INFECTION AND IMMUNITY, JUlY 1987, p. 1580-15870019-9567/87/071580-08$02.00/0Copyright X) 1987, American Society for Microbiology

Serotype Specificity and Immunogenicity of the Capsular Polymer ofHaemophilus pleuropneumoniae Serotype 5

THOMAS J. INZANAt* AND BRUCE MATHISONDepartment of Veterinary Microbiology-Pathology, Washington State University, Pullman, Washington 99164-7040

Received 29 December 1986/Accepted 25 March 1987

Serotyping of Haemophilus pleuropneumoniae and serologic assays for detection of serotype-specific antibodyare problematic due to the potential cross-reactivity of the crude antigens used for raising immune serum orfor serology. The capsular polymer (CP) of H. pleuropneumoniae serotype 5 was investigated for serotype-specific activity with antiserum to whole cells or with antiserum made monospecific to CP by adsorption witha capsule-deficient mutant. When antiserum to whole cells or monospecific antiserum to CP was tested againstpurified CP from serotypes 1 to 7 by immunodiffusion or enzyme-linked immunosorbent assay, only capsulesof serotype 5 were reactive. In addition, only encapsulated serotype 5 cells reacted with serum monospecific toCP in an indirect immunofluorescent-antibody assay. Serotype-specific antibody was completely inhibited ineach assay by preincubation of purified CP with the serum. Antiserum to whole cells of H. pleuropneumoniaeserotype 5 contained antibodies to proteins and lipopolysaccharide; these antibodies cross-reacted with antigensof heterologous serotypes by dot-blot enzyme-linked immunosorbent assay and immunoblotting. The antigenicactivity of CP was stable after heating for at least 30 min at 100°C. High titers of antibody to CP were presentin the sera of rabbits immunized intravenously with whole log-phase cells or in the convalescent sera of pigsexperimentally infected with H. pleuropneumoniae serotype 5. However, the purified CP was poorlyimmunogenic in rabbits and swine. Our results indicate that the capsule is the serotype-specific antigen of H.pleuropneumoniae and that a monospecific antiserum to capsule or purified capsule should be used forserotyping or serologic assays, respectively.

Haemophilus pleuropneumoniae may be classified into 1of 10 serotypes or may be nontypable (20, 32). Serotype-specific antigens have been described as heat stable and heatlabile and have been proposed to be capsule, lipopolysac-charide (LPS), or both (6, 8, 9, 14, 18, 26, 27, 32). A widevariety of assays have been developed for serotyping or forserologic diagnosis of H. pleuropneumoniae, including com-plement fixation (7), indirect fluorescent antibody (26, 27),coagglutination (16), indirect hemagglutination (15), slideagglutination (14), tube agglutination (8), immunodiffusion(6, 18), ring precipitation (14), enzyme-linked immunosor-bent assay (ELISA) (19), and latex agglutination (17).

Bacterial serotyping and determination of serotype-specific antibodies in swine are important tools for epidemi-ology and control of disease. Crude antigens, or antibodiesto crude antigens, have been used in diagnostic tests becauseit was believed that convalescent serum was generallyserotype specific (31). However, as diagnostic proceduresbecome more sensitive, the possibility of false-positive re-sults due to cross-reacting antibodies or antigens becomesgreater. Reports from other laboratories have indicated thatthe complement fixation, slide agglutination, and latex ag-glutination tests may give false-positive or cross-reactiveresults with H. pleuropneumoniae serotypes, other Haemo-philus species, or Actinobacillus species (17, 23, 28). Mittalet al. (16) reported that about 12% of the pigs they testedwere infected with more than one serotype of H.

* Corresponding author.t Present address: Department of Pathobiology, Virginia-

Maryland Regional College of Veterinary Medicine, Virginia Poly-technic Institute and State University, Blacksburg, VA 24061.

pleuropneumoniae, because serum from these animals re-acted with saline extracts of more than one serotype bycoagglutination. The possibility that these results were dueto cross-reactions rather than to multiple infections was notinvestigated. Thus, reports of questionable multiple infec-tions and cross-reactions indicate that there is a need toconfirm the identity of the serotype-specific antigen and touse a purified preparation for production of antiserum andfor use in serologic assays.The capsular polymer (CP) of H. pleuropneumoniae

serotype 5 (the most common serotype isolated in the UnitedStates [22, 30]) and the CPs of other serotypes have recentlybeen purified to a high degree in our laboratory (10). Inaddition, a high-titered, monospecific rabbit antiserum to CPwas obtained by adsorption of hyperimmune antiserum towhole cells with a capsule-deficient mutant of the serotype 5parent. These reagents were used to demonstrate that the CPpreviously purified was the serotype-specific antigen of H.pleuropneumoniae serotype 5 and that some protein andLPS antigens cross-reacted with heterologous serotypes.(A preliminary report of this work was presented at the

87th Annual Meeting of the American Society for Microbi-ology, Atlanta, Ga., March, 1987.)

MATERIALS AND METHODS

Bacterial strains and culture conditions. H. pleuropneumo-niae serotypes 1 to 5 were obtained from the NationalVeterinary Services Laboratory (Ames, Iowa). Serotype 2strain 27089, serotype 3 strain 27090, serotype 4 strain 33378,and serotype 6 strain 33590 were obtained from the Ameri-can Type Culture Collection (Rockville, Md.). Serotype 5

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strain 178 and serotype 7 strain 53 were obtained fromMartha Mulks (Department of Microbiology and PublicHealth, Michigan State University, East Lansing). Serotype5 strains K17 and J45 and serotype 1 strain 4045 wereobtained from Bradley Fenwick (Department of Pathology,School of Veterinary Medicine, University of California,Davis). A capsule-deficient mutant of strain K17 (K17-C)was isolated by six passages of a nonirridescent colony onbrain heart infusion agar supplemented with 5 ,ug of NADper ml (BHI-N). This mutant produced 0.17% of the capsuleproduced by the parent strain as determined by competitiveradioimmunoassay (T. Inzana, manuscript in preparation).Bacteria were grown in BHI-N broth or in bufferedCasamino Acids (Difco Laboratories, Detroit, Mich.)-yeastextract medium (3) to 109 CFU/ml, as previously described(10).

Preparation of protein-enriched outer membranes, cap-sules, and LPS. Protein-enriched outer membranes wereprepared from sonicated cells by extraction with N-lauroylsarcosine (Sigma Chemical Co., St. Louis, Mo.) asdescribed by Barencamp et al. (4). The CP was purified aspreviously describe (10). LPS was extracted with hot 45%phenol and purified by ultracentrifugation as previouslydescribed (11, 35).

Antisera. New Zealand White rabbits were immunizedintravenously with live or Formalin-fixed H. pleuropneumo-niae serotype 5 strain K17. Bacteria were grown to 109CFU/ml in BHI-N, centrifuged at 10,000 x g, and suspendedin phosphate-buffered saline (pH 7.4) (PBS) at 109 CFU/ml.The bacteria were not washed to avoid excess loss of CP.Bacteria that were Formalin fixed were incubated in PBSwith 1% Formalin for 30 to 60 min at 0°C. Rabbits wereinoculated in the marginal ear vein with 2.5 x 108 CFU/ml ofPBS. Rabbits were immunized every 3 to 4 days, and theconcentration of bacterial inoculum was doubled until itreached 4 x 109 CFU/ml. Injections of 4 x 109 CFU/ml wererepeated weekly until the ELISA titer to purified CP wasgreater than 1:10,000. Rabbits were exsanguinated by car-diac puncture, and the serum was collected and stored inaliquots at -20°C.

Monospecific rabbit antiserum to CP was obtained byadsorption of hyperimmune antiserum to whole cells withstrain K17-C. The mutant was grown in 2 liters of BHI-N,harvested at 10,000 x g, washed twice in PBS, and dividedinto two pellets (each about a 5-ml packed cell volume). Onepellet was suspended in 5 to 10 ml of immune serum, and thesuspension was incubated for 1 h at 0°C with occasionalmixing. The bacteria were removed by centrifugation at10,000 x g, and the adsorption was repeated with theremaining bacterial pellet. After centrifugation, the serumwas filter sterilized and stored at 4°C. Alternatively, rabbitswere immunized with 100 ,ug of purified CP in Freundcomplete adjuvant in two sites intramuscularly (i.m.). After3 weeks the rabbits were immunized i.m. with 100 ,ug of CPin Freund incomplete adjuvant. Three weeks later, therabbits were immunized i.m. with 50 ,ug of CP in saline. Therabbits were exsanguinated by cardiac puncture 1 week afterthe last immunization, and the serum was collected andstored at -20°C.

Yorkshire pigs (6 to 10 weeks old) were immunized withpurified CP in Freund complete or Freund incomplete adju-vant. Animals given CP in Freund incomplete adjuvant wereimmunized in four sites subcutaneously with 250 ,ug ofantigen. The animals were boosted 2 weeks later with thesame concentration of antigen in Freund incomplete adju-vant, and again 3 weeks later with 250 p.g of CP in saline in

several sites i.m. Animals given CP in Freund completeadjuvant were immunized i.m. in at least two sites with 50 ,ugof antigen three times at 1-week intervals. Two weeks later,the pigs were boosted i.m. three times at 1-week intervalswith 100 ,ug of CP in saline. Blood samples were taken onceor twice weekly throughout the immunization schedule andassayed by ELISA for antibody to CP and whole cells.Preimmune and convalescent sera from swine experimen-tally infected intratracheally with H. pleuropneumoniaeserotype 5 were provided by Martha Mulks.

Antibodies to CP or LPS were inhibited by the addition of100 ,ug of the respective antigen to 1 ml of serum, followedby at least overnight incubation at 4°C. The sera were thencentrifuged at 10,000 x g and filter sterilized.ELISA and dot-blot ELISA. The direct ELISA assay for

CP was a modification of that described by Voller andBidwell (34). Assays were performed in polystyrene micro-titer plates (Costar, Cambridge, Mass.). Antiserum used wasfrom hyperimmunized rabbits or from swine experimentallyinfected with H. pleuropneumoniae serotype 5 cells or

purified CP from serotype 5. Goat antiserum to rabbitimmunoglobulin G (IgG), IgM, and IgA or goat antiserum toswine IgG (heavy and light chain), conjugated to horseradishperoxidase, were obtained from Cooper Biomedical, Inc.(West Chester, Pa.). Serum incubations were for 1 h at 37°C.The absorbance was measured after 1 h of incubation withsubstrate (5-aminosalicylic acid in 0.005% H202) with a

dual-wavelength ELISA reader (Dynatech Laboratories,Inc., Alexandria, Va.) at 490 nm, and the A630 was automat-ically subtracted.The ELISA for LPS was similar to that for CP except the

coating buffer was PBS containing 2 mM magnesium chlo-ride. The LPS was incubated overnight at 37°C, and thewashing buffer and antiserum diluent were PBS containing10% fetal bovine serum (Flow Laboratories, Inc., McLean,Va.). The optimal concentration of antigens and antiserumreagents for all ELISAs were previously determined bycheckerboard titration.The dot-blot ELISA was a modification of that described

by Afshar et al. (1). Nitrocellulose sheets (Bio-Rad Labora-tories, Richmond, Calif.) were washed for 10 min each indistilled water and then PBS, with shaking. The sheets wereplaced in a dot-blot apparatus (Bio-Rad), and the wells werewashed with PBS. CP (100 Pdl; 10 ,ug/ml in PBS) or bacterialcells (1:16 dilution of a 109-CFU/ml broth culture) wereallowed to drain through the wells by gravity, and vacuumwas applied to dry the wells. The sheets were removed fromthe apparatus and incubated with shaking in blocking buffer(PBS containing 0.05% Tween 20 and 5% dry skim milk) for30 to 60 min at room temperature. The sheets were thenwashed three times for 5 min each in PBS containing 0.05%Tween 20 (PBS-T) and incubated with a 1:200 dilution ofadsorbed or unadsorbed heat-inactivated rabbit antiserum toK17 cells with shaking for 1 h at room temnperature. Thesheets were washed three times as described above andshaken for 1 h at room temperature with a 1:4,000 dilution ofgoat anti-rabbit IgG, IgM, and IgA antibody conjugated tohorseradish peroxidase (Cooper Biomedical) in PBS-T. Thesheets were washed three times for 5 min each in PBSwithout Tween and rinsed three times in distilled water.Horseradish peroxidase color development reagent (4-chloro-1-naphthol in 0.02% H202; Bio-Rad) in PBS wasadded to the sheets, the sheets were shaken for up to 15 min,and the reaction was stopped by washing the sheets indistilled water. The sheets were dried, and the intensity ofstaining was determined with a densitometer in the reflec-

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Maine) in saline. Purified CP (10 ,u; 1 mg/ml) was placed inperipheral wells, and 10 RI of undiluted, adsorbed, or unad-sorbed antiserum was placed in the center well. Theseconcentrations were previously found to be optimal formaximum precipitation in gels. The slides were incubated atroom temperature in a moist environment overnight, washedthoroughly in distilled water, stained with Coomassie blue,and dried.3

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FIG. 1. Immunodiffusion of purified CP with unadsorbed andadsorbed antiserum to H. pleuropneumoniae serotype 5. CP (10 ,ul;1 mg/ml) was placed in the peripheral wells, and 10 ,ul of undilutedantiserum was placed in the center well. Diffusion was allowed totake place overnight at room temperature. The gels were thenwashed, stained with Coomasie blue, and dried. Wells: 1, serotype1 CP; 2, serotype 2 CP; 3, serotype 3 CP; 4, serotype 4 CP; 5,serotype 5 CP from strain K17; 6, serotype 5 CP from strain J45; A,unadsorbed rabbit antiserum to strain K17; B, antiserum to strainK17 preincubated with 100 ,ug of serotype 5 CP per ml; C, antiserumto strain K17 adsorbed with K17-C; D, antiserum to strain K17adsorbed with K17-C and then preincubated with 100 p.g of serotype5 CP per ml.

tance mode (model GS 300; Hoefer Scientific Instruments,San Francisco, Calif.). The optimal concentrations of anti-body reagents were previously deterrmined.

Electroimmunoblotting. A modification of the Westernblotting procedure described by Towbin et al. (33) was usedfor electroimmunoblotting. Protein (30 jig) from protein-enriched outer membranes was electrophoresed on 10%polyacrylamide gels and transferred to nitrocellulose on aBio-Rad transfer blot apparatus following the instructions ofthe manufacturer. The efficiency of transfer was determinedby staining the gel with Coomassie blue after transfer. Thenitrocellulose sheets were tested for reactivity with antise-rum to K17 cells as described above for dot blots, except theblocking step was omitted.

Indirect fluorescent antibody and immunodiffusion. Theindirect fluorescent-antibody assay was a modification of theprocedure described by Rosendal et al. (27). Briefly, bacteriawere grown to 109 CFU/ml in BHI-N and washed, and a dropof bacterial suspension was dried onto a microscope slide.The slides were fixed in acetone and incubated with a 1:200dilution (in PBS) of adsorbed or unadsorbed antiserum toK17 cells. The slides were washed, and a 1:1,000 dilution ofgoat antiserum to rabbit IgG (heavy and light chain) conju-gated to fluorescein isothiocyanate (Tago, Burlingame,Calif.) was added. Fluorescence was examined with anUltraphot II microscope (Carl Zeiss, Inc., Thornwood,N.Y.) by epifluorescence.

Immunodiffusion was performed in 1% SeaKem LEagarose (FMC Corp., Marine Colloids Div., Rockland,

RESULTS

Serotype specificity of CP. Monospecific antiserum to CPwas best obtained by complete adsorption of hyperimmuneantiserum to whole cells with the capsule-deficient mutant,K17-C. The LPS electrophoretic profiles of the mutant andparent were identical, and only small quantitative differencesin some outer membrane proteins were evident (data notshown). When fixed cells were incubated with monospecificserum followed with fluorescein-labeled goat antiserum torabbit immunoglobulin, the cell surface of each serotype 5strain examined fluoresced (data not shown; see reference27). When monospecific antiserum to CP was incubated withK17-C or serotypes 1 to 4, 6, or 7, no binding of antibodycould be detected.

Purified CP was assayed for reactivity with antiserum towhole cells or with adsorbed serum by immunodiffusion(Fig. 1). Unadsorbed antiserum to whole cells and antiserumadsorbed with K17-C reacted with CP from both of theserotype 5 strains tested with a single precipitin line ofcomplete identity. No reactivity was evident against the CPof serotypes 1 to 4, 6, or 7 (immunodiffusion with CP ofserotypes 6 and 7 is not shown). The negative results werenot due to nonequivalent proportions of antigen and anti-body because precipitin lines did not form even when theconcentrations of antiserum and heterologous CPs werevaried by a checkerboard-type titration. If purified serotype5 CP was preincubated with adsorbed or unadsorbed antise-rum to K17 cells, no precipitin lines formed.Antiserum to K17 cells was also tested for reactivity with

purified CP of each serotype by ELISA (Table 1). Thepurified CP of serotype 5 strains K17 and J45 reactedstrongly with antibody to K17 cells, whereas purified CPfrom all other serotypes was negative (i.e., absorbance wasbelow the linear portion of the antibody dilution curveobtained with the K17 capsule). Heating the K17 CP for 30min at 100°C had no effect on antigenic activity in theELISA. Therefore, antiserum to whole bacteria reacted onlywith purified CP antigens of the homologous serotype, andserotype-specific activity could not be removed by adsorp-

TABLE 1. ELISA of rabbit antiserum to K17 cells againstpurified CP antigens of H. pleuropneumoniae serotypes 1 to 7a

Serotype of purified CP A49b

Serotype 5 strain K17 (heated)c .....................0.295 ± 0.018Serotype 5 strain K17 ...................... 0.296 ± 0.032Serotype 5 strain J45 ...................... 0.226 ± 0.013Serotype 1 strain 4045 ...................... 0.034 ± 0.004Serotype 2 strain 27089...................... 0.022 ± 0.003Serotype 3 strain 27090...................... 0.019 ± 0.006Serotype 4 strain 33778...................... 0.008 ± 0.002Serotype 6 strain 33590...................... 0.027 ± 0.009Serotype 7 strain 53 ...................... 0.011 ± 0.004

a Rabbit antiserum to K17 cells was used at a 1:200 dilution.b Nonspecific absorbance was subtracted from total absorbance, and the

standard deviation was calculated from triplicate samples.c The CP antigen was heated at 100°C for 30 min before testing by ELISA.

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Hpl Hp2 Hp3 Hp4 Hp6 Hp7 Hp5 Hp5 Hp5 Hp54045 27089 27090 33378 33590 53 J45 178 K17 K17-C

FIG. 2. Dot-blot ELISA of whole cells of H. pleuropneumoniae serotypes 1 to 7 with nonadsorbed antiserum to K17, serum inhibited withK17 LPS, or serum adsorbed with K17-C. Bacteria were grown in BHI-N to 109 CFU/ml and filtered through nitrocellulose paper. Afterwashing, the paper was incubated with a 1:200 dilution of antiserum, washed, incubated with a 1:4,000 dilution of goat antiserum to rabbitIgG, IgM, and IgA conjugated to horseradish peroxidase, and washed again, and colorimetric substrate was added. The intensity of colordevelopment was determined by reflectance densitometry, and the height of the peaks is reported in centimeters. Open bars, Unadsorbedantiserum; slashed bars, serum preincubated with K17 LPS; closed bars, serum adsorbed with K17-C. Bars on top show standard deviation.

tion with a capsule-deficient mutant, but was inhibited bypurified CP.

Cross-reactivity of antibodies to non-CP antigens. Antise-rum to K17 was tested for reactivity with whole bacteria ofhomologous and heterologous serotypes by dot-blot ELISA(Fig. 2). Reactivity was variable, but was positive for eachserotype tested. Other strains of serotypes 1 to 3, not shownin Fig. 2, also reacted with antiserum to strain K17. Thegreatest activity with unadsorbed serum was directed toserotype 5 strains, including the capsule-deficient mutant.Strong reactivity with serotypes 1, 2, 6, and 7 also occurred.When antiserum to K17 cells was adsorbed with K17 LPS,there was some loss of reactivity to each serotype. The losswas minimal, however, with serotype 5 strains. When anti-serum was adsorbed with K17-C, additional loss of activityoccurred with serotypes 2, 3, 6, and 7 and with serotype 5strains. Adsorption of antiserum to K17 cells with K17-C didnot reduce activity beyond that inhibited by K17 LPS for

TABLE 2. ELISA of rabbit antiserum to K17 cells againstpurified LPS from H. pleuropneumoniae serotypes 1 to 5a

ReciprocalPurified LPS endpoint

titerb

Serotype 5 strain K17 ................................. 25,600Serotype 5 strain 178 .................................. 6,400Serotype 1 strain 4045 ................................. 200Serotype 2 strain 27089 ................................ 1,600Serotype 3 strain 27090 ................................ <1:50Serotype 4 strain 33778 ................................ 200

a Antiserum was serially diluted from 1:50 to 1:51,200.bThe endpoint titer was considered the highest dilution of antiserum that,

in the ELISA, resulted in an absorbance on the linear portion of a standardantibody dilution curve.

serotypes 1 and 4. Inhibition with LPS or adsorption withK17-C resulted in only slight loss of activity to encapsulatedserotype 5 strains, suggesting that most of the remainingantibodies were probably to CP.

Purified LPS from serotypes 1 to 5 was assayed by ELISAfor reactivity with antiserum to K17 cells (Table 2). Anti-bodies to K17 reacted with purified LPS from each serotypeexcept serotype 3. Reactivity was the greatest with LPSfrom K17, but LPS from serotype 5 strain 178 and fromserotype 2 strain 27089 also reacted strongly. Concentrationsof antiserum greater than 1:800 reacted with greater affinityfor serotype 2 LPS than for LPS from serotype 5 strain 178,as determined by absorbance. However, reactivity toserotype 2 diluted out more quickly than did that to strain178. Antiserum to K17 cells also reacted with LPS fromserotypes 1 and 4, but reactivity was weaker.

Antibodies in K17 antiserum to outer membrane proteinsof each serotype were identified by electroimmunoblotting(Fig. 3). The strongest reactivity occurred with proteins ofthe serotype 5 strains. Antiserum to K17 also recognized oneor more proteins in most serotypes tested. Four strains(serotype 7 strain 53, serotype 2 strain 27089, and bothserotype 1 strains) reacted with antibody to a 78,000-daltonprotein. Seven strains (serotype 6 strain 33590 and bothstrains from serotypes 1, 2, and 3) also reacted with antibodyto proteins of about 52,000 molecular weight. Serotype 3strain 27090 reacted with antibody to a protein of about42,000 daltons. Although not clearly visible in Fig. 3, allstrains reacted very faintly with an antibody to a protein ofabout 16,000 molecular weight. In addition, a doublet ofbands at about 40,000 molecular weight appeared in eachlane. However, since no proteins were identified at thismolecular weight by direct staining of outer membranepreparations and because the doublet occurred in the lane

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1 2 3 4 5 6 7 8 9 10 11 12 13 14FIG. 3. Electroimmunoblot of outer membrane preparations of H. pleuropneumoniae serotypes 1 to 7 with antiserum to whole cells of

serotype 5 strain K17. Outer membranes were electrophoresed and transferred to nitrocellulose, and antigenically common proteins wereidentified as described in the legend to Fig. 2. Lanes: 1, serotype 7 strain 53; 2, serotype 6 strain 33590; 3, serotype 4 strain 33378; 4, serotype3 strain 27090; 5, serotype 3 from the National Veterinary Services Laboratory (NVSL); 6, serotype 2 from NVSL; 7, serotype 2 strain 27089;8, serotype 1 strain 4045; 9, serotype 1 from NVSL; 10, serotype 5 from NVSL; 11, serotype 5 strain 178; 12, serotype 5 strain K17, 13, K17-C;14, serotype 5 strain J45. The migration of molecular weight markers is indicated at right (K, x 103). The doublet bands at about 40,000molecular weight were considered artifactual.

containing the molecular weight standards, the 40,000-molecular-weight doublet was considered artifactual.Immunogenicity of purified CP antigen. The capacity of the

purified CP to raise an immune response in rabbits and pigsin comparison with that of whole bacteria is shown in Fig. 4and 5, respectively. The purified CP was capable of inducing

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only a poor response in rabbits and pigs even in the presenceof Freund complete adjuvant. In contrast, rabbits hyperim-munized intravenously with live or Formalin-killed wholecells, or pigs recovering from experimental challenge, raisedhigh titers of antibody to CP in the absence of adjuvant.There was no substantial difference in the antibody response

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serotype 5 CP in Freund complete adjuvant (A---A) or intravenously with whole bacteria (A-). There was no substantial difference in theimmune response to capsule by rabbits immunized with live bacteria or Formalin-killed bacteria. Antibody to serotype 5 was measured byELISA. Each curve represents the mean antibody response from two rabbits. Bars show standard deviation.

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of rabbits to CP after immunization with live or Formalin-killed bacteria.

DISCUSSION

Polysaccharide capsules and LPS are recognized as

serotype-specific antigens in many bacterial species (12).The identity of the serotype-specific antigen of H. pleuro-pneumoniae has been proposed to be the capsule, the LPS,or both, but this has not been confirmed because of the crudenature of the antigens used for serotyping or for raisingimmune serum (6-8, 14-19, 26, 27). Gunnarsson et al. (9)considered the capsule to be serotype specific becausebacteria that underwent a smooth-to-rough conversion lostserotype specificity. However, enteric bacteria that lose 0side chains from their LPS also undergo a smooth-to-roughconversion and lose 0-antigen specificity (13). Electropho-retic analysis of H. pleuropneumoniae LPS in our laboratory(unpublished data) and by Fenwick et al. (5) has demon-strated a ladderlike appearance with some strains of H.pleuropneumoniae, indicative of an LPS containing 0 sidechains. Thus, mutations affecting LPS antigens could beresponsible for a smooth-to-rough conversion in H. pleuro-pneumoniae. Gunnarsson (6, 7) also reported that the largestamounts of serotype-specific antigens were extracted withphenol-water, which extracts LPS and nucleic acids as wellas CP antigens (35). Immunodiffusion of antiserum withantigens extracted with aqueous phenol demonstrated twoconstituents that were considered to be LPS and capsule (6).Immunodiffusion experiments in this communicationshowed that antiserum to K17 cells formed a single precipitinline of complete identity to CP of serotype 5 strains, but did

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Reciprocal Serum TiterFIG. 5. Swine immune response to the CP antigen of H.

pleuropneumoniae serotype 5. Pigs (10 to 12 weeks old) were

immunized with purified CP in Freund complete adjuvant (0 0)or Freund incomplete adjuvant (0-----) or were experimentallychallenged intratracheally with live H. pleuropneumoniae (0-0).Antibody to purified serotype 5 CP was measured by ELISA. Eachcurve represents the mean antibody response of three pigs immu-nized with serotype 5 CP, or three serum samples taken at 5, 7, and9 weeks postimmunization from a pig experimentally challengedwith H. pleuropneumoniae serotype 5. Bars show standarddeviation.

not react with CP of other serotypes. Furthermore, therewas no loss of reactivity with CP after adsorption with acapsule-deficient mutant. However, complete inhibition ofactivity occurred if CP was added to the serum beforeimmunodiffusion. ELISA experiments also showed that an-tiserum to K17 cells reacted with purified CP from serotype5 strains, but not with purified CP of other serotypes. Inaddition, when monospecific antibody to CP antigen wastested with whole cells by immunofluorescence, surfacefluorescence was observed only with encapsulated serotype5 strains.Rosendal et al. (27) confirmed that hot aqueous phenol

extracted the most serotype-specific antigen and suggestedthat this antigen was LPS (26). However, the extractscontained antigens that cross-reacted with several H. pleu-ropneumoniae serotypes, indicating that non-serotype-specific antigens were present (26). Fenwick et al. (5)showed that a capsular-enriched polysaccharide preparationcontained substantial amounts of LPS, as determined bypolyacrylamide gel electrophoresis and silver staining.Nicolet et al. (19) demonstrated that antigens extracted withaqueous phenol were low in reactivity in an ELISA but thatantigen extracted with EDTA and further purified withSephacryl 200 was serotype specific by ELISA. The natureof the antigen purified, however, was not determined. Pre-vious results from our laboratory (10) demonstrated thataqueous phenol extracts of serotype 5 culture supernatantsprecipitated with hexadecyltrimethylammonium bromidecontained CP as well as substantial amounts of LPS, someprotein, and some nucleic acid. Ultracentrifugation and gelfiltration were required to reduce endotoxin contaminationto 0.01%. Current experiments indicated that when antise-rum to K17 was tested by ELISA against purified LPS fromserotypes 1 to 5, reactivity occurred with LPS from eachserotype except serotype 3. Furthermore, incubation ofpurified K17 LPS with antiserum to K17 inhibited somereactivity to whole cells of each serotype of H. pleuro-pneumoniae, as tested by dot-blot ELISA. Therefore, LPSantigens were cross-reactive among different serotypes of H.pleuropneumoniae.

It is well recognized that antiserum to H. pleuro-pneumoniae raises antibodies to species-specific as well asserotype-specific antigens (6, 8, 9, 14, 18, 32). Dot-blotELISA data confirmed that antibodies to serotype 5 cellscross-reacted to some degree with cells of all other serotypestested. The cross-reacting antibodies were directed to so-matic antigens, because cross-reactivity was removed afteradsorption of the serum with a capsule-deficient mutant. Inaddition to LPS, whole cell antiserum contained antibodiesto several proteins cross-reactive with one or more sero-types of H. pleuropneumoniae. Rapp et al. (21) recentlycharacterized the major outer membrane proteins ofserotypes 1 to 9. Major proteins with molecular weights of39,000 to 43,500, 29,000, and 16,000 to 16,500 were identi-fied, but several proteins ranging in molecular weight fromabout 45,000 to about 95,000 were also identified in eachserotype. In our experiments, electroimmunoblotting indi-cated that the strongest cross-reactivity was present inproteins with molecular weights of 52,000 and 78,000. Aprotein with a molecular weight of 42,000 was cross-reactivein serotype 3, and a protein of about 16,000 molecular weightwas weakly cross-reactive in each serotype. The differencesseen in electroimmunoblotting and in outer membrane pro-tein profiles are likely to be due to differences in theimmunogenicity or accessibility or both of some proteins.Adsorption experiments would be useful in determining

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which of these proteins are exposed on the cell surface.Because of the limited number of strains of each serotypethat were available, we could not determine the extent ofcross-reactivity of strains within a particular serotype.Electroimmunoblotting did demonstrate variation in proteinantigens in both of the serotype 2 and serotype 3 strains wetested. Variation in the extent of cross-reactivity in LPSantigens was also likely.

Capsules are important virulence factors due to theircapacity to prevent activation of the complement system andinhibit phagocytosis of bacteria. In addition, antibody tosome capsular antigens has been shown to protect the hostagainst disease (24, 25). The role of H. pleuropneumoniaecapsule in virulence, or the protective capacity of antibodyto capsule, is not clear. Some capsules are immunogenic andare capable of raising protective antibodies in purified form,while other capsules are not (24, 25). The immunogenicity ofpurified H. pleuropneumoniae serotype 5 CP in rabbits andpigs was very poor in comparison with that of CP on thebacteria. Although the protective capacity of serotype 5 CPwas not investigated and the capsule is nontoxic (5; T.Inzana, unpublished data), the lack of immunogenicity of thepurified serotype 5 CP makes it a poor vaccine candidate. Ifantibody to the CP antigen is protective, however, it may bepossible to improve immunogenicity of the capsule by con-jugation to a protein carrier (2, 29).

ACKNOWLEDGMENTS

We thank Porter Anderson for review of the manuscript, MarthaMulks and Bradley Fenwick for donation of serum or bacterialstrains or both, and Ma Jianneng and Theresa Workman for techni-cal assistance.

This work was supported, in part, by a grant from Praxis Biologicsand by the Washington State University Grant-in-Aid program.

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