Veterinary Microbiology, 14 (1987) 393-409 393 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

P r o t e c t i o n of S h e e p aga i ns t Footrot w i t h a R e c o m b i n a n t D N A - B a s e d F imbr ia l Vacc ine

J.R. EGERTON, P.T. COX, B.J. ANDERSON, C. KRISTO, M. NORMAN and J.S. MATTICK 1

Department of Veterinary Clinical Studies, The University of Sydney, Sydney, N.S. W. 2006 (Australia)

~Division of Molecular Biology, Commonwealth Scientific and Industrial Research Organization, North Ryde, N.S. W.2113 (Australia)

(Accepted for publication 13 January 1987)

ABSTRACT

Egerton, J.R., Cox, P.T., Anderson, B.J., Kristo, C., Norman, M. and Mattick, J.S., 1987. Protec- tion of sheep against footrot with a recombinant DNA-based fimbrial vaccine. Vet. Microbiol., 14: 393-409.

Recombinant Pseudomonas aeruginosa cells containing the Bacteroides nodosus fimbrial sub- unit gene under the transcriptional control of a strong promoter produce large amounts of B. nodosus-type fimbriae. We have carried out vaccination trials which show that these fimbriae are just as effective as either natural fimbriae or whole cell preparations of B. nodosus in inducing protective immunity against homologous footrot challenge. The recombinant-produced fimbriae are also effective therapeutically in accelerating the rate of healing of pre-existing footrot lesions. These results confirm that the structural subunit of the fimbrial strand is a primary protective antigen against footrot, and demonstrate the practicality and potential of recombinant DNA approaches to the development of new vaccines against B. nodosus and other Type 4 fimbriate pathogens.

INTRODUCTION

Ovine footrot is a mixed bacterial infection, with Bacteroides nodosus, a gram- negative non-sporing anaerobe, being the essential etiological component of the infective synergy (Beveridge, 1941; Egerton et al., 1969; Roberts and Eger- ton, 1969 ). One characteristic of virulent isolates ofB. nodosus is the presence of fine surface filaments termed fimbriae (or pili) (Stewart, 1973; Walker et al., 1973; Every and Skerman, 1983 ), which are thought to play a major role in the colonization of the epidermal matrix of the hoof (for a recent review, see Mattick et al., 1985). Sheep can be immunized against experimental and field challenge with footrot by vaccination either with whole B. nodosus cells ( Eger-

0378-1135/87/$03.50 © 1987 Elsevier Science Publishers B.V.

394

ton and Burrell, 1970; Egerton and Roberts, 1971) or with isolated fimbrial preparations (Stewart, 1978; Stewart et al., 1982; Every and Skerman, 1982; Lee et al., 1983). The fimbriae also appear to be directly involved in the ser- ological K-agglutination reaction (Egerton, 1973; Walker et al., 1973; Every, 1979 ), which has been used to classify field isolates into at least 8 major sero- groups and a number of subtypes (Egerton, 1974; Schmitz and Gradin, 1980; Claxton et al., 1983). Correspondingly, the range of protection conferred by vaccination with either whole cells or isolated fimbriae is largely restricted to the serogroup involved (Egerton, 1974; Every and Skerman, 1982; Stewart et al., 1982). Based on these studies, a number of traditional whole cell vaccines, comprising mixtures of representatives of each of the serogroups, are now com- mercially available. However, these vaccines are relatively expensive, appar- ently not only because of their multivalency, but also because of the fastidious growth requirements of B. nodosus and the difficulty of maintaining stable fimbrial production under liquid culture conditions (see e.g. Skerman, 1975; Skerman et al., 1981).

We have attempted to bypass some of the problems associated with conven- tional footrot vaccines by developing a recombinant DNA-based fimbrial vac- cine (Mattick et al., 1985, 1987 ). B. nodosus fimbriae consist of a single repeated structural subunit of about 17 000 molecular weight, which varies among dif- ferent serotypes and serogroups (Every, 1979; McKern et al., 1985; Anderson et al., 1986). These fimbriae belong in the category of Type 4 (Ottow, 1975; Mattick et al., 1987), which are characterized by their polar location on the cell, the phenomenon of "twitching motility" (Henrichsen, 1983), and the conserved amino-terminal sequence of the structural subunit (McKern et al., 1983). Similar fimbriae are observed in a range of other (potentially) patho- genic bacteria, such as a number of species classified within the genera Mor- axeUa, Neisseria and Pseudomonas (see Henrichsen, 1983; Mattick et al., 1987).

The genes encoding the fimbrial subunits representative of most of the B. nodosus serogroups have been cloned and expressed in Escherichia coli (Ander- son et al., 1984; Elleman et al., 1984; Mattick et al., 1985). However, the expressed subunits are not assembled into the mature fimbriae in E. coli, but rather remain embedded within the cell membrane (Anderson et al., 1984; Elleman et al., 1986). This is clearly inappropriate for vaccine production, both because of the impracticalities of antigen recovery and purification, and because intact fimbriae appear to be required to elicit an effective immune response (see Mattick et al., 1985 ). This problem has recently been overcome by placing the B. nodosus fimbrial subunit gene into a compatible Type 4 tim- briate host, Pseudomonas aeruginosa, which (under the control of a strong transcriptional promoter) expresses high levels of fimbriae that are structur- ally and antigenically indistinguishable from those produced by B. nodosus itself (Mattick et al., 1987). In this paper, we examine the efficacy of these recombinant-produced fimbriae in eliciting prophylactic and therapeutic immune responses against footrot in sheep.

395

MATERIALS AND METHODS

Sheep

Adult Merino wethers, free of prior experience of B. nodosus infection, were used.

Bacteria

The prototype of B. nodosus Serogroup A, isolate VCS1001 (ATCC 25549) (Claxton et al., 1983), was used as the source of whole cell and B. nodosus fimbrial vaccines, for agglutination test antigen, and as the challenge strain in vaccination trials.

The multifimbriate P. aeruginosa strain PAK/2Pfs (ATCC 53308) (Brad- ley, 1974) was used as the source of normal P. aeruginosa fimbriae, as well as the host for the production of B. nodosus-type fimbriae from the recombinant plasmid pJSM202 (Mattick et al., 1987). Plasmid pJSM202 (ATCC 40203) contains the B. nodosus fimbrial subunit gene, isolated from VCS1001 (Ander- son et al., 1984), under the transcriptional control of the strong promoter, PL, from bacteriophage 2, inserted into the broad-host-range vector pKT240 (Bagdasarian et al., 1983). For a complete description of these constructions and the characterization of the fimbriae produced by P. aeruginosa cells con- taining pJSM202, see Mattick et al. (1987).

Preparation of vaccine antigens

Bacteroides nodosus whole cell preparation B. nodosus VCS1001 cells were grown on hoof agar plates (Thomas, 1958)

for 4 days at 37°C in an anaerobic atmosphere of 90% H2/10% CO2 and har- vested by scraping into cold phosphate-buffered saline (PBS) (140 mM NaC1, 10 mM Na phosphate, pH 7.2 ) (Mattick et al., 1984). Formalin was added to a final concentration of 0.25%.

Bacteroides nodosus fimbrial preparation B. nodosus fimbriae were purified from the supernatant of PBS-suspended

cells by isoelectric precipitation with 0.1 M sodium acetate, pH 4.5, as described previously (Mattick et al., 1984).

Pseudomonas aeruginosa fimbrial preparation Normal P. aeruginosa fimbriae were isolated from PAK/2Pfs cells cultured

aerobically at 37 ° C overnight on nutrient ( Luria broth) agar plates. The cells were harvested by scraping into cold PBS, and the resulting suspension blended for 1 min using a Silverson Mixer-Emulsifier. All steps were carried out at

396

4 ° C. The cells and cell debris were removed by centrifugation (24000 x g for 30 min ) and the supernatant adjusted to pH 4.5 by the addition of acetic acid. After standing overnight, the fimbriae were collected by centrifugation (24000 ×g for 30 min). The fimbriae were then further purified by a second round of dissolution in PBS and precipitation with sodium acetate, pH 4.5.

Recombinant fimbrial preparation P. aeruginosa PAK/2Pfs cells containing pJSM202 were cultured aerobi-

cally at 37 °C overnight on nutrient (Luria broth) agar plates supplemented with the antibiotic carbenicillin (1 mg ml-1), to ensure the maintenance of the recombinant plasmid. The cells were harvested in PBS and subjected to mechanical blending as described above. The fimbriae were then purified from the (cell-free) supernatant by two rounds of isoelectric precipitation with sodium acetate, pH 4.5, as described above, or by the independent method of two rounds of precipitation with 0.1 M MgC12 (Mattick et al., 1987).

Antigen analysis and characterization

The contents and purity of the various antigen preparations were checked by electrophoretic display and Western transfer analyses. Whole cell or fim- brial samples were displayed on sodium dodecyl sulphate-urea gradient (8-15%) polyacrylamide gels using a modified Laemmli buffer system (Mat- tick et al., 1984), and either stained for protein using Coomassie Blue R250, or electrophoretically transferred to nitrocellulose paper (Towbin et al., 1979), followed by incubation with anti-fimbrial antiserum, and 125I-labelled protein A, as detailed in Anderson et al. (1986). Diagnostic anti-fimbrial antisera were raised in rabbits vaccinated with purified fimbriae as described previously ( Mattick et al., 1984 ). Anti-fimbrial antisera were used at a dilution of 1 : 1000.

Vaccine formulation

Vaccines were formulated in an alum-oil adjuvant system as described below. In the case of the B. nodosus whole cell vaccine, the concentration of the cell suspension was measured spectrophotometrically and adjusted so that after adsorption onto alum (see below; Thorley and Egerton, 1981), there were approximately 101° cells ml- 1. One hundredth volume of Tween 80 was then added and the suspension emulsified with an equal volume of Freund's incom- plete adjuvant. Vaccine doses were 2 ml.

In the case of fimbrial preparations, the fimbriae were redissolved in PBS and the protein concentration measured using a Coomassie Blue dye-binding assay (Bradford, 1976). The protein concentration was then adjusted to yield a standard dose of 250 fig (or in one case, 500 pg) fimbriae m1-1 after adsorp- tion with alum and the addition of Tween 80. Alum adsorption was carried out

397

by the addition of one seventh volume of 10% AIK (SO4)2 and adjustment of the pH to 6.2. After standing on ice for 1 h, one hundredth volume of Tween 80 was added and the mixture emulsified with an equal volume of Freund's incomplete adjuvant. Again vaccine doses were 2 ml.

All vaccine antigens were treated with 0.25% formalin and checked for ste- rility by plating 100~l of the preparations (prior to emulsification in adjuvant) on appropriate media.

Vaccination schedules, challenge and assessment

Prophylactic trial Sheep were randomly allocated to 7 groups of 10 or 11 individuals and inoc-

ulated subcutaneously with various vaccine preparations as follows: Group 2, whole cells (10 TM) of B. nodosus; Group 3, purified fimbriae (250 ~g) from B. nodosus; Group 4, normal P. aeruginosa PAK/2Pfs fimbriae ( 250 ~g) ; Groups 5 and 6, fimbriae isolated from recombinant P. aeruginosa cells containing pJSM202 (250 and 500 zg, respectively); Group 7, recombinant fimbriae pur- ified by the alternative method of MgC12 precipitation (250 ~g). Group 1 remained uninoculated. The dose was repeated 28 days later. The individuals from the various groups remained mixed throughout the duration of the experiment.

One week after the second vaccine dose, the vaccinated animals and the uninoculated controls were placed (at random) in a series of neighbouring pens, the floors of which were covered with sponge mats and kept wet. After 5 days predisposition in this environment, the sheep were challenged with B. nodosus VCS1001 cells by the method described by Egerton and Roberts (1971). Twenty-one days after challenge, the feet of all sheep in the experiment were closely examined and allocated a score of lesion severity on a scale of 2 to 4 (Egerton and Roberts, 1971 ), as follows: 2, obvious interdigital dermatitis; 3, underrunning lesions of the soft horn of the heel of the hoof; 4, underrunning lesions of the hard horn of the hoof. A lesion score of 1 is normally applied to feet exhibiting symptoms of mild interdigital dermatitis. This may be due to other causes, especially in the damp environment used in these experiments, and hence was not considered in the assessment of footrot (cf. Stewart et al., 1982 ). Such feet, and those that were unaffected, were given a score of 0.

Therapeutic trial Twenty-one sheep severely affected with footrot as a result of B. nodosus

challenge in the prophylactic trial were used to assess the curative effects of the recombinant fimbrial vaccine. These animals, which were derived from the two negative control groups (1, uninoculated; 4, vaccinated with normal P. aeruginosa fimbriae), were divided into 3 groups (8, 9 and 10) of 7 animals, such that the animals in each group had similar scores of severity of footrot.

398

In addition, given this restriction, the animals from the original Groups I and 4 were distributed as evenly as possible among the 3 new groups.

The sheep in Groups 8 and 9 were vaccinated at monthly intervals with whole B. nodosus cells, or with recombinant fimbriae, respectively. The objec- tive was to raise the serum agglutinin titre to a level considered adequate for the expression of therapeutic activity. Altogether, Group 9 received four 2-ml doses of vaccine, each containing 250 pg of fimbriae isolated from P. aeruginosa containing pJSM202, and Group 8 received three 2-ml doses of whole cell (10 l°) B. nodosus VCS1001 vaccine. The third group (10), received no treatment. All sheep were housed together for the 4-month duration of the trial in a damp environment conducive to the persistence of footrot. One week prior to the conclusion of the trial, all sheep were run together on a dry pasture. All feet were examined at regular intervals and the severity of footrot lesions assessed as described above.

Serology

The serum agglutinin titre against B. nodosus was measured in the sheep prior to and at regular intervals after vaccination and challenge, using the tube agglutination test described by Egerton (1973). The agglutination test antigen was unwashed VCS1001 cells harvested from hoof agar plates in PBS. Sera (prepared from jugular vein blood samples) were initially diluted 1 in 10, and thereafter in 2-fold increments, and the titre was recorded as the inverse of the highest dilution at which the K-agglutination reaction was visible.

RESULTS

Characterization of vaccine antigens

The content and purity of the various whole cell and fimbrial antigens used in the vaccination trials were examined by electrophoretic display and immu- nological analyses (Fig. 1 ). The Coomassie profiles (Fig. 1A) show that each of the fimbrial preparations was essentially pure, and also that there was an approximately equivalent amount of fimbriae present in the whole B. nodosuz cell preparation. The fimbriae clearly represent the major protein component of this fraction. The fimbrial subunit from normal P. aeruginosa PAK/2Pfs cells has an apparent molecular weight of about 16000, which is smaller than that of the fimbrial subunit ofB. nodosus VCS1001 (apparent molecular weight about 17000). The actual sizes of these proteins, calculated from sequence (Pasloske et al., 1985; Elleman and Hoyne, 1984), are 15082 (145 amino-acids) and 16104 (151 amino-acids), respectively. The fimbriae produced by the recombinant P. aeruginosa cells containing the plasmid pJSM202 are com- posed of a subunit of the same size as that from B. nodosus. There was little

399

A .....

W

w t

i ¸ W

~ ~ ~iill ̧ O

B

/

C

1 2 3 4 5 6 7

Fig. 1. Characterization of vaccine antigens. Samples of the antigen preparations used in the vaccination trials, equivalent in each case to 2% of the standard vaccine dose (see Text), were displayed by electrophoresis on sodium dodecyl sulphate-urea gradient 8-15% polyacrylamide gels and stained for protein with Coomassie Blue R250 (panel A). Identical displays were subjected to Western transfer analyses using either anti-P, aeruginosa fimbrial antiserum (panel B ) or anti- B. nodosus fimbrial antiserum (panel C), as described in Materials and Methods. The entire gel display is shown in panel A, whereas only the lower portions of the corresponding autoradiograms (containing the fimbrial subunits ) are shown in panels B and C. Lanes: 2, whole cells ofB. nodosus VCS1001; 3, fimbriae isolated from B. nodosus VCS1001; 4, fimbriae purified from P. aeruginosa PAK/2Pfs; 5 and 6, fimbriae isolated from P. aeruginosa PAK/2Pfs containing pJSM202 using either isoelectric or MgCl2 precipitation, respectively; 1 and 7, standard protein molecular weight markers (Bethesda Research Laboratories), from top to bottom; myosin H chain (Mr 200 000 ), phosphorylase B (Mr 97 400), bovine serum albumin (Mr 68 000), ovalbumin (Mr 43 000), ~- chymotrypsinogen (Mr 25 700), fl-lactoglobulin (Mr 18 400) and lysozyme (Mr 14 300).

400

TABLE I

Prophylactic vaccination against footrot ~

Group

1 2 3 4 5 6 7

Vaccine Ni l B. nodosus B. nodosus P. aeruginosa Recombinant Recombinant Recombinant cells fimbriae fimbriae fimbriae fimbriae fimbriae

( MgCl2 ) (10 lo ) 250 #g 250 ttg 250 ttg 500 pg 250 #g

Underrunning infections 25/40 7/44 6/40 29/44 9/44 6/40 8/40

GMFS 2.2 0.7 1.1 2.5 1.0 0.75 0.9

GMAT 200 7000 3400 200 8000 6800 3200

Groups contained either 10 or 11 animals, and were vaccinated and challenged as described in Materials and Methods. The clinical and serological responses shown were those measured 3 weeks after challenge. Under- running infections were those observed in feet with lesion scores of 3 or 4, expressed as the proportion of total feet in the group. GMFS is the group mean footseore, and was calculated as the sum of the lesion scores of all animals in the group, divided by the number of feet in the group. GMAT is the group mean agglutinin titre, expressed as the geometric mean value for the group, rounded off to the nearest 100.

difference in the quality of the preparations obtained by the two independent methods of fimbrial recovery, isoelectric or MgC12 precipitation (Fig. 1A).

The production of B. nodosus-type fimbriae by recombinant P. aeruginosa cells was then confirmed by reciprocal Western transfer analyses using specific anti-P, aeruginosa (Fig. 1B) or anti-B, nodosus (Fig. 1C) fimbrial antisera. These fimbriae reacted strongly with the B. nodosus antiserum, whereas within the limits of detection, no reaction was observed with the P. aeruginosa anti- serum. This antiserum reacted strongly with the natural fimbriae of P. aeru- ginosa PAK/2Pfs.

Prophylactic vaccination trial

This trial was carried out using 7 groups of 10 or 11 animals, organized as follows (see Table I ) : Groups 1 and 4 were negative controls, being untreated or vaccinated with normal P. aeruginosa fimbriae, respectively; the positive controls, Groups 2 and 3, were immunized with B. nodosus whole cells or with fimbriae, respectively; the experimental Groups 5, 6 and 7 were vaccinated with fimbriae from the recombinant P. aeruginosa, using standard dose, double dose or fimbriae purified by a different procedure, respectively (see Table I and Materials and Methods). The standard dose of fimbriae used in these experiments ( 250 ]xg) was selected after reference to previous vaccination trials. Vaccination doses as low as 38/~g (Stewart et al., 1983 ) and as high as 400-1000 /xg (Every and Skerman, 1982) have been reported to elicit good protective

401

responses. The level of 250/lg lies well within this range. Two doses were given 1 month apart, a protocol which approximates normal field usage.

The response of groups of sheep to the various vaccines is summarised in Table I. The sheep in Groups 2, 3, 5, 6 and 7, i.e. those receiving B. nodosus whole cells, purified fimbriae, or fimbriae derived from recombinant cells, all developed substantial serum agglutinin titres against B. nodosus VCS1001 cells, with geometric mean values of between about 3000 and 8000. Statistically, the agglutinin levels recorded in these various groups were not significantly differ- ent from each other. However, these values were (all) somewhat less than had been expected for fimbriate vaccines in alum-oil adjuvant ( Thorley and Eger- ton, 1981; cf. Stewart et al., 1986 ), but similar to those reported using oil-based vaccines (see e.g. Every and Skerman, 1982; Stewart et al., 1982, 1983, 1986). Infection with footrot itself does not usually induce a significant rise in homol- ogous agglutinin levels (Thorley and Egerton, 1981 ) and this was evident again in the 2 negative control Groups 1 and 4, both of which exhibited low serolog- ical titres (Table I ). This suggests that the immune system of the sheep remains relatively naive or insensitive to the invasion of the epithelial matrix of the hoof by B. nodosus (see Discussion). There were no obvious signs of systemic reactions following vaccination with fimbriae isolated from P. aeruginosa, even in animals from Group 6 which received the higher (500/~g) dose. No differ- ences were observed in the granulomas which developed at sites inoculated with different vaccines.

The response of each group of sheep to experimental footrot was measured by the following criteria: (i) the proportion of feet which developed severe infections, i.e. infections which resulted in invasion of the epidermal matrix of the hoof by the bacterial flora of footrot; (ii) the mean foot score, which was derived from the sum of scores (on the scale 0-4) assigned to each challenged foot divided by the number of feet challenged in each group. The results (Table I) clearly show that the recombinant fimbriae confer the same level of protec- tion as B. nodosus whole cells or fimbriae against homologous footrot chal- lenge. Again, there was no significant difference between the positive controls and experimental groups. Complete immunity against footrot is rarely observed in such vaccination trials, especially when a highly virulent strain such as VCS1001 is involved. The response of individual animals to vaccination and/or challenge is quite variable (see below; Table II) . Nevertheless, and although neither the natural nor the recombinant material conferred full immuni ty in this trial, there was a large and, in practical terms, important difference between the vaccinated groups and negative controls. All animals in the latter category (i.e. untreated or vaccinated with normal P. aeruginosa fimbriae) became affected with severe footrot, with about 65% of all feet having underrunning lesions. The average lesion score for these groups was 2.4. Such lesions were relatively rare in the groups vaccinated with B. nodosus cells or fimbriae, or fimbriae produced by recombinant cells, severe clinical signs being observed

402

TABLE II

Comparison offootscore and agglutinin t i t res of individual sheep in selected g roups f romprophy- lactic trial I

Animal Group 2 Group 4 Group 5 B. nodosus whole cells P. aeruginosa f imbriae Recombinan t fimbriae (101°) (250 ~g) (250#g)

Footscores Ti t re Footscores Ti t re Footscores Ti t re

1 0030 10 240 3333 80 2333 10 240 2 0003 10 240 0303 40 0332 2 560 3 0000 5 120 2033 80 0000 10 240 4 0003 5 120 0023 80 0000 20 480 5 2023 2 560 3433 640 0000 20 480 6 2000 10 240 4442 80 0223 2 560 7 0000 5 120 4432 640 3323 5 120 8 3330 5 120 2020 160 0022 5 120 9 0020 10 240 4433 1 280 0020 10 240

10 0000 20 480 4432 640 0020 5 120 11 0000 5 120 3033 1 280 0000 20 480

Group mean 0.7 7 000 2.5 200 1.0 8 000

1 Footscores and serum agglutinin t i t res were measured as described in Materials and Methods. The data were taken 3 weeks after challenge ( see Table I ). Individual footscores were recorded in the following order: f ront left, f ront right, rear left, rear right. The group mean footscores and agglutinin t i t res are the a r i thmet ic and geometric values, respectively.

in only 15-20% of all feet. The average lesion score among these groups was 0.9; this effectively means the difference between sheep which are debilitated by the disease, and those which are mildly affected, albeit with some exceptions ( see e.g. Table II ). This practical difference is important in the field.

As has been observed in previous vaccination trials (Thorley and Egerton, 1981; Every and Skerman, 1982; Stewart et al., 1982; Lee et al., 1983), there was a positive correlation between the mean agglutinin titre and the clinical response of each of the groups of sheep involved (Table I) . However, it was also equally clear that at an individual level this correlation breaks down, there being no strict relationship between the agglutinin titre and the severity of lesions, irrespective of whether natural or recombinant material was used. Representat ive data are shown in Table II. It has been suggested that this phenomenon is a consequence of differences in individual susceptibility to footrot or the severity of challenge (Stewart et al., 1983). However, the more obvious implication is that the serum agglutinin titre is not directly related to the immunoprotect ive status of the animal. This view was reinforced by exam- ining the average footscores of animals recording various levels of agglutinin titre (Fig. 2 ). Beyond a threshold value (titre 2560 ), there was a rapid decline

403

2 . 5 -

w 2 7

O 0 2-0- Or) b.-

0 0 1.5- IJ.

z "~ I"O- I,U =Z

0-5-

5 _// •

i i i I i i 2 3 4 ; ( i 7 8 9 1 ; 1'1

LOG2[AGGLUTININ TITRE (xlO-1l]

Fig. 2. Relationship between the agglutinin titre against B. nodosus and the clinical severity of footrot following vaccination and challenge. The agglutinin titre is expressed as the inverse of the maximum serum dilution at which the K-agglutination reaction (Egerton, 1973) was visible, and is plotted on a logarithmic scale. The clinical signs of footrot were assessed on the basis of foot- scores, on a scale 0-4, as described in Materials and Methods. The data was pooled from all 8 groups (84 sheep) involved in the prophylactic vaccination trial, 3 weeks after challenge (see Tables I and I I ) . The number of animals recording a given agglutinin titre is indicated beside each point.

in the number of affected feet. This threshold is consistent with that value (3000-5000) previously suggested as the minimum commensurate with group protective immunity (Thorley and Egerton, 1981; Stewart et al., 1982; Lee et al., 1983). Nevertheless, beyond this threshold there appeared to be little fur- ther gain in protection as the titre increased, up to levels close to the maximum elicited by current vaccination procedures ( noting also that relatively few ani- mals fell into these upper classes). Similar trends are evident in earlier vacci- nation trials (see e.g. Stewart et al., 1983 ).

Therapeutic vaccination trial

Vaccination of sheep with B. nodosus cells has been shown to accelerate healing in affected animals (Egerton and Burrell, 1970; Egerton and Roberts, 1971 ), presumably as a consequence of alerting an otherwise unaware immune system to the presence of epidermal B. nodosus infection. The antigen(s) responsible for this phenomenon have not been identified.

The prophylactic vaccination trial was therefore extended to examine the therapeutic potential of the recombinant-produced fimbriae. Twenty-one ani- mals affected with severe footrot from the negative control groups were divided into 3 equivalent groups of 7 animals (see Materials and Methods). These groups were left untreated or were vaccinated either with B. nodosus whole

404

TABLE III

Therapeutic vaccination (with GMFS or GMAT) against footrot ~

Time (months)

0 1 2 3 4

GMFS GMAT GMFS GMAT GMFS GMAT GMFS GMAT GMFS GMAT

B. nodosus whole cells (10 l° ) 2.9 N.D. 1.4 1400 1.6 6200 1.5 7600 0.7 6900 Recombinant fimbriae (250/~g) 2.9 N.D. 1.5 300 1.4 1100 1.4 2300 0.9 3100 Nil 2.7 N.D. 2.1 200 1.9 300 2.3 400 1.9 600

Groups contained 7 animals each, derived from the negative control Groups 1 and 4 of the prophylactic trial ( see Table I and Materials and Methods ). GMFS and GMAT refer to the group mean footscore and agglutinin titre, respectively, as described in the legend to Table I. N.D., not determined.

cells, or with fimbriae isolated from recombinant P. aeruginosa cells. The results are shown in Table III. In this case, aggutinin titres were slow to develop in the vaccinated animals, especially in the recombinant group, although the reason for this is unknown. Adequate levels were obtained after additional doses. However, despite the relatively low levels of agglutinins recorded, there was clear evidence that footrot was reduced in severity in the vaccinated groups, compared to that persisting in the untreated animals. No real difference was observed between the group vaccinated with B. nodosus and those with recom- binant fimbriae. The most marked difference between these groups and the controls followed a period of 1 week when sheep were held on pasture rather than in an artificially moist environment. At this point, the group mean foot- scores were 0.7, 0.9 and 1.9 for the whole cell, recombinant fimbriae and control groups, respectively. Hence it does appear that homologous fimbriae are capa- ble of accelerating healing in footrot-affected sheep.

D I S C U S S I O N

The results presented in this paper indicate that the recombinant-produced fimbriae are as effective as either whole cells or isolated fimbriae from B. nodo- sus in inducing protective immunity against homologous footrot infection. This finding also provides unequivocal evidence that important protective epitopes against footrot are located on the structural subunit of the fimbrial s trand itself. A variety of studies have indicated that isolated fimbriae are as good as, if not superior to, whole cells in eliciting protective immunity to the disease (Stewart , 1978; Every and Skerman, 1982; Lee et al., 1983), but there has always been the interpretational caveat that, despite the degree of purification, it was not possible to guarantee that such fractions were free of contaminat ion with other cellular material of antigenic significance. This problem was recently

405

highlighted by the finding that isolated B. nodosus fimbriae almost invariably contain a second polypeptide, of about 80000 molecular weight, which appears to represent the basal protein linking the fimbrial strand to the surface of the cell (Mattick et al., 1984; Anderson et al., 1986). This protein is a prominent antigen in vivo and in vitro. Subsequent electrophoretic and immunological surveys of the structural subunit and basal protein antigens in the context of the serological system have shown that it is the structural subunit which is the primary serotype-specific, and by implication protective, antigen (Mattick et al., 1985; Anderson et al., 1986). This has now been confirmed: the recombi- nant P. aeruginosa cells contain only 576 nucleotide pairs of B. nodosus DNA, encoding one polypeptide (Mattick et al., 1987). Clearly, and provided it is expressed in the proper form, this protein is sufficient on its own to establish protective immunity against footrot.

The results of this study also provide the first evidence that it is the fimbriae which are responsible for the accelerated healing of footrot. The therapeutic potential of whole cell vaccines against footrot is an unusual but well-estab- lished feature of the system (Egerton and Roberts, 1971 ). Most data have been derived from field studies (Egerton and Burrell, 1970; Egerton et al., 1978) where there had been some evidence that the curative effects of vaccination may not have been strictly serogroup-specific, i.e. that an antigen or anti- gen (s) other than fimbriae may have been involved. The present results sug- gest that this might not be the case, and that the fimbriae do play a central role in both prevention and treatment of footrot by vaccination. The use of immunotherapy should perhaps be more broadly considered, as an important adjunct to immunoprophylaxis in the management of chronic infections. This is particularly relevant for superficial infections affecting epithelial tissues, not only footrot but also such conditions as bovine keratoconjunctivitis, which is caused by the Type 4 fimbriate bacterium M. bovis, and for which antibiotic therapy is not practical under extensive field conditions.

Our results also suggest that the nature of the relationship between the agglutinin titre against B. nodosus and protective immunity against footrot should be reassessed. While there is clearly a correlation, this appears to be more qualitative than quantitative. It should be noted that high agglutinin titres are only engendered by vaccination (normally with adjuvants ) and that, in this sense, we are dealing with an artificial response. There seems little doubt that serological relationships (or differences) among strains, as defined by the K-agglutination test, are closely linked to the range of immunoprotec- tion conferred by vaccination. Nevertheless, closer examination of the data does suggest that the agglutinin titre should be viewed simply as an index of exposure and the capacity to recognize the correct (fimbrial) epitopes from the homologous strain and related variants, and not as a reliable measure of the magnitude of the actual responses which act against B. nodosus infection. These responses may themselves be a subset of those involved in the K-agglu- tination reaction.

406

The recombinant antigens examined in this trial represent the prototype for the development of a multivalent vaccine for use against footrot in the field. Fimbriae representative of the other major serogroups of B. nodosus may be produced simply by substituting the relevant subunit gene in the Pseudomonas/PL promoter-expression vector system developed for this pur- pose (Mattick et al., 1987). Such genes are comparatively easy to clone, by screening with either antibody or sequence probes, and indeed most have already been obtained (Mattick et al., 1985). Furthermore, there is no a priori reason to expect that this system will not be equally applicable to the production of similar vaccines against related Type 4 fimbriate pathogens, such as N. gon- orrhoeae and M. boris ( Mattick et al., 1987). There is evidence that fimbrial preparations are immunoprotective against these infections ( Pugh et al., 1977; Brinton et al., 1978; Lehr et al., 1985), and it seems that the main barrier to the development of recombinant DNA-based vaccines against these pathogens may be the present lack of serological classification data upon which to base a multivalent formulation. Furthermore, since morphogenetic assembly and the interaction between the subunits in the fimbrial strand are unlikely to be altered by antigenic changes, especially given that interspecific differences in the pro- tein are happily accepted by the recombinant Pseudomonas host (Mattick et al., 1987 ), there is the attractive possibility that the co-insertion of two or more fimbrial subunit genes into the same host cell will result in the formation of hybrid fimbriae, which may then provide immunological coverage against more than one serotype, or even perhaps against more than one species. This pos- sibility is currently being tested.

Footrot itself is an unusual disease in that the infection is relatively remote from host immune surveillance and does not generally provoke immunopro- tective responses, although such responses can be aroused by vaccination. Thus, in the absence of intervention, the condition is chronic or semi-chronic, depending on environmental factors (see Egerton, 1977), although natural healing or recovery has been observed (Egerton et al., 1983 ). Intervention by vaccination may occur either in advance of or during infection, and in this context it is unlikely that there are any real differences between prophylactic and therapeutic responses. In any event, it is essentially a one-way street, and experience shows that, unlike many other pathogens, B. nodosus infection does not stimulate the immune system, even if already primed by vaccination. Hence the duration of protection is more limited than usual. Since the means for efficient and economic production of the protective fimbrial antigens against footrot are now available via recombinant DNA technology (Mattick et al., 1987), the contemporary issues in footrot vaccine development would now seem to be to identify the immunological mechanisms which are involved in dealing with B. nodosus infection, and to design strategies for vaccine formulation or delivery which will increase the intensity and duration of the relevant responses.

407

ACKNOWLEDGEMENTS

This work was supported in part by grants from the Australian Wool Research Trust Fund (J.R.E.) and the Australian Meat and Livestock Research and Development Corporation (J.S.M.). P.T.C. and B.J.A. are supported by Post- graduate Studentships from the Australian Wool Research Trust Fund.

REFERENCES

Anderson, B.J., Bills, M.M., Egerton, J.R. and Mattick, J.S., 1984. Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae. J. Bacteriol., 160: 748-754.

Anderson, B.J., Kristo, C.L., Egerton, J.R. and Mattick, J.S., 1986. Variation in the structural subunit and basal protein antigens ofBacteroides nodosus fimbriae. J. Bacteriol., 166:453-460.

Bagdasarian, M.M., Amann, E., Lurz, R., Ruckert, B. and Bagdasarian, M., 1983. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene, 26:273-282.

Beveridge, W.I.B., 1941. Foot-rot in sheep: a transmissable disease due to infection with Fusifor- mis nodosus. Bulletin 140, Council for Scientific and Industrial Research, Melbourne.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254.

Bradley, D.E., 1974. The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili. Virology, 58: 149-163.

Brinton, C.C., Bryan, J., Dillon, J.-A., Guerina, N., Jacobsen, L.T., Labik, A., Lee, S., Levine, A., Lim, S., McMichael, J., Polen, S., Rogers, K., To, A.C.-C. and To, S.C.-M., 1978. Uses of pili in gonorrhea control: role of bacterial pili in disease, purification and properties of gonococcal pili, and progress in the development of a gonococcal pilus vaccine for gonorrhea. In: G.F. Brooks, Jr., E.C. Gotschlich, K.K. Holmes, W.D. Sawyer and F.E. Young (Editors), Immu- nobiology ofNeisseria gonorrhoeae. American Society for Microbiology, Washington, DC, pp. 155-178.

Claxton, P.D., Ribeiro, L.A. and Egerton, J.R., 1983. Classification of Bacteroides nodosus by agglutination tests. Aust. Vet. J., 60:331-334.

Egerton, J.R., 1973. Surface and somatic antigens of Fusiformis nodosus. J. Comp. Pathol., 83:151-159.

Egerton, J.R., 1974. Significance ofFusiformis nodosus serotypes in resistance of vaccinated sheep to experimental foot-rot. Aust. Vet. J., 50:59-62.

Egerton, J.R., 1977. Footrot of sheep---pathogenesis and immunity. Prog. Immunol., 3: 645-650. Egerton, J.R. and Burrell, D.H., 1970. Prophylactic and therapeutic vaccination against ovine

foot-rot. Aust. Vet. J., 46:517-522. Egerton, J.R. and Roberts, D.S., 1971. Vaccination against ovine foot-rot. J. Comp. Pathol.,

81:179-185. Egerton, J.R., Roberts, D.S. and Parsonson, I.M., 1969. The aetiology and pathogenesis of ovine

foot-rot. I. A histological study of the bacterial invasion. J. Comp. Pathol., 79:207-216. Egerton, J.R., Laing, E.A. and Thorley, C.M., 1978. Effect of Quil A, a saponin derivative, on the

response of sheep to alum precipitated Bacteroides nodosus vaccines. Vet. Sci. Commun., 2:247-252.

Egerton, J.R., Ribiero, L.A., Kieran, P.J. and Thorley, C.M., 1983. Onset and remission of ovine foot-rot. Aust. Vet. J., 60: 334-336.

408

Elleman, T.C. and Hoyne, P.A., 1984. Nucleotide sequence of the gene encoding pilin of Bacter- oides nodosus, the causal organism of ovine foot-rot. J. Bacteriol., 160:1184-1187.

Elleman, T.C., Hoyne, P.A., Emery, D.L., Stewart, D.J. and Clark, B.L., 1984. Isolation of the gene encoding pilin from Bacteroides nodosus (strain 198 ), the causal organism of ovine foot- rot. FEBS Lett., 173:103-107.

Elleman, T.C., Hoyne, P.A., Emery, D.L., Stewart, D.J. and Clark, B.L., 1986. Expression of the pilin gene from Bacteroides nodosus in Escherichia coli. Infect. Immun., 51:187-192.

Every, D., 1979. Purification of pill from Bacteroides nodosus and an examination of their chem- ical, physical and serological properties. J. Gen. Microbiol., 115:309-316.

Every, D. and Skerman, T.M., 1982. Protection of sheep against experimental foot-rot by vacci- nation with pill purified from B. nodosus. N.Z. Vet. J., 30:156-158.

Every, D. and Skerman, T.M., 1983. Surface structure of Bacteroides nodosus in relation to vir- ulence and immunoprotection in sheep. J. Gen. Microbiol., 129:225-234.

Henrichsen, J., 1983. Twitching motility. Annu. Rev. Microbiol., 37:81-93. Lee, S.W., Alexander, B. and McGowan, B., 1983. Purification, characterization, and serologic

characteristics of Bacteroides nodosus pill and use of a purified pill vaccine in sheep. Am. J. Vet. Res., 44:1676-1681.

Lehr, C., Jayappa, H.G. and Goodnow, R.A., 1985. Serologic and protective characterization of Moraxella bovis pill. Cornell Vet., 75:484-492.

Mattick, J.S., Anderson, B.J., Mott, M.R. and Egerton, J.R., 1984. Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens. J. Bacteriol., 160:740-747.

Mattick, J.S., Anderson, B.J. and Egerton, J.R., 1985. Molecular biology and footrot of sheep. In: R.A. Leng, J.S.F. Barker, D.G. Adams and K.R. Hutchinson (Editors), Biotechnology and Recombinant DNA Technology in the Animal Production Industries. Proc. Syrup., 27-30 November 1984, Armidale, N.S.W., Australia, Rev. Rural Sci., 6:79-91.

Mattick, J.S., Bills, M.M., Anderson, B.J., Dalrymple, B., Mott, M.R. and Egerton, J.R., 1987. Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa. J. Bacteriol., 169: 33-41.

McKern, N.M., O'Donnell, I.J., Inglis, A.S., Stewart, D.J. and Clark, B.L., 1983. Amino acid sequence of pilin from Bacteroides nodosus ( strain 198 ), the causative organism of ovine foot- rot. FEBS Lett., 164:149-153.

McKern, N.M., O'Donnell, I.J., Stewart, D.J. and Clark, B.L., 1985. Primary structure of pilin protein from Bacteroides nodosus strain 216: Comparison with corresponding protein from strain 198. J. Gen. Microbiol., 131:1-6.

Ottow, J.C.G., 1975. Ecology, physiology and genetics of fimbriae and pill. Annu. Rev. Microbiol., 29:79-108.

Pasloske, B.P., Finlay, B.B. and Paranchych, W., 1985. Cloning and sequencing of the Pseudo- monas aeruginosa PAK pilin gene. FEBS Lett., 183:408-412.

Pugh, G.W., Hughes, D.E. and Booth, G.D., 1977. Experimentally induced infectious bovine ker- atoconjunctivitis: effectivenss of a pilus vaccine against exposure to homologous strains of MoraxeUa bovis. Am. J. Vet. Res., 38:1519-1522.

Roberts, D.S. and Egerton, J.R., 1969. The aetiology and pathogenesis of ovine foot-rot. II. The pathogenic association ofFusiformis nodosus and F. necrophorus. J. Comp. Pathol., 79:217-227.

Schmitz, J.A. and Gradin, J.L., 1980. Serotypic and biochemical characterization of Bacteroides nodosus isolates from Oregon. Can. J. Comp. Med., 44:440-446.

Skerman, T.M., 1975. Determination of some in vitro growth requirements of Bacteroides nodo- sus. J. Gen. Microbiol., 87:107-119.

Skerman, T.M., Erasmuson, S.K. and Every, D., 1981. Differentiation of Bacteroides nodosus biotypes and colony variants in relation to their virulence and immunoprotective properties in sheep. Infect. Immun., 32:788-795.

409

Stewart, D.J., 1973. An electron microscope study ofFusiformis nodosus. Res. Vet. Sci., 14:132-134. Stewart, D.J., 1978. The role of various antigenic fractions of Bacteroides nodosus in eliciting

protection against foot-rot in vaccinated sheep. Res. Vet. Sci., 24:14-19. Stewart, D.J., Clark, B.L., Peterson, J.E., Griffiths, D.A. and Smith, E.F., 1982. Importance of

pilus-associated antigen in Bacteroides nodosus vaccines. Res. Vet. Sci., 32:140-147. Stewart, D.J., Clark, B.L., Peterson, J.E., Griffiths, D.A., Smith, E.F. and O'Donell, I.J., 1983.

Effect of pilus dose and type of Freund's adjuvant on the antiboidy and protective responses of vaccinated sheep to Bacteroides nodosus. Res. Vet. Sci., 35: 130-137.

Stewart, D.J., Clark, B.L., Peterson, J.E., Griffiths, D.A., Smith, E.F. and O'Donnell, I.J., 1986. Cross-protection from Bacteroides nodosus vaccines and the interaction of pili and adjuvants. Aust. Vet. J., 63:101-106.

Thomas, J.H., 1958. A simple medium for the isolation and cultivation of Fusiformis nodosus. Aust. Vet. J., 34:411.

Thorley, C.M. and Egerton, J.R., 1981. Comparison of alum-absorbed or non-alum-absorbed oil emulsion vaccines containing either pilate or non-pilate Bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot-rot. Res. Vet. Sci., 30:32-37.

Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from poly- acrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A., 76:4350-4354.

Walker, P.D.,Short, J., Thompson, R.O. and Roberts, D.S., 1973. The fine structure of Fusiformis nodosus with special reference to the antigens associated with immunogenicity. J. Gen. Micro- biol., 77:351-361.