, A., 1986a. Partial purification of an osteolytic toxin from Pasteurella multocida. J. Gen. Microbiol., 132: 1089-1097. Chanter, N., Rutter, J.M. and Mackenzie, A., 1986b. Partial purification of an osteolytic toxin from Pasteurella multocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 228. Chanter, N., Magyar, T. and Rutter, J.M., 1989. Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Res. Vet. Sci., 47: 48-53. Collings, L.A. and Rutter, J.M., 1985. Virulence ofBordetella bronchiseptica in the porcine respiratory tract. J. Med. Microbiol., 19: 247-258. Dominick, M.A. and Rimler, R.B., 1986. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am. J. Res., 47: 1532-1536. Foged, N.T., Nielsen, J.P. and Jorsal, S.E., 1989. Protection against progressive atrophic rhinitis by vaccination with Pasteurella multocida toxin purified by monoclonal antibodies. Vet. Rec., 125:7-11. Kobisch, M. and Pennings, A., 1986. An evaluation ofNobi-vac AR and an experimental atrophic rhinitis vaccine, containing Pasteurella rnultocida DNT-toxoid and Bordetella bronchiseptica, in pigs. Proc. the ninth Congress of the International Pig Veterinary Society, p. 246. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 681-685. Magyar, T., Chanter, N., Lax, A.J., Rutter, J.M. and Hall, G.A., 1988. The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica. Vet. Microbiol., 18:135-146. Nagy, L.K., Mackenzie, T. and Scarnell, J., 1986. Serum antibody values to Pasteurella multocida type D toxin and susceptibility of piglets to experimental challenge with toxigenic type D P. rnultocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 224. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K., 1984. Purification of dermonecrotic toxin from a sonic extract ofPasteurella multocida SP-72 serotype D. Infect. Immun., 46: 429-434. Nielson, J.P., Pedersen, K.B. and Willeberg, P., 1986. Epidemiology of atrophic rhinitis and toxin producing Pasteurella multocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 239.
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Pedersen, K.B. and Barfod, K., 1981. The aetiological significance of Bordetella bronchiseptica and Pasteurella multocida in atrophic rhinitis of swine. Nord. Veterinaermed., 33:513-522. Pedersen, K.B. and Barfod, K., 1982. Effect on the incidence of atrophic rhinitis of vaccination of sows with a vaccine containing Pasteurella multocida toxin. Nord. Veterinaermed., 34: 293-302. Pedersen, K.B. and Elling, F., 1984. The pathogenesis of atrophic rhinitis induced by toxigenic Pasteurella m ultocida. J. Comp. Pathol., (4) 203-214. Rimler, R.B. and Brogden, K.A., 1986. Pasteurella multocida isolated from rabbits and swine; serologic types and toxin production. Am. J. Vet. Res., 47: 730-737. Rutter, J.M., 1983. Virulence of Pasteurella multocida in atrophic rhinitis of gnotobiotic pigs infected with Bordetella bronchiseptica. Res. Vet. Sci., 34: 287-295. Rutter, J.M., 1985. Atrophic rhinitis in swine. Adv. Vet. Sci. Comp. Med., 29: 239-279. Rutter, J.M. and Rojas, X., 1982. Atrophic rhinitis in gnotobiotic piglets: differences in the pathogenicity of Pasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec., 110: 531-535. Rutter, J.M., Taylor, R.J., Crighton, W.G., Robertson, I.B. and Benson, J.A., 1984. Epidemiological study of Pasteurella multocida and Bordetella bronchiseptica in atrophic rhinitis. Vet. Rec., 115: 615-619. Semjen, G. and Magyar, T., 1985. A bovine haemagglutinin ofBordetella bronchiseptica responsible for adherence. Acta Vet. Hung., 33: 129-136. Schoss, P. and Theil, P., 1983. Occurrence of toxin producing strains of Pasteurella rnultocida and Bordetella bronchiseptica in pig herds affected with atrophic rhinitis and in unsuspicious herds. In: Atrophic rhinitis in pigs, CEC report EUR 9643 EN, p. 105. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat. Acad. Sci., New York, 76: 4350-4354. Voets, M.Th., 1988. Challenge experiments to test the protective effect ofAR-vaccines. 9th Congress of the International Pig Veterinary Society, p. 42.
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Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin N. Chanter and J.M. Rutter* AFRC, Institute for Animal Health, Compton, Newbury, Berkshire RG16 ONN, Great Britain (Accepted 11 April 1990)
ABSTRACT Chanter, N. and Rutter, J.M., 1990. Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin. Vet. Microbiol., 25: 253-265.
Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain ofPasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated ( 1 ) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.
INTRODUCTION
Atrophic rhinitis is a disease of growing pigs which is characterised in its severest form by complete or near complete destruction of the nasal turbinate bones (Rutter, 1985). The severe disease has been reproduced experimentally only by infection with toxigenic isolates of Pasteurella multocida (Pedersen and Barfod, 1981; Rutter and Rojas, 1982), which are normally isolated from herds with the disease (Schoss and Theil, 1983; Rutter et al., 1984; Nielsen et al., 1986), or by inoculation with a protein toxin purified from *Present address: Veterinary Medicines Directorate, Central Veterinary Laboratory, Weybridge, Surrey, Great Britain.
0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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these bacteria (Chanter et al., 1986a; Dominick and Rimler, 1986). Multiplication of P. multocida to produce enough toxin to cause severe atrophic rhinitis requires either prior infection with Bordetella bronchiseptica (Rutter and Rojas, 1982 ) or pretreatment of the nasal cavity with dilute acetic acid (Pedersen and Elling, 1984). The factors associated with B. bronchiseptica infection or acetic acid treatment that enhance colonisation by toxigenic P. multocida are unknown but are thought to represent an important part of the complex set of events in natural cases that ends in the elaboration of sufficient toxin to damage the nasal turbinate bones. The purified toxoid has been shown to be immunogenic (Nakai et al., 1984; Chanter et al., 1986b) even though antitoxin can be detected in few experimental (Rutter, 1983 ) or natural (Rutter et al., 1984) cases of disease. Vaccines containing purified toxoid have been used to protect against natural or experimental disease (Pedersen and Barfod, 1982; Baars et al., 1986; Kobisch and Pennings, 1986; Nagy et al., 1986; Voets, 1988; Foged et al., 1989) and claims have been made of the ability of a toxoid vaccine to eliminate infection (Baars et al., 1986 ). Most isolates oftoxigenic P. multocida belong to capsule type D but toxigenic isolates occur which produce capsule type A. The antigenic relationship between toxins from isolates with different capsule types is not known nor has the cross-protection provided by different toxoids been compared. B. bronchiseptica is widely prevalent in pig herds, experimentally it causes a mild turbinate atrophy and predisposes pigs to colonisation by toxigenic P. multocida (Rutter, 1985 ). This has meant that commercial vaccines for atrophic rhinitis include a bordetella bacterin but the effects of this on an antibody response to the toxin ofP. multocida have not been reported. The objectives of this study were to (1) assess the protective and cross protective properties of antibody to toxin from different capsule types compared with the protective capacity of antisera to crude lysate or bacterin which contained little antitoxin; and (2) examine the role of toxin as a colonisation factor. The results showed that antibody to toxin proved to be protective and because of the use ofbordetella bacterins in commercial atrophic rhinitis vaccines, a preliminary investigation was made to see if a bordetella bacterin enhanced or reduced an antibody response to the pasteurella toxin. A key element of this study was the use of gnotobiotic or specific pathogen free pigs and the acetic acid model of challenge with P. multocida alone; this protocol excluded interference from superinfection with B. bronchiseptica or other P. multocida and maternal or actively aquired antibodies to these bacteria. MATERIALS AND METHODS
Bacteria P. multocida strain LFB3 was a capsule type D toxigenic isolate from a British pig with clinical atrophic rhinitis (Rutter, 1983 ) and is known to pro-
PASTEURELLA MULTOCIDA IN ATROPHIC RHINITIS OF PIGS
2 55
duce disease. P. multocida strain 4661 was a toxigenic capsule type A isolate from Denmark (provided by K.B. Pedersen, Serum Laboratorium, Copenhagen). Nontoxigenic P. multocida strain P37 was isolated from a pig in a herd without a history of atrophic rhinitis; strains LFB3, 4661 and P37 were grown on 5% horse blood agar at 37 ° C overnight. B. bronchiseptica strain B58 was isolated in Hungary from a herd with clinical atrophic rhinitis (Magyar et al., 1988); B58 was grown on Bordet-Gengou agar containing 20% (v/v) sheep blood at 37°C for 72 h. Numbers of P. multocida recovered from the experimental infections (see below) were counted by spread plate inoculation of 0.1 ml of serial dilutions of nasal washings in phosphate buffered saline (PBS) at pH 7.0 on each of three 5% horse blood agar plates/dilution (0.1 ml/plate). Inoculated plates were incubated for 24 h at 37 °C and numbers ofP. multocida in nasal washes were calculated from the dilution used to inoculate the plates on which discrete colonies grew (within the range 30-300) and from the number of colonies counted.
Purified toxin, crude toxin, bacterin and inactivation Crude lysates ofP. multocida strains LFB3 and 4661 were prepared by the lysis method of Rimler and Brogden (1986). Crude lysates, treated with RNase, DNase, benzamidine and phenylmethylsulphonylfluoride were sequentially fractionated by DEAE Sephacel chromatography and preparative polyacrylamide gel electrophoresis (Chanter et al., 1986a). In the final step purified toxin was electroeluted from the polyacrylamide gel. Purity of preparations was assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels (SDS-PAGE), (Laemmli, 1970) stained with silver (Chanter et al., 1986a). Toxins purified from LFB3 or 4661 had indistinguishable molecular weights and were at least 99% pure as judged by silver strains of serial dilutions of toxins separated by SDS-PAGE. Toxoids were prepared by treatment of purified toxins or crude lysate from LFB3 with 1% formaldehyde at 37 °C for 1 h. A bacterin of LFB3 or B58 was prepared by incubation of bacteria suspended to approximately 109/ml in 1% formaldehyde at 37 °C for 1 h. Immunoassays In an ELISA, microtitre plates (Falcon - Becton Dickinson) were coated overnight with 100/tl of 1 #g/ml of purified toxin or 10 mg/ml of crude lysate of LFB3 in 50 mM carbonate/bicarbonate buffer, pH 9.6. Plates were washed in PBS containing 0.03% Tween 20 (PBS/Tween) and incubated at 37 °C for 1 h with serial dilutions of pig antiserum in PBS/Tween with 1% dehydrated skimmed milk (Marvel, Cadbury, Ltd. ). Plates were washed again three times in PBS/Tween and 100/d of 1:5000 rabbit anti-swine Ig conjugated to horseradish peroxidase (Nordic Immunological Laboratories) in PBS/Tween/
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skimmed milk were added to each well and incubated at 37 °C for 3 h. Plates were washed again and developed with 100 #1 of 0.04% O-phenylenediamine in 0.015% hydrogen peroxide buffered with 0.15 M citrate/hydrogen orthophosphate, pH 5.0 for 15 min when the reaction was stopped with 10 #1 of 2 N sulphuric acid. Control cells were treated similarly, excluding either the antigen or the pig antiserum. Optical densities (492 n m ) of the wells were measured with a Titretec Microplate Reader; the titre of a serum was taken as the highest dilution which gave an optical density of at least twice that for both controls. Proteins separated by SDS-PAGE were immunoblotted by the method of Towbin et al. ( 1979 ) using a Transblot apparatus and the recommended protocols of the manufacturer (Biorad), biotinylated anti-pig immunoglobulin and ~25I-streptavidin (Amersham).
e/gs Gnotobiotic pigs were reared in plastic isolators in pairs and remained free from accidental contamination with other bacteria or fungi. Culture of regular faecal swabs onto blood agar prior to experimental infection did not yield growth and after infection bacteria or fungi, other than the inoculum, did not grow on nasal samples inoculated onto horse blood agar. Specific pathogen free pigs were derived from gnotobiotic pigs by oral inoculation of a preparation of a 'standard flora'. This was produced by serial passage of a cows faeces through two gnotobiotic calves which remained healthy (J.H. Morgan, personal communication). Faeces from the last calf was then serially passaged through two gnotobiotic pigs without signs of disease or infection with P. multocida or B. bronchiseptica. Faeces from the last pig were mixed and stored at - 7 0 ° C diluted to 20% (w/v) in Robertsons cooked meat m e d i u m containing 20% glycerol. Pigs 4 days of age were given 0.2 ml of stored faeces in 5 ml of canned condensed milk. At 4 weeks of age pigs were transferred to the high security building at IAH where extraneous infection with P. multocida or B. bronchiseptica could be prevented.
Gnotobiotic pig ant&era Pig (a) was inoculated twice subcutaneously with 36 #g of purified toxoid from LFB3 in 2.5 ml of Freunds incomplete adjuvant (FIA) with 35 days between injections, and finally 35 days later with 114 #g of toxoid in 3 ml of saline. Pig (b) was inoculated subcutaneously with 44 #g of purified toxoid from 4661 in 2 ml of FIA and with 56 #g in 4 ml of saline 38 days later. Differences in the amounts of toxoid used to immunise pigs (a) and (b) were attributable to the m a x i m u m amounts of toxin available at the time. Pig (c) was inoculated subcutaneously with 2.7 mg of crude lysate from LFB3 in 2 ml of FIA and again with 5.2 mg in 2 ml of saline 38 days later. Pig (d) was inoculated subcutaneously with LFB3 bacterin, corresponding to approxi-
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mately 10 9 cfu before inactivation, in 2 ml of FIA and again 38 days later with twice the dose of antigen in saline. All pigs were bled out at slaughter 10 days after the last injection. Blood was allowed to clot and the serum fraction collected. Serum was inactivated at 56 °C for 30 min, sterilised using an 0.22/tm cellulose filter and stored at - 20 ° C. Pigs (a) or (b) had titres of 500 against crude lysate of LFB3 but titres of 12 500 and 62 500, respectively, when purified toxin from LFB3 was used as antigen. Pigs (c) or (d) had titres of greater than 625 000 when crude lysate of LFB3 was used as antigen in an ELISA; antibody was not detected when purified toxin was used as antigen. Turbinate bones of all pigs were undamaged at slaughter.
Passive protection of gnotobiotic pigs by antisera and the role of toxin in colonisation Thirty two gnotobiotic pigs derived from five litters were divided into eight groups and kept as pairs without mixing groups. Group A (seven pigs, at least one from each litter) served as an uninfected and unprotected control for the calculation of % turbinate atrophy of other pigs at slaughter. Group B (four pigs from two litters ), given a total of 100 ml of PBS each, were infected but not given antiserum. Group C (four pigs from three litters) were each given 100 ml of antiserum, as described below, to the purified toxoid of LFB3. Each m e m b e r of group D (four pigs from two litters) was given 12.5, 25, 50 or 100 ml of antiserum to the purified toxoid of 4661; where pigs were given less than 100 ml the injection was made up to 100 ml by dilution in PBS (see below). Group E (four pigs from two litters) were given 100 ml of antiserum to the crude lysate (two pigs) or to the bacterin (two pigs). Group F (two pigs from the same litter) were given 50 ml of antiserum to the crude lysate plus 50 ml of antiserum to the bacterin. Pigs in groups B to F were infected with toxigenic P. multocida LFB3 using the acetic acid model of infection ( Pedersen and Elling, 1984 ). Group G (four pigs from two litters ) and group H (three pigs from two litters) were infected with nontoxigenic P. multocida P37 but only group G was pretreated with acetic acid. Pigs were injected intraperitoneally, where appropriate, with a total of four doses of 25 ml ( 100 ml in total for each pig) of serum, serum diluted in PBS or PBS, each dose given separately at 8, 10, 12 or 14 days of age. They were instilled intranasally, where applicable, with 0.5 ml of 1% acetic acid in PBS at 9 and 10 days of age in each nostril and infected with approximately 108 cfu ofP. multocida at 11 days of age. A nasal wash was collected 2 days before and 2 days after infection, and thereafter weekly until slaughter at 10 weeks of age. Nasal washes were made by instillation of 10 ml of PBS into one nostril and collection of sample from the other nostril. After weighing and slaughter of pigs a visual estimate was made of % turbinate atrophy by examination of snouts sectioned longitudinally with the
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septum removed. Damage was also measured by removal and weighing of the ventral turbinates and calculation of % turbinate atrophy from turbinate to body weight ratio compared with that of the mean for the uninfected group of controls (group A).
Effect of a bordetella bacterin on an antitoxoid response Ten specific pathogen free pigs derived from one litter were divided into five groups (two pigs per group) and injected subcutaneously at 29 and 56 days of age with vaccines made with an adjuvant. Pigs in group 1 were given 2 ml of either 25% alhydrogel (Superfos a / s ) in Tris/HC1, pH 7.4, (Tris buffer) or FIA emulsified in Tris buffer. Group 2 were given 25 #g of toxoid from LFB3 in FIA/Tris buffer and group 3 were given the same preparation also containing bordetella bacterin equivalent to 109 cfu. Groups 4 and 5 were treated as groups 2 and 3, respectively, but FIA was replaced with alhydrogel. All pigs were re-immunised with the same antigens at 84 days of age and bled at slaughter 7 days later. Alhydrogel vaccines were prepared by overnight incubation with constant mixing at room temperature of a combination of one part toxoid plus two parts Tris buffer, or where appropriate bacterin in Tris buffer, and one part alhydrogel. Statistical analyses An analysis of variance model was fitted to the numbers of bacteria recovered in nasal washings from each pig throughout the experiment to identify any differences between groups. An analysis of variance was also used, where applicable, to test the significance of differences in turbinate damage between groups. RESULTS
Passive protection of gnotobiotic pigs by antisera All control uninfected pigs (group A) had undamaged turbinate bones at slaughter. In contrast, in control infected pigs given PBS (group B ) turbinate bones were estimated to be reduced by 80-90% at slaughter; this was confirmed when the reduction was calculated (Table 1 ). Protection was seen in pigs in group C given antiserum to the purified toxoid from LFB3 when damage to turbinate bones was not visible at slaughter. However, the calculated turbinate to body weight ratio for one pig indicated 30% turbinate atrophy; the same calculation based solely on its uninfected littermate control gave a result of 12%. This resulted in the small calculated turbinate atrophy for group C (Table 1 ). Cross-protection was seen in group D given antiserum to the purified toxoid of 4661 (group D ) when damage to the turbinate bones was not visible at slaughter in three pigs. Damage was seen in one pig of group D but this was
PASTEURELLA MULTOCIDA IN ATROPHIC RH1NITIS OF PIGS
259
TABLE 1
Percentage turbinate damage in pigs infected with toxigenic P. multocida LFB3 calculated from the turbinate to body weight ratio compared with the mean value for the uninfected control group A Treatment and values for individual pigs
Mean turbinate wt/body wt X 10 - 4 (range)
% turbinate atrophy
Group A ( n = 7 ) , uninfected control Group B ( n = 4 ) , given PBS Group C ( n = 4 ) , given antiserum to LFB3 toxoid Group D ( n = 4 ) , given antiserum to 4661 toxoid Group E ( n = 4 ) , given anti-crude lysate or anti-bacterin Group F ( n = 2 ) , given mixture of antisera to crude lysate or bacterin
2.71 (2.46-3.7)
0
0.44 (0.082-0.9)
83
2.59 (1.92-2.98)
4
2.37 (1.67-2.8)
13
0.34 (0.19-0.43)
87
1.11 (0.93-1.29)
59
confined to the left turbinates only (estimated to be reduced by 40%); this pig was given 50 ml of antiserum. Otherwise there was no correlation between the amount of antiserum given or protection; pigs given 25 or 12.5 ml of antitoxin were as equally protected as the pig given 100 ml. Protection was not seen in pigs given antiserum to the crude lysate or bacterin of LFB3 (group E) because turbinate damage seen at slaughter was severe (turbinate bones estimated to be reduced by 90%); this was confirmed when the reduction was calculated (Table 1 ). However, some protection was seen in pigs given a mixture of antisera to crude lysate and bacterin from LFB3 (group F) when damage was significantly less than that seen in pigs in group E given antiserum to crude lysate or to bacterin (P was less than 0.01 ). An immunoblot of antisera to crude lysate or bacterin, using crude lysate of LFB3 separated by SDS-PAGE as antigen, showed that a combination of these sera would give a product which contained antibodies to more antigens of P. multocida than each serum alone (Fig. 1 ); both antisera to the purified toxoids in immunoblots with crude lysate reacted with a single polypeptide of the same molecular weight as the toxin.
The role of toxin in colonisation The numbers of P. multocida which colonised the nasal cavities of groups B to F were compared with those recovered from pigs given acetic acid and infected with nontoxigenic P. multocida P37 (group G) or infected with P37
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N. C H A N T E R AND J.M. R U T T E R
A
13
Fig. 1. Immunoblot of crude lysate ofP. multocida LFB3 separated by SDS-PAGE and probed with antiserum to crude lysate (lane A), antiserum to bacterin (lane B). Markers indicate polypeptides uniquely detected by one or other antiserum. without acetic acid treatment (group H). Statistical comparison of each group with the others showed that colonisation by toxigenic or nontoxigenic P. multocida took the form o f one of two patterns into which all groups fell (Fig. 2 ). There was no significant difference in colonisation by toxigenic P. multocida in pigs given PBS, antiserum to crude lysate or bacterin or a mixture of anticrude lysate and anti-bacterin (groups B, E and F). Also, there was no significant difference in colonisation by toxigenic or nontoxigenic P. multocida in pigs given antiserum to a purified toxoid or given nontoxigenic strain P37, with or without prior acetic acid treatments (groups C, D, G and H ) . However, colonisation by P. multocida in groups C, D, G and H was by significantly fewer bacteria than in groups B, E and F (Fig. 2); the value of P for differences between group F and groups D or G was < 0.05 and the value of P for differences between the remaining groups was < 0.01.
PASTEURELLA MULTOCIDA IN A T R O P H I C R H I N I T I S O F PIGS
8
6
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261
B
A
t
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t
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Weeks after infection
Weeks after infection
Fig. 2. P. multocida in nasal washings of pigs. Graph (A): ( I ) , group B pigs given PBS and infected with toxigenic P. multocida LFB3; ( O ) , group E pigs given antiserum to the crude lysate or bacterin and infected with LFB3; ( • ) , group F pigs given a mixture ofantisera to crude lysate and bacterin and infected with LFB3. Graph (B): ( • ), group C pigs given antiserum to LFB3 toxoid and infected with LFB3; ( • ) , group D pigs given antiserum to 4661 toxoid and infected with LFB3; ( 0 ) , group G pigs treated with acetic acid and infected with nontoxigenic P. multocida P37; ( I ) , group H pigs infected with P37. Inset graph: ( ~ ) , mean of values for graph A (groups B, E, and F); (El), mean of values for graph B (groups C, D, G and H). Colonisation by each group in graph B was significantly different from each group in graph A (P at least less than 0.05 for each group in one line tested against each group in the other line).
Effect o f a bordetella bacterin on an antitoxoid response
Antitoxin was produced by pigs immunised with toxoid in alhydrogel without bordetella bacterin (group 4; log~o titres of 2 and 5), or with toxoid in FIA with bordetella bacterin (group 3; log~o titres of 4 and 4) or without bacterin (group 2; log~o titres of 3 and 3). However, pigs immunised with adjuvant alone (group 1 ) or with toxoid and bordetella bacterin in alhydrogel (group 5 ) did not produce antitoxin. DISCUSSION
The importance of antitoxin production after vaccination was illustrated when seven out of eight pigs given antitoxin were completely protected against turbinate damage. Pigs given antiserum to the crude lysate or bacterin were not protected even though these sera contained large amounts of antibody to bacteria detected by ELISA using crude lysate ofP. multocida as antigen. The slight mean turbinate atrophy detected from turbinate to body weight ratios in pigs given antitoxoid in groups C and D may be due to variation inherent
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N. CHANTERANDJ.M. RUTTER
in this method (see range of values for control group A). Alternatively, incomplete protection may have been detected in some pigs which was not detectable by visual examination. Nonetheless, visual examination did not suggest turbinate damage except in the one pig in group D. This pig given 50 ml of antiserum to the toxin from the type A strain was not fully protected because one of the ventral turbinates was reduced by 40%. Why this was so when the other turbinate was normal, and pigs given either 25 or 12.5 ml of the same serum where protected even though colonised by indistinguishable numbers of toxigenic bacteria, is unclear. The general protection against turbinate atrophy caused by infection with a toxigenic strain of capsule type D, in pigs given antibody to the toxoid from a capsule type A isolate, suggested a very close similarity or identity between toxins from these bacteria. The intermediate damage in pigs passively immunised with a mixture of antisera to crude lysate and bacterin suggested a degree of protection. One possible explanation may come from the observation that these sera when combined contained antibodies to more antigens than each serum alone. The partial protection could not be explained by the presence of undetectable amounts of antibody to toxin because if either antisera had contained antitoxin, then protection would have been seen in pigs given either antiserum to bacterin or to crude lysate (group E). Consequently, there may be a mechanism of protection based on a combination of antibodies to different antigens. If this occurred it was apparently unrelated to a reduction in colonisation; perhaps a different mechanism of protection may either alter colonisation by P. multocida away from a precise site where toxin can cause damage, or reduce the expression of toxin. Toxigenic P. multocida colonised pigs, treated with acetic acid and given antiserum to purified toxoid, to a degree indistinguishable from that seen with pigs infected with nontoxigenic P. multocida, with or without prior acetic acid treatment. Colonisation in these animals was also significantly less than that seen with infections of toxigenic P. multocida in pigs unprotected by PBS or antiserum to crude lysate or bacterin. These observations suggested that the mechanism by which acetic acid enhanced colonisation by P. multocida was dependent on the action of the toxin in an additive or synergistic fashion. This possibility was supported because colonisation by nontoxigenic P. multocida was not enhanced by treatment of the nasal cavity with acetic acid. However, this line of evidence is dependent on the assumption that nontoxigenic P. multocida possess the same colonisation factors as toxigenic P. rnultocida, other than the toxin itself. The cytotoxin of B. bronchiseptica has been shown to enhance colonisation by toxigenic P. multocida (Chanter et al., 1989) but the mechanism for this, like the action of acetic acid treatment, is unknown. There was, however, evidence that the stimulatory effect of the bordetella cytotoxin was dependent on, or synergistically enhanced, by the action of the pasteurella toxin. It has
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been speculated that adhesion to turbinate mucosal cells may be a necessary step in the pathogenesis of atrophic rhinitis. However, several authors (reviewed by Chanter and Rutter, 1989) using in vitro tests have not consistently detected adhesion to these or other cells by toxigenic P. multocida. The toxin does not, therefore, presumably act as an adhesin although attempts to detect adhesion in vivo or compare turbinate mucosa colonised by toxigenic or nontoxigenic P. multocida have not been published. There remains speculation that the toxin enhances colonisation by causing damage and the release of nutrients more favourable for growth or that it interferes with nonspecific or specific immunological defences to reduce clearance of bacteria. However, in the experiment described here, even when the action of the toxin was not eliminated by antitoxin, numbers of toxigenic P. multocida still declined at a time when damage to the mucosa would have been at its most severe. Consequently, colonisation by toxigenic P. multocida seems to be a complex and dynamic process with much still to be learnt. The reduction in colonisation caused by antitoxin nonetheless did not reduce the mean numbers of bacteria to much below 103 c f u / m l of nasal wash. This reduction was much less than the complete elimination of infection observed by others (Baars et al., 1986). Nontoxigenic P. multocida do not seem to require toxin to establish colonisation so it would seem unlikely that antibody to toxin alone could eliminate colonisation by toxigenic bacteria from pig herds. Failure by others to recover toxigenic P. multocida from pigs containing antitoxin may have been due to low sensitivity of isolation techniques because of the background flora in conventional animals. Alternatively it is still possible that these bacteria were not present because of competition from the normal flora. The failure of pigs to respond to the toxoid in alhydrogel with a bacterin of B. bronchiseptica was unexpected, in view of the well-recognised adjuvant properties of bacterins made from the closely related organism B. pertussis. The mechanism explaining these results is not known but it is particularly relevant to vaccination against atrophic rhinitis. The wide spread prevalence ofB. bronchiseptica in pig herds and experimental evidence that these bacteria can both cause mild turbinate damage themselves and also predispose pigs to colonisation by toxigenic P. multocida, will ensure that atrophic rhinitis vaccines are likely to contain a bordetella component. Colonisation by B. bronchiseptica requires an adhesin (Collings and Rutter, 1985; Semjen and Magyar, 1985 ) and the cytotoxin of B. bronchiseptica is a key factor which enhances colonisation by toxigenic P. multocida (Chanter et al., 1989). Currently, atrophic rhinitis vaccines contain a bordetella bacterin rather than important subunit components. A P. multocida toxoid vaccine containing important subunit components ofB. bronchiseptica may not only provide better protection against bordetella and pasteurella infections but may also reduce the likelihood of failed responses to the P. multocida toxoid. This would be
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true so long as any of the isolated bordetella components did not interfere with an antitoxin response. ACKNOWLEDGEMENTS
The authors would like to thank Mr. M. Dennis and his staff for the production and care of pigs, Miss K. Walker, Miss H. Williamson and Miss C. Thomson for technical assistance and Mrs. K. Bunch for statistical analyses. REFERENCES Baars, J.C., Pennings, A. and Storm, P.K., 1986. Challenge and field experiments with an experimental atrophic rhinitis vaccine, containing Pasteurella multocida DNT-toxoid and Bordetella bronchiseptica. Proc. ninth Congress of the International Pig Veterinary Society, p. 247. Chanter, N. and Rutter, J.M., 1989. Pasteurellosis in pigs and the determinants of virulence of toxigenic Pasteurella multocida. In: C. Adlam and J.M. Rutter (Editors), Academic Press, Harcourt Brace Iovanovich, London, pp. 161-196. Chanter, N., Rutter, J.M. and Mackenzie, A., 1986a. Partial purification of an osteolytic toxin from Pasteurella multocida. J. Gen. Microbiol., 132: 1089-1097. Chanter, N., Rutter, J.M. and Mackenzie, A., 1986b. Partial purification of an osteolytic toxin from Pasteurella multocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 228. Chanter, N., Magyar, T. and Rutter, J.M., 1989. Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Res. Vet. Sci., 47: 48-53. Collings, L.A. and Rutter, J.M., 1985. Virulence ofBordetella bronchiseptica in the porcine respiratory tract. J. Med. Microbiol., 19: 247-258. Dominick, M.A. and Rimler, R.B., 1986. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am. J. Res., 47: 1532-1536. Foged, N.T., Nielsen, J.P. and Jorsal, S.E., 1989. Protection against progressive atrophic rhinitis by vaccination with Pasteurella multocida toxin purified by monoclonal antibodies. Vet. Rec., 125:7-11. Kobisch, M. and Pennings, A., 1986. An evaluation ofNobi-vac AR and an experimental atrophic rhinitis vaccine, containing Pasteurella rnultocida DNT-toxoid and Bordetella bronchiseptica, in pigs. Proc. the ninth Congress of the International Pig Veterinary Society, p. 246. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 681-685. Magyar, T., Chanter, N., Lax, A.J., Rutter, J.M. and Hall, G.A., 1988. The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica. Vet. Microbiol., 18:135-146. Nagy, L.K., Mackenzie, T. and Scarnell, J., 1986. Serum antibody values to Pasteurella multocida type D toxin and susceptibility of piglets to experimental challenge with toxigenic type D P. rnultocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 224. Nakai, T., Sawata, A., Tsuji, M., Samejima, Y. and Kume, K., 1984. Purification of dermonecrotic toxin from a sonic extract ofPasteurella multocida SP-72 serotype D. Infect. Immun., 46: 429-434. Nielson, J.P., Pedersen, K.B. and Willeberg, P., 1986. Epidemiology of atrophic rhinitis and toxin producing Pasteurella multocida. Proc. ninth Congress of the International Pig Veterinary Society, p. 239.
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