Biochemical characterization of an antigenic saline extract of Actinobacillus pleuropneumoniae serotype 5 and identification of a serotype-specific antigen for ELISA serodiagnosis

Veterinary Microbiology, 30 (1992) 369-385 Elsevier Science Publishers B.V., Amsterdam

369

Biochemical characterization of an antigenic saline extract ofActinobacillus pleuropneumoniae serotype 5 and identification of a serotypespecific antigen for ELISA serodiagnosis Sophie Radacovici, Rral Lallier, Serge Larivirre and J. Daniel Dubreuil ~ Groupe de Recherche sur les Maladies Infectieuses du Porc, FacultO de mOdecine vkt~rinaire, Universit~ de MontrOal, C.P. 5000, Saint-Hyacinthe, Quebec, J2S 7C6 Canada (Accepted 12 August 1991 )

ABSTRACT Radacovici, S., Lallier, R., Larivi~re, S. and Dubreuil, J.D., 1992. Biochemical characterization of an antigenic saline extract ofActinobacillus pleuropneumoniae serotype 5 and identification of a serotype-specific antigen for ELISA serodiagnosis. Vet. Microbiol., 30: 369-385. A saline extract of boiled-formalinized whole cells from a local strain (81-750; Quebec, Canada) of

Actinobacillus pleuropneumoniae, serotype 5b was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of swine pleuropneumonia. Characterization of this crude extract was done and proteins, neutral sugars, hexosamines, and 2-keto-3-deoxyoctonate (KDO) were evaluated. On phenol extraction of the crude extract a serotype-specific antigen of polysaccharidic nature was recovered from the aqueous phase. This antigen was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue, silver and Schiff stainings. Immunoblots were done using sera of experimentally infected pigs that showed serotype specificity and cross-reactivity. Overall, the results indicate that the O-chain of lipopolysaccharides is a specific antigen that could be used in ELISA for the serodiagnosis of serotype 5 ofA. pleuropneumoniae.

INTRODUCTION

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. This disease causes important economic losses in many countries where swine production is important (Nicolet, 1986). Presently, 12 A. pleuropneumoniae serotypes have been described (Nielsen, 1986a) with serotype 5 divided into 5a and 5b (Nielsen, 1986b). Various serological assays for A. pleuropneumoniae have been described: the complement fixation test (CFT) (Nicolet et al., 1971 ), the enzyme-linked immunosorbent assay (ELISA) (Nicolet et al., 1981; Goyette et al., 1986) ~To w h o m correspondence should be addressed.

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and the 2-mercaptoethanol tube agglutination test (Mittal et al., 1984). Out of the three assays, the CFT has been most often used for routine diagnosis (Nicolet, 1970; Nielsen, 1988 ). The ELISA has also been evaluated for serodiagnosis of pleuropneumonia (Nicolet et al., 1981; Goyette et al., 1986; Willson et al., 1987; Bosse et al., 1990). Up to now, the results obtained suggested the use of a more purified antigenic preparation in order to improve the specificity of the test. For A. pleuropneumoniae, serotype-specific antigens have been reported to be capsular polysaccharides (Fenwick and Osburn, 1986; Inzana and Mathison, 1987; Altman et al., 1988). Smooth lipopolysaccharides have also been shown to be serotype specific and serotype cross-reactive in ELISA, co-agglutination and 2-mercaptoethanol tube agglutination tests (Fenwick and Osburn, 1986; Mittal et al., 1989 ). It was also shown that proteins were responsible in a large part for cross-reactions between serotypes (Rapp and Ross, 1986; MacInnes and Rosendal, 1987 ). In our study, we first characterized the crude extract of a local field isolate ofA. pleuropneumoniae belonging to serotype 5b, strain 81-750, presently used in ELISA for the detection of serotype 5 carrier pigs. Then, using phenol extraction, we isolated a specific fraction of serotype 5 that could be used advantageously for ELISA serodiagnosis of porcine pleuropneumonia. MATERIALSAND METHODS

Bacterial strain and growth conditions A. pleuropneumoniae 81-750, a local field isolate, was obtained from the clinical diagnostic laboratory, Faculty of Veterinary Medicine, Saint-Hyacinthe, Que., Canada. This strain belongs to serotype 5b (Dr R. Nielsen, Denmark, personal communication) and was isolated from the lung of a pig that died of pleuropneumonia. Cultures were grown at 37 °C aerobically on PPLO (Difco Laboratories, Detroit, Michigan) agar plates supplemented with 0.1% glucose, 5% horse serum and 10% yeast extract. Crude extract After 6 h of growth on PPLO agar, cells were harvested with 3 ml per plate of 0.01 M phosphate-buffered saline (pH 7.3) (Oxoid Ltd., Basingstoke, England) (PBS) containing 0.05% (v/v) formaldehyde. This cell suspension was allowed to stand overnight at 4°C. The bacterial suspension was then adjusted to 1X 109 CFU/ml, boiled for l h and centrifuged at 12 000 g for 50 min. The supernatant was collected and filtered on a 0.22 #m pore size filter (Millipore Corp, Bedford, Massachussetts). This crude extract was stored at - 2 0 ° C until used. Crude extract of strain 81-750 was dialyzed against distilled water at 4 °C for 24 h.

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Proteinase K treatment Proteinase K (Boehringer Mannheim, Dorval, Quebec) was used to digest the proteins in the crude extract. First the pH of the crude extract was adjusted to 8.0, then proteinase K was added to a final concentration of 0.5 m g / ml, and CaC12 to a final concentration of 2 mM. This solution was incubated in a water bath for 2 h at 55 °C. Proteinase K was then inactivated by boiling for 30 min. The digested extract was dialyzed against distilled water and concentrated to the initial volume of crude extract using an Amicon system (Amicon Corp, Danvers, Massachussetts) with a YM 10 000 membrane. Detoxi-GeffM column chromatography The crude extract was processed on a Detoxi-Gel T M column (Pierce Chemical Company, Rockford, Illinois), an affinity gel for LPS chromatography, as follows. A 20 ml column of Detoxi-Gel T M was equilibrated with 0.05 M phosphate buffer (pH 7.2 ) containing 0.1 M NaC1 (PBS-NaC1). The sample containing 10 m M 3- [ (3-cholamidopropyl) dimethylammonio ] 1-propanesulfonate (CHAPS) (Sigma Chemical, St-Louis, Michigan ) and 0.2 M NaC1 was applied to the column and left in contact for 2 h. The column was then washed with three vols of PBS-NaC1. This constituted the washed fraction. The column was eluted with three vols of 1% sodium desoxycholate (w/v) (Fisher Scientific, Fair Lawn, New Jersey) and two vols of PBS-NaC1. This constituted the eluted fraction. Both fractions were dialyzed against PBS for 24 h at 4 °C to remove the detergent and concentrated to the initial volume of crude extract as previously described. Phenol extraction Phenol extraction of the crude extract was done at room temperature. An equal volume of 90% phenol (w/v) (Fisher Scientific) was added to a volume of crude extract and homogenized. The solution was allowed to stand at room temperature for 30 min. After centrifugation at 12 000 g for 30 rain, two phases were obtained with an interface of insoluble material. The upper aqueous phase and the lower phenol phase were collected. The interface was discarded. The aqueous phase was extracted twice more in the same manner. No interface was visible after those extractions. Both, aqueous and phenol phases were dialyzed against distilled water at 4 °C to remove phenol. After dialysis, all phases were concentrated to initial volume of crude extract as previously described. Those extracts were stored at - 70 ° C until used. Analysis of extracts The protein content was measured by the m e t h o d of Lowry et al. (1951 ) as modified by Markwell et al. ( 1978 ) using bovine serum albumin (BioRad Laboratories Ltd, Mississauga, Ontario) as the standard. Neutral sugars were measured by the m e t h o d of Dubois et al. ( 1956 ) using D-glucose (Fisher Sci-

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entific) as the standard. Analysis of extracts for hexosamines was done by the method of Smith and Gilkerson ( 1979 ) using N-acetyl-D-glucosamine (Sigma Chemical) as the standard. The extracts were also assayed for 2-keto-3-deoxyoctonate (KDO) by the method of Weisbach and Hurwitz ( 1959 ) as modified by Osborn ( 1963 ) using KDO (Sigma Chemical ) as the standard.

Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (1970) in an SE 200 vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, California). All samples were previously evaporated on a Savant Speed Vac Concentrator (Emerston Instruments, Richmond Hill, Ontario) and rehydrated in solubilization buffer. When needed, samples were treated with proteinase K (Hitchcock and Brown, 1983 ). Whole cells were prepared by mixing 25 mg ofa 6 h culture grown on PPLO plates with 500/~1 of solubilization buffer (Laemmli, 1970). Whole cells treated with proteinase K were prepared according to the method of Hitchcock and Brown (1983). Samples were stacked in 4.5% acrylamide (100 V; constant voltage) and separated by using 12.5% acrylamide (200 V; constant voltage). Minigels were stained with Coomassie blue R-250 for proteins and with silver nitrate for lipopolysaccharides (LPS) as described by Tsai and Frasch ( 1982 ). Polysaccharides were stained using the periodate-Schiff procedure as described by Fairbanks et al. ( 1971 ). Capsular polysaccharides were also stacked in 3% acrylamide and separated by using 5% acrylamide. All gel loadings were done on the dry weight basis of each sample except for whole cells.

Western blotting After SDS-PAGE, separated material was transferred from the slab gel to nitrocellulose membrane by the methanol-Tris-glycine system described by Towbin et al. ( 1979 ). Electroblotting was performed in a transblot apparatus (Hoefer Scientific Instruments) for 18 h at 60 V. Unreacted sites on the nitrocellulose paper (NCP) were blocked with a 2% (w/v) solution of casein in 10 mM Tris-HC1, 0.9% NaC1 (pH 7.4) (CTS) for 1 h at room temperature. The NCP was then incubated with an appropriate dilution of antiserum in the same buffer for 2 h. The NCP was washed with Tris-saline ( 10 mM TrisHC1, 0.9% NaC1, pH 7.4). Then, rabbit anti-porcine immunoglobulin (ICN ImmunoBiologicals, Lisle, Illinois) or goat anti-rabbit immunoglobulin (Prince Laboratories, Toronto, Ontario) conjugated to horseradish peroxidase was added in CTS buffer and incubated at room temperature for 90 min. After washings in Tris-saline, the reactive bands were visualized by reacting NCP with 10 ml of 0.3% 4-chloro-l-naphthol (Sigma Chemical) in cold methanol mixed with 50 ml of 0.06% HzOz in Tris-saline (Hawkes, 1982 ).

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Antisera Pigs (8-10 weeks old) were obtained from a minimal disease herd free of A. pleuropneumoniae. All sera were tested by ELISA and CFT for serotypes 1, 2, 3, 5, and 7 before the infection. Antisera raised against the different serotypes were produced by inoculating 3 ml of a bacterial suspension followed by 3 ml of sterile physiological saline solution directly into the lung of a pig under anaesthesia. Cells grown on PPLO agar for 18 h were harvested with cold sterile saline solution and adjusted to 1 × 10 6 CFU/ml. Sera were then collected and tested by ELISA and CFT at days 7, 14, 21, and 28 against homologous and heterologous serotypes in order to evaluate their titer and specificity. At days 30-32, sera were collected and pigs sacrificed. Serum from one uninfected animal served as negative control. Serum 382 was chosen since it gave the highest value in ELISA of all the sera tested from uninfected swines. Sera from experimentally and naturally infected pigs were kindly provided by France De La Salle from the pleuropneumonia diagnostic laboratory, Faculty of Veterinary Medicine, Saint-Hyacinthe, Que., Canada. Rabbit antiserum R-31 to strain 81-750 was kindly provided by Dr K.R. Mittal and was produced according to a method previously described (Mittal et al., 1982). ELISA The ELISA was performed according to the method developed for the diagnosis of pig pleuropneumonia at the Faculty of Veterinary Medicine, SaintHyacinthe, Que., Canada. This indirect ELISA was done using 96 well Ushape Cooke Microtiter Polystyrene plates (001-010-2201 ) (Dynatech Laboratories Inc, Chantilly, Virginia). Dilutions of the antigens were done in 0.1 M carbonate buffer (pH 9.6) and a volume of 50/zl was added to each well. Plates were incubated overnight at 4°C. The plates were then washed three times with 0.02 M phosphate-buffered saline (pH 7.4) containing 0.05% Tween20 (Sigma Chemical) (PBS-Tween). Sera diluted 1:200 in PBSTween were added to the wells ( 50/zl/well ) and incubated for 15 min at room temperature. After incubation, the plates were washed as before. Horseradish peroxidase-labelled immunoglobulin G fraction of rabbit antiserum raised against porcine IgG (ICN ImmunoBiologicals) diluted 1:2400 in PBS-Tween was added to each well ( 50/~1). After 15 min incubation at room temperature, plates were washed as before and 100/tl of a chromogenic solution was added to each well. The solution contained 2 mM hydrogen peroxide and 0.4 mM 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) (Sigma Chemical ) in a 50 mM citrate solution (pH 4.0). Optical density (O.D.) at 414 nm was read using an automated plate reader (Titertek Multiskan, Flow Laboratories, Mississauga, Ontario ) when three target sera reached definite values against the crude extract of strain 81-750. These values were between 1.6 and 1.7 for serum 380 (serotype 5b), between 1.2 and 1.3 for serum 398 (sero-

374

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TABLE 1 Characteristics of swine antisera used for ELISA Serum number

Antiserum to strain

Serotype

Characteristics

382

-

-

Uninfected swine from a minimal disease herd

250 398

K17

5a

Positive for serotype 5 No cross-reaction with other serotypes

392 396

L20

5b

Positive for serotype 5 No cross-reaction with other serotypes

380 381 391

81-750

5b

Positive for serotype 5 No cross-reaction with other serotypes

386

1421

3

Positive for serotype 3 Cross-reaction with serotype 5

264

Natural infection with unidentified serotype ELISA positive (crude extract) and CFT negative for serotype 5

type 5a) and 0.5 to 0.6 for serum 382 (normal serum). A serum was arbitrarily declared negative in ELISA ifO.D, was ~<0.7. ELISA was specifically used to evaluate the activity of the different fractions obtained. The characteristics of the sera are listed in Table 1.

Complement fixation test CFT was performed in 96 well U-shaped Microtiter Polyvinyl plates (001010-2401) (Dynatech Laboratories Inc.) according to the method of the Center for Disease Control, US Department of Health and H u m a n Services, Atlanta, USA. Calf serum was added as an accessory factor as described by Nicolet (1970). Whole cells were used as the antigen. Capsular polysaccharides Purified capsular material from strain 81-750 was kindly provided by Dr. E. Altman (National Research Council of Canada, Ottawa). The material had been purified according to the method ofAltman et al. ( 1986, 1987 ). RESULTS

Proteinase K treatment The crude extract of strain 81-750 contained an average of 2.12 mg of protein per ml. Dialysis of that crude extract removed approximately 1 m g / m l

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375

of protein (Table 2 ). The fraction obtained after treatment with proteinase K and dialysis contained less than 0.04 mg of protein per ml. Proteinase K treatment reduced markedly the protein and KDO content of the crude extract. Chemical interference with KDO determination was suspected. The hypothesis of interference was supported by the results of immunoblot with the dialysed proteinase K-treated extract and serum 380 (serotype 5b) showing a positive reaction in the 60 kDa region observed by SDS-PAGE (Data not shown). Dialysed and dialysed proteinase K-treated crude extract were assayed in ELISA and the values obtained with the different sera did not show any marked differences with the results obtained with the crude extract (Table 3 ). Dialysis of the crude extract alone showed an important reduction in neutral sugars and hexosamines and a reduction of approximately half the content in KDO (Table 2 ).

Detoxi-GelT M column chromatography The crude extract was chromatographed on a Detoxi-Gel T M column, containing an affinity gel for LPS. Two fractions were obtained, the washed fraction which contained protein as the major constituent and the eluted fraction. TABLE2 Results of the colorimetric assays for proteins, neutral sugars, hexosamines and KDO ofA. pleuropneumoniae, strain 81-750 (serotype 5b ) Fractions Crude extract Dialyzed crude extract Proteinase K-treated crude extract Dialyzed proteinase K-treated crude extract Crude extract Detoxi-Gel T M column washed fraction eluted fraction Crude extract phenol extraction aqueous phase phenol phase

Proteins' mg/ml

Neutral mg/ml

sugars 2

Hexosamines 3 mg/ml

KDO 4

pg/ml

2.125

2.06

2.02

22.59

1.22

0.49

0.47

10.63

1.46

1.84

1.67

2.58

< 0.04

0.23

0.33

< 0.12

0.26 0.05

0.17 0.18

0.07 0.04

< 0.12 2.14

0.08 0.35

0.20 0.03

0.05 0.04

1.22 0.49

1Determined by the method of Lowry et al. ( 1951 ) as modified by Marwell et al. ( 1978 ). 2Determined by the method of Dubois et al. (1956). 3Determined by the method of Smith and Gilkerson ( 1979 ). 4Determined by the method of Weisbach and Hurwitz ( 1959 ) modified by Osborn ( 1963 ). Walues are the mean of at least two determinations.

376

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TABLE 3 ELISA results obtained following treatments of the crude extract ofA. pleuropneumoniae strain 81-750 (serotype 5b )

Fraction

Antisera Serotype 5b Strain 81-750

380

Crude extract 1.75 ~ Dialyzed crude extract 1.75 Dialyzed proteinase K-treated crude extract 1.62 Crude extract Detoxi-Gel T M column washed fraction 0.52 elutedfraction 1.81 Crude extract phenol extraction aqueous phase 1.83 phenolphase 0.66

381

391

Serotype 5b Strain L20

Serotype 5a Strain K l 7

Serotype 3 Reacting Negative Strain 1421 with control serotype 5 - -

392

398

386

264

382

396

250

1.70 1.25 1.35 1.31

1.24 1.67

1.70

1.25

0.60

1.70 1.20 1.40 N / D 2

1.23 N / D

1.57

1.18

0.46

1.46 1.02 1.30 N / D

0.79 N / D

1.27

0.93

0.27

0.35 0.40 0.41 N / D 1.72 1.16 1.38 N / D

0.29 N / D 1.35 N / D

0.35 1.13

0.50 1.10

0.21 0.53

1.64 1.11 1.20 1.15 0.48 0.67 0.43 0.68

1.25 1.60 0.46 1.14

0.22 1.60

0.30 1.42

0.34 0.52

IValues are the mean of at least two determinations. 2N/D: not done.

The protein content was higher in the washed fraction containing a low KDO value ( < 0.12 #g K D O / m l ). The eluted fraction, containing the LPS, had an average of 2.14 # g / m l KDO. Both fractions were low in neutral sugars and hexosamines compared to the crude extract. The ELISA values obtained with the eluted fraction (LPS fraction ) were equivalent to those obtained with the crude extract except for serum 386 (serotype 3) which gave a lower value. The washed fraction, low in KDO, gave negative ELISA results with all the sera tested (Table 3 ). Phenol extraction The third aqueous and the first phenol phases resulting from the phenol extraction of the crude extract were assayed for proteins, neutral sugars, hexosamines and KDO. The extraction reduced the quantity of proteins from 2.12 m g / m l in the crude extract to 0.08 m g / m l in the aqueous phase and 0.35 m g / m l in the phenol phase. The KDO content was almost three times higher in the aqueous phase ( 1.22 # g / m l ) than in the phenol phase (0.49/2g/ml)

(Table 2 ). The ELISA results for the aqueous phase showed a positive reaction with homologous sera (250, 398 (serotype 5a); 380, 3 8 1 , 3 9 1 , 3 9 2 , 396 (serotype 5b) ) and a negative reaction for the cross-reacting serum 386 (serotype 3)

BIOCHEMICALCHARACTERIZATIONOFANEXTRACTOFA.PLEUROPNEUMONIAESEROTYPE5

377

and serum 264 (unidentified serotype). The opposite results were observed with the phenol phase except for serum 250 (serotype 5a) (Table 3 ).

Analysis by SDS-PAGE SDS-PAGE o f whole cells stained with Coomassie blue showed numerous protein bands. Fewer bands were present in the crude extract compared to whole cell protein profile. Treatment with proteinase K greatly reduced the protein content o f the crude extract. ( D a t a not shown). Silver nitrate staining of LPS showed a major band with an apparent molecular weight o f 60 k D a for strain 81-750 in proteinase K-treated whole cells and crude extract. The b o t t o m of the gel representing the core-lipid A region was also stained with silver nitrate. This was observed with whole cells ( ~ 10-20 k D a region ) and crude extract ( ~ 10-14 k D a region) treated with proteinase K (Fig. 1 ). Electrophoresis of the LPS fraction obtained by Detoxi-Gel T M column chromatography revealed a 60 k D a band and a ~ 10-14 k D a b a n d at the bott o m of the gel (Fig. 1 ). The phenol extraction procedure resulted in a partition between the 60 k D a and the ~ 10-14 k D a components. The 60 k D a component was recovered in the aqueous phase and the ~ 10-14 k D a in the phenol phase (Fig. 1).

94.0

="

67.0

=','-

43.0

30.0

20.1

~"

~--

14.4

1

2

3

4

5

6

Fig. 1. Silver stained SDS-PAGE, 12.5% acrylamide gel using A. pleuropneumoniae strain 81750 (serotype 5b). Lane 1, proteinase K-treated whole cells, 250 #g; lane 2, proteinase K-treated crude extract, 550 gg; lane 3, proteinase K-treated eluted fraction (LPS fraction) of DetoxiGelT M column, 450 #g; lane 4, proteinase K-treated washed fraction of Detoxi-GelT M column, 550 #g; lane 5, aqueous phase of the phenol extraction, 30/lg; lane 6, phenol phase of the phenol extraction, 100/lg. Molecular weight standards in thousands are indicated on the left.

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Western blotting Different fractions resulting from crude extract treatments were assayed in immunoblots with sera 380 (serotype 5b) and 386 (serotype 3). Immunoblots with whole cells and crude extract, despite the same ELISA value, showed that different components reacted with the two sera (Figs. 2A, 2B). The aqueous phase and the phenol phase of the phenol extraction were assayed in immunoblots with sera 380 (serotype 5b) and 386 (serotype 3). Serum 380 (serotype 5b) reacted with a 60 kDa band present in the aqueous phase and serum 386 (serotype 3) with the ~ 10-14 kDa region present in the phenol phase (Figs. 2A, 2B). A weak reaction was observed between components of the phenol phase and serum 380 (serotype 5b) but no reaction was noticed between components of the aqueous phase and serum 386 (serotype 3 ) (Data not shown). Both fractions obtained with the Detoxi-Gel T M column after treatment with proteinase K were assayed for immunoblots with sera 380 (serotype 5b) and 386 (serotype 3 ). No reaction was seen with the washed fraction, with a low KDO content. No reaction was observed between the eluted fraction containing LPS and serum 386 (serotype 3), but a reaction was observed between this fraction and serum 380 (serotype 5b) in the 60 kDa region previously described (Data not shown). Immunoblots were done using whole cells, crude extract and aqueous phase of the phenol extraction, and homologous sera for serotype 5 (380, 392, 398 ). The same pattern was observed for all sera. The crude extract gave a major reaction with a 60 kDa band and much weaker reactions with other components of lower molecular weight. The aqueous phase of the phenol extraction gave a strong reaction with the 60 kDa band with serum 380 (serotype 5b). The reaction was weaker towards that same band with sera 398 (serotype 5a) and 392 (serotype 5b) (Fig. 2B, 2C, 2D).

Capsular polysaccharides Gel electrophoresis of purified capsular material and crude extract was done. The gel was stained by the periodate-Schiff procedure. The purified capsular polysaccharides did not enter the 12.5% separating gel but remained in the 4.5% stacking gel. No band corresponding to capsular polysaccharide was observed with the crude extract. However, a band in the 60 kDa region and one in the ~ 10-14 kDa region were stained in the crude extract. Aqueous phase of the phenol extraction and the eluted fraction of the Detoxi-Gel T M column (LPS fraction ) also showed a band in the 60 kDa region when stained by the periodate-Schiff procedure. Whole cells, crude extract and purified capsular polysaccharides were separated using a 5% polyacrylamide gel. Immunoblots were done using swine antiserum 380 and rabbit antiserum R-31, both produced with strain 81-750 (serotype 5b) ofA. pleuropneumoniae. A positive reaction was observed be-

BIOCHEMICAL CHARACTERIZATIONOF AN EXTRACT OF A. PLEUROPNEUMONIAE SEROTYPE 5

379

94.0 67.0

43.0

30.0

~'-

20,1

I,.-

14.4

1

94.0

D,-

67.0

~"

43.0

~"

2

3

!

2

3

3

i

2

3

30.0

20.1

14.4

I

2

Fig. 2. Immunoblots using A. pleuropneumoniae strain 81-750 (serotype 5b) as antigen. A: swine antiserum 386 (serotype 3, strain 1421 ); lane 1, whole cell, 175/lg; lane 2, crude extract, 200 pg; lane 3, phenol phase of the phenol extraction, 100 pg. B: swine antiserum 380 (serotype 5, strain 81-750 ). C: swine antiserum 398 (serotype 5, strain K 17 ). D: swine antiserum 392 (serotype 5, strain L20). B, C and D: lane 1, whole cell, 175 pg; lane 2, crude extract, 250 #g; lane 3, aqueous phase of the phenol extraction, 35/tg. Molecular weight standards in thousands are indicated on the left.

380

S. RADACOVICI ET AL.

tween purified capsular polysaccharides and serum R-31. No reaction was observed between whole cells or crude extract with rabbit serum R-31 where purified polysaccharides migrated. No reaction was noticed between whole cells, crude extract or purified capsular polysaccharides with swine serum 380 where purified polysaccharides migrated (Data not shown). DISCUSSION

A saline extract of boiled, formalinized whole cells of strain 81-750 belonging to serotype 5b ofA. pleuropneumoniae was characterized. This crude extract was found to contain proteins, neutral sugars, hexosamines and KDO. Different treatments were performed on the crude extract in order to separate it into its different chemical components. Thus, proteins of various molecular weights, rough and smooth LPS, and polysaccharides were partitioned. Then, components constituting the different fractions were tested to identify any serotype 5 specific antigen (s); the quality of the antigen preparation being important in order to have a better specificity and sensitivity in ELISA. Proteins in the crude extract were shown to cause cross-reaction. Boiling the crude extract denatured most of the proteins. Nevertheless, these proteins were still part of the crude extract and antibodies contained in swine sera could react with linear epitopes. This was observed in the immunoblot performed with whole cells and crude extract of strain 81-750 using heterologous serum 386 (serotype 3). This was substantiated with the ELISA test which gave lower values for the same serum with the crude extract after digestion with proteinase K indicating that cross-reactions are, in part, due to the presence of proteins. Previous reports have also indicated that whole cell and outer membrane proteins contributed to cross-reactions between serotypes (Rapp and Ross, 1986; Maclnnes and Rosendal, 1987 ). The Detoxi-Gel T M column, containing an affinity gel for LPS, provided us with a fraction of LPS almost free of proteins. Schiffreagent was used to stain the LPS fraction and a band in the 60 kDa region was observed indicative of the presence of polysaccharidic components. In ELISA, with the exception of serum 386 (serotype 3), this fraction gave a positive reaction equivalent to the reaction observed with the crude extract. A lower value was observed for serum 386 since a large part of the proteins of the crude extract responsible for cross-reactivity was not present in the LPS fraction of the Detoxi-Gel TM column. As a whole, this indicated that the LPS present in the crude extract were responsible for the positive reaction in ELISA. On the other hand, non-specific reactions were caused by LPS with no or short polysaccharidic chains. The presence of rough-type LPS was previously demonstrated for A. pleuropneumoniae (Fenwick et al., 1986; Altman et al., 1990) and LPS profiles by SDS-PAGE showed the core-lipid A at the bottom of the gel. On phenol extraction, we could isolate these components in the

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phenol phase on the basis of their hydrophobic nature. In ELISA, the phenol phase gave a positive reaction with sera 264 (unidentified serotype) and 386 (serotype 3 ), but a negative reaction with serotype 5 sera 380, 38 l, 39 l, 392, 396, 398 except for serum 250. Immunoblots done with this fraction using serum 386 (serotype 3 ) showed a reaction in the ~ 10-14 kDa region. A weak reaction in the same region of the gel was also observed with sera raised against serotype 5. This reaction was negligible compared to the one observed between components of the aqueous phase and antisera for serotype 5. The phenol phase was treated with proteinase K and the same reaction was observed with serum 386 (serotype 3 ), indicating that the reaction was not due to proteins. Cross-reactions between serotypes ofA. pleuropneumoniae due to rough-type LPS were reported (Fenwick and Osburn, 1986) and also between gram-negative bacteria sharing c o m m o n epitopes located in the corelipid A region (Mutharia et al., 1984). The aqueous phase of the phenol extraction containing the LPS with long polysaccharidic chains was the fraction in our crude extract that was specific for serotype 5 of A. pleuropneumoniae. This was shown by ELISA, in which all the antisera for serotype 5 gave a positive reaction, even if they were produced with different strains belonging to serotype 5. Immunoblots using aqueous phase of the phenol extraction and serotype 5 sera showed a reaction with the 60 kDa band identified by SDS-PAGE. Something similar was observed by Chang and Lai (1988) for serotype 1 ofA. pleuropneumoniae. Using phenol extraction, SDS-PAGE and immunoblotting, they were able to identify a band of 70 kDa specific for serotype 1 and of lipopolysaccharidic nature. The surface exposure of A. pleuropneumoniae LPS was demonstrated by Jacques et al. (1988), who showed that numbers of outer membrane blebs crossed the capsular layer. For A. actinomycetemcomitans and Haemophilus aphrophilus, those blebs were shown to contain LPS (Holt et al., 1980). LPS are strongly antigenic and immunogenic molecules (Munford, 1991 ). In addition, immunogenicity and antigenicity of LPS are known to be preserved for many days after inoculation of LPS or bacteria containing LPS into the animal (Munford, 1991 ). These antigens can trigger the i m m u n e response and thus establish humoral immunity. A. pleuropneumoniae strain 81-750 appeared to be different from other serotype 5 strains. Using SDS-PAGE, Rapp and Ross ( 1986 ) showed that some serotype 5 strains are of rough type. More recently, Byrd and Kadis (1989 ) demonstrated the presence of a semirough-type LPS for serotype 5. Semirough-type LPS appeared to be composed of the core-lipid A region at the bottom of the gel with an additional band located, for serotype 5, just above the core-lipid A region. Strain 81-750 appeared to be different but can still be classified as semirough according to the definition of Byrd and Kadis. In fact, instead of having the extra band just above the core-lipid A region, this band

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was observed in the 60 kDa region indicating that it contained a highly substituted O chain. Nielsen (1986b) have demonstrated that serotypes 5a and 5b share comm o n antigenic determinants located in the capsular polysaccharides and lipopolysaccharides. Capsular polysaccharides bear also some specific determinants either for serotype 5a or 5b. More recently, Airman et al. (1987) described the chemical composition of capsular polysaccharides for serotype 5a which are repeating disaccharide units of 2-acetamido-2-deoxy-D-glucose and 3-deoxy-D-manno-2-octulonic acid (dOclA). Serotype 5b capsular polysaccharides have the same basic structure but possess an additional lateral chain of one unit offl-D-glucose (Altman et al., 1988 ). On the other hand, the O-chain portions of all the LPS of the strains, belonging to serotype 5a and 5b, examined consist of unbranched linear chain offl-D-galactopyranosyl residues, a structure unique to serotype 5 (Altman et al., 1990). In summary, some capsular polysaccharides epitopes are specific either to serotype 5a or 5b. However serotypes 5a and 5b share the same LPS structure. In the crude extract of strain 81-750 we were unable to detect the presence of capsular polysaccharides. When SDS-PAGE were stained with Schiff reagent, no polysaccharides were seen in the crude extract where purified capsular polysaccharides migrated. In addition, the washed fraction, with low KDO content obtained with the Detoxi-Gel T M column chromatography, which should have contained capsular polysaccharides, did not show any positive reaction in ELISA with serotype 5 antisera. As well, rabbit antiserum reacted in immunoblots with purified capsular polysaccharides, whereas no reaction was observed with the crude extract. This may indicate that either capsular polysaccharides were in low quantities in our extract or that a low level of antibodies was raised against capsular polysaccharides by swine following experimental infections. It is noteworthy that the strain used and the growth conditions may have not favored the production of capsular polysaccharides. Inzana ( 1987 ) noticed an increase in capsular material production using broth cultures and longer incubation periods. On the other hand, some capsular polysaccharides are recognized to act as a camouflage for the bacteria by possibly showing structural similarities with the host material. This is the case for E. coli K1 and K5 capsules (Jann and Jann, 1985). The encapsulated bacteria cannot be recognized as foreign by the i m m u n e system of the host and antibodies against that structure are not produced properly. To our knowledge, this aspect has not yet been studied for A. pleuropneumoniae. In conclusion, our study provided a simple method of obtaining a specific fraction from a crude saline extract for serotype 5 of A. pleuropneumoniae. This fraction could probably be used advantageously in ELISA for the serodiagnosis ofA. pleuropneumoniae serotype 5.

BIOCHEMICALCHARACTERIZATIONOFAN EXTRACTOF,4. PLEUROPNEUMONIAE SEROTYPE5

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ACKNOWLEDGMENTS F i n a n c i a l s u p p o r t for this r e s e a r c h w a s p r o v i d e d by C o n s e i l de R e c h e r c h e s c n P ~ c h e s et A l i m e n t a t i o n d u Q u 6 b e c .

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