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obtained from G. R. Carter, Virginia-Maryland Regional College of Veterinary. Medicine, and those of 4275 serotype D and its noncapsulated variant (4275B),.
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1995, p. 952–957 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 4

Identification and Characterization of Outer Membrane Proteins of Pasteurella multocida Serotype D by Using Monoclonal Antibodies M. VASFI MARANDI

AND

K. R. MITTAL*

De´partement de Pathologie et Microbiologie, Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montre´al, C.P. 5000, Saint-Hyacinthe, Que´bec, Canada J2S 7C6 Received 4 August 1994/Returned for modification 15 December 1994/Accepted 17 January 1995

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida. Pasteurella multocida is characterized serologically by identification of capsular antigens by passive hemagglutination (5) and somatic antigens by gel diffusion precipitin tests (14). Among the five capsular serotypes, serotypes A, B, D, E, and F (5, 30), serotypes A, D, and F are responsible for diseases in cattles, pigs, rabbits, and turkeys in North America (9, 30). Atrophic rhinitis is a worldwide disease of pigs. P. multocida serotypes A and D are often isolated from the nasal cavities of piglets from farms with atrophic rhinitis. However, the prevalence of serotype D strains remains higher in pigs with atrophic rhinitis (20). The interest in the potential use of outer membrane proteins (OMPs) for serotyping is rapidly increasing (1, 4, 12, 28). Lugtenberg et al. (24) analyzed cell membrane preparations from pig isolates of P. multocida of unknown capsular serotypes by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Three different cell envelope profiles were distinguished on the basis of the electrophoretic mobilities of the H (heavy) and W (weak) proteins. Johnson et al. (16) determined the protein profiles of capsular serotype B and E strains isolated from animals with hemorrhagic septicemia and placed the isolates in two distinct groups on the basis of the molecular masses (32 to 37 kDa) of the major proteins. Ireland et al. (15) showed that the major difference among avian isolates of serotype A was the location of a major protein band in the 34- to 38-kDa region. The purpose of the investigation described here was to identify and characterize the OMP of P. multocida by using monoclonal antibodies (MAbs) with a view to subclassifying serotype D strains.

MATERIALS AND METHODS Bacterial strains and culture media. Reference strains of P. multocida 1062 serotype A, 656 serotype B, P210 serotype D, and Bunia II serotype E were obtained from G. R. Carter, Virginia-Maryland Regional College of Veterinary Medicine, and those of 4275 serotype D and its noncapsulated variant (4275B), P2153 serotype D, P4673 serotype D, and P4679 serotype F were obtained from R. B. Rimler, National Animal Disease Center, Ames, Iowa. A total of 37 field strains of P. multocida isolated locally from pigs with clinical signs of atrophic rhinitis were obtained from our stock culture. Bacteria were grown in brain heart infusion (BHI) broth (Difco) at 378C for 18 h before they were grown on blood agar base containing 5% calf blood for 18 to 24 h. The cultures were harvested and were washed three times with 0.01 M phosphate-buffered saline (PBS; pH 7.2) and centrifuged at 10,000 3 g for 20 min. The bacterial pellet was suspended and adjusted with PBS to an A540 of 1.0 and is referred to as the whole-cell suspension (WC). The WC was boiled in a water bath for 10 min and is referred as the boiled cell suspension (BC). Antigen preparation. (i) Preparation of OMPs. The method of Thomson et al. (32) was followed for the preparation of OMPs of P. multocida P210, 3001, and 1703, with some modifications. The WC was disrupted in a French press at 20,000 b/in2 in 1 mM EDTA–20 mM Tris HCl (pH 7.8). After centrifugation at 10,000 3 g for 20 min, (wt/vol) 1% Sarkosyl (N-lauroylsarcosine; Sigma) was added to the supernatant, and the mixture was incubated at 48C overnight. After centrifugation, the pellet was resuspended in distilled water and was stored at 2208C. The protein concentration was determined by a modified Lowry technique (3). (ii) Preparation of OMVs. Outer membrane vesicles (OMVs) of P. multocida P210 were isolated by the method of Lugtenberg et al. (24), with slight modifications. Briefly, bacteria grown in BHI medium overnight at 378C with agitation were removed by centrifugation at 10,000 3 g for 20 min and filtration through a 0.45-mm-pore-size membrane (Nalge). Trichloroacetic acid was added to a final concentration of 10% to precipitate the OMVs, which were harvested by centrifugation and resuspended in diethyl ether to remove the remaining trichloroacetic acid. (iii) Preparation of LPS. Lipopolysaccharide (LPS) was extracted from P. multocida P210 by using a hot phenol-water procedure described by Rebers et al. (29). The LPS concentration was determined by the method of Hanson and Phillips (13). Immunization procedure and production of MAbs. Five 6-week-old female BALB/c mice were immunized intraperitoneally with 50 mg of the OMP of P. multocida P210 mixed with Freund’s incomplete adjuvant (Difco) on days 0, 14, and 21. Blood was taken from each mouse, and the antibody response was measured by enzyme-linked immunosorbent assay (ELISA) and Western blotting (immunoblotting). The mouse with the highest antibody titer in its serum was selected as the spleen donor and was given a booster injection of 15 mg of OMP in PBS intravenously 3 days before fusion. Sera collected from the unim-

* Corresponding author. Mailing address: De´partement de Pathologie et Microbiologie, Faculte´ de Me´decine Ve´te ´rinaire, Universite´ de Montre´al, C.P. 5000, Saint-Hyacinthe, Que´bec, Canada J2S 7C6. Phone: (514) 773-8521. Fax: (514) 773-5633. 952

VOL. 33, 1995 munized and immunized mice served as negative and positive controls, respectively. SP2/0-Ag 14 murine myeloma cells were grown in Dulbecco modified Eagle medium (Gibco) supplemented with 10% heat-inactivated bovine fetal serum, 100 U of penicillin-streptomycin per ml, and 2 mM L-glutamine (Gibco). The spleen cells from the immunized mouse were fused with SP2/0-Ag 14 myeloma cells as described by Ko ¨hler and Milstein (18) by using 50% (wt/vol) polyethylene glycol (molecular weight, 3,000 to 3,700; Sigma). The fused cells were cultured in 5 microtiter plates in the presence of hypoxanthine, aminopterin, and thymidine (Sigma), and the plates were incubated at 378C in a humid atmosphere of 5% CO2. Hybridoma culture supernatants were examined for the presence of antibody by ELISA by using the OMP of P. multocida P210 as the antigen. Hybridoma cells producing the desired antibodies were cloned twice by limiting dilution (11). Antibodies were purified by treatment with caprylic acid and ammonium sulfate (27) and were stored at 2208C. Isotype determination. The isotypes of the MAbs were determined by an ELISA with a mouse monoclonal subisotyping kit containing rabbit anti-mouse immunoglobulin G1 (IgG1), IgG2a, IgG2b, IgG3, IgM, and IgA by following the procedure provided by the manufacturer (Bio-Rad). ELISA. Hybridoma culture supernatants were screened for antibodies by ELISA by using the OMP as the antigen. A 96-well microtiter plate (Linbro) was coated with 1 mg of OMP per well in carbonate buffer (pH 9.6) and was kept overnight at 48C. The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T). Optimally diluted hybridoma culture supernatants, purified MAbs, and serum from unimmunized and immunized mice were added in 100-ml volumes. The plate was incubated at 378C for 1 h and washed. The goat antimouse IgG conjugated to horseradish peroxidase (Sigma) optimally diluted in PBS was added to each well, and the plate was incubated at 378C for 1 h and washed. o-Phenylenediamine color development reagent was added thereafter. The absorbance of the peroxidase reaction product in the ELISA was read on an automated microplate reader (EAR 400 AT; SLA-Labinstruments). Since OMP and BC antigen preparations gave similar results in the ELISA, the reactivities of the MAbs with field strains of P. multocida were determined by using BC as the antigen. Protease digestion of P. multocida OMP. Each well of the plate coated with the OMP of P. multocida P210 was treated with 30 mg of each of proteinase K, chymotrypsin, or trypsin (all from Sigma) in PBS (pH 7.2) at 378C for 4 h. Following four washes, ELISA was performed as described above. A nontreated OMP was used as a control. Periodate oxidation of P. multocida OMP. The OMP of P. multocida P210 was treated with sodium periodate (Sigma) by the method described by Woodward et al. (35). Briefly, the plate coated with OMP was washed with PBS-T and was then rinsed with 50 mM sodium acetate buffer (pH 4.5) before exposure to various concentrations of sodium periodate (1 to 20 mM) in sodium acetate buffer for 1 h in the darkroom. Following a brief rinse with sodium acetate buffer, the plate was incubated in 50 mM sodium borohybride in PBS for 30 min. After washing with PBS-T, ELISA was performed as described above. Nontreated OMP was used as a control. SDS-PAGE and Western blotting. Various antigen preparations such as OMP (12 mg), OMV (12 mg), LPS (10 mg), and WC (25 mg) of P. multocida P210 as well as the OMPs of P. multocida field strains 3001 and 1703 were separated by SDS-PAGE by the method of Laemmli (19). The antigens were solubilized in sample buffer containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 1M Tris base (pH 8.3), and 0.025% bromophenol blue, and the solution was heated at 1008C for 5 min. In addition, solubilization of the OMP of P. multocida P210 in sample buffer was also carried out at 378C for 20 min, a condition which may leave some complexes intact. Proteins were separated on a 4% polyacrylamide stacking gel and a 12 or 10% separating gel. Gels were stained with 0.1% (wt/vol) Coomassie brilliant blue (R-250; Sigma) to detect the separated proteins. For Western blotting, samples were transferred to a nitrocellulose membrane (Bio-Rad) in a Trans-blot apparatus (Bio-Rad) by the method of Towbin et al. (33). Nonspecific binding was blocked with 3% bovine serum albumin (BSA) in 50 mM Tris-buffered saline (TBS; 0.02 M Tris, 0.5 M NaCl [pH 7.5]). The sheets were washed three times by soaking them in TBS containing 0.05% Tween 20 (TBS-T), cut into strips, and placed into the troughs of a plastic tray. MAbs optimally diluted in TBS-BSA were added and incubated at room temperature for 90 min with gentle rocking. The strips were washed three times in TBS-T and were incubated for 90 min in goat anti-mouse IgG horseradish peroxidase conjugate (Sigma). The strips were washed and developed in 4-chloro-1-naphthol substrate for 1 to 5 min, and the color reaction was terminated by flooding the strips with distilled water. The blotted OMP of P. multocida P210 was treated with various concentrations of sodium periodate (1 to 20 mM) in sodium acetate buffer for 1 h (35) or with 100 mg of proteinase K, chymotrypsin, or trypsin in PBS per ml at 378C for 4 h. Immunoelectron microscopy. Immunogold labelling was performed as described by Li et al. (21). Single drops of P. multocida 4275 and 4275B cell suspensions were placed on Formvar-coated grids and were blocked for 5 min with 1% egg albumin. The grids were incubated for 30 min with a suitable dilution of MAbs and were rinsed five times with distilled water. The grids were then incubated for 30 min with goat anti-mouse IgG conjugated with 10-nm gold particles (Sigma), rinsed, and negatively stained with 1% phosphotungstate for

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953

FIG. 1. SDS-PAGE and Western blots of OMP of P. multocida P210. A Coomassie blue-stained OMP (lane 1), OMP detected by serum from mouse immunized with OMP (lane 2), MAb MT1 (lane 3), MAb MT2 (lane 4), and SP2/0-Ag 14 supernatant (lane 5) are shown. Molecular mass markers (in kilodaltons) are indicated on the left.

10 s. The grids were observed with a Philips model 201 electron microscope at an accelerating voltage of 60 kV. Capsular antigen determination. Capsular antigen types were determined for all 37 field strains of P. multocida. Capsular antigen serotype A was identified by the hyaluronidase test (6), and capsular antigen serotype D was identified by the acriflavine test (7). Isolates that were nontypeable by the nonserological methods were examined for their capsular antigen types by a passive hemagglutination test (5).

RESULTS Isolation of serotype D-specific hybridomas. Of 480 hybridomas tested by ELISA, only 2 hybridomas produced serotype D-specific antibodies, designated MT1 and MT2. The immunoglobulin class of both MAbs MT1 and MT2 was IgG2b. In the Western blot with a 12% separating gel, MAb MT1 reacted with a major band with an estimated molecular mass of 32 kDa (Fig. 1, lane 3), whereas MAb MT2 reacted with a weak band with a similar molecular mass (Fig. 1, lane 4). However, with a 10% separating gel, MAbs MT1 and MT2 reacted with bands with molecular masses of 32 kDa (Fig. 2A) and 31 kDa (Fig. 2B), respectively. With WC as the antigen, MAb MT1 reacted with a single major band (Fig. 2A, lane 1); however, MAb MT2

FIG. 2. Identities of epitopes reactive with MAbs MT1 (A) and MT2 (B) determined by testing various P. multocida P210 antigen preparations by Western blotting. The reactivities of MAbs MT1 and MT2 were determined with WC (lane 1), OMP (lane 2), OMV (lane 3), and LPS (lane 4) as antigens. Molecular mass markers (in kilodaltons) are indicated on the left.

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FIG. 3. SDS-PAGE and Western blotting of OMP of P. multocida P210 by using two different temperatures, 1008C (lane 1) and 378C (lane 2), for solubilization in sample buffer prior to electrophoresis. After electrophoresis, one slice was stained with Coomassie brilliant blue (A), whereas the others were transferred to a nitrocellulose membrane and were incubated with MAbs MT1 (B) or MT2 (C). Molecular mass markers (in kilodaltons) are indicated on the left.

reacted with multiple bands (Fig. 2B, lane 1). Both MAbs reacted equally well with OMV (Fig. 2A and B, lanes 3), whereas neither of the MAbs reacted with LPS (Fig. 2A and B, lanes 4). When solubilization of the OMPs was carried out at 378C for 20 min in place of boiling for 5 min in a sample buffer, the results of SDS-PAGE analysis showed that the OMPs almost had similar gel patterns except that the major protein band of 32 kDa was replaced by multiple bands of higher molecular mass (Fig. 3A, lanes 1 and 2). Subsequent analysis of epitopes reactive with MAb MT1 in Western blots also showed that the single major band was replaced similarly with multiple bands of higher molecular mass (Fig. 3B, lanes 1 and 2). The binding of MAb MT2 to OMP was not affected when OMP was solubilized at 378C (Fig. 3C, lanes 1 and 2). Protease digestion of P. multocida OMP. The epitopes reactive with MAb MT1 were partially sensitive to all the proteolytic enzymes used, whereas those reactive with MAb MT2 were completely sensitive to proteinase K but were only partially sensitive to chymotrypsin or trypsin in the ELISA (Fig. 4). Similar results were obtained in Western blots (Fig. 5),

FIG. 4. Effect of proteinase K (Prt-K), chymotrypsin (chy-trypsin), and trypsin treatment of OMP of P. multocida P210 on their binding with MAbs MT1 and MT2 in the ELISA. OD, optical density.

J. CLIN. MICROBIOL.

FIG. 5. Western blot of OMP of P. multocida P210 with MAbs MT1 (A) and MT2 (B) before and after treatment with 100 mg of proteolytic enzymes per ml or 20 mM sodium periodate. The reactivities of MAbs before treatment of the OMP (lane 1) and after treatment of the OMP with proteinase K (lane 2), chymotrypsin (lane 3), trypsin (lane 4), and sodium periodate (lane 5) are shown. Molecular mass markers (in kilodaltons) are indicated on the left.

except that MAb MT1 did not react with the OMP treated with proteinase K (Fig. 5A, lane 2). Periodate oxidation of P. multocida OMP. The binding of MAb MT1 to OMPs in the ELISA was not affected by periodate oxidation, whereas about 80% loss of binding of MAb MT2 occurred at a concentration of 1 mM sodium periodate and complete loss occurred at 20 mM (Fig. 6). Similar results were obtained in Western blots (Fig. 5A and B, lane 5). Immunoelectron microscopy. Surface localization of OMP was visualized by immunoelectron microscopy with the MAbs. The labelling obtained with MAbs MT1 and MT2 on capsulated strain 4275 (Fig. 7B and D) was weaker than that obtained on noncapsulated strain 4275B (Fig. 7A and C). The labelling of MAbs MT1 and MT2 on the capsulated strain was concentrated on OMVs. Serological specificities of MAbs. Seven of 37 field isolates of P. multocida examined in the study belonged to capsular serotype A and 30 belonged to capsular serotype D (Table 1). The OMPs of P. multocida 3001 and 1703 serotype D were analyzed with a 12% separating gel. Two patterns of OMPs designated types I and II were distinguished on the basis of

FIG. 6. Effect of sodium periodate treatment of OMP of P. multocida P210 on binding with MAbs MT1 and MT2 in ELISA.

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FIG. 7. Immunoelectron microscopy of P. multocida 4275B (A and C) and 4275 (B and D). Labelling of P. multocida 4275B by MAbs MT1 (A) and MT2 (C) and P. multocida 4275 by MAbs MT1 (B) and MT2 (D) is shown. Bars, 200 nm.

major protein bands with molecular masses of 32 and 37 kDa, respectively (Fig. 8A, lanes I and II, respectively). Seventy percent of serotype D isolates belonged to type I and 30% belonged to type II. Only type I isolates reacted with MAb MT1, whereas both type I and II isolates reacted with MAb MT2 in ELISA and Western blots (Table 1). Neither MAb MT1 nor MAb MT2 reacted with either reference strains of serotypes A, B, E, and F or any field isolates of capsular serotype A (Table 1). MAb MT2 recognized both P4673 and P2153 serotype D strains of P. multocida isolated from a rabbit and a turkey, respectively (Table 1). DISCUSSION Lugtenberg et al. (24) compared the cell envelope proteins of P. multocida strains presumably of serotypes A and D associated with atrophic rhinitis by using SDS-PAGE. On the basis of the electrophoretic mobilities of the H and W proteins, they distinguished three groups of cell envelope proteins designated types I, II, and III. Comparative study of the cell envelope

protein types and their pathogenicities revealed that all strains of type I and some of type III were pathogenic, whereas all strains of type II were nonpathogenic (25). MAbs directed against the OMP of P. multocida serotype A have been reported earlier (17, 22, 34). In the present report we have described MAbs MT1 and MT2 directed against the H and W proteins of P. multocida serotype D, respectively. Both MAbs MT1 and MT2 recognized an OMP with a molecular mass of 32 kDa (Fig. 1, lanes 3 and 4). However, when SDS-PAGE was carried out with 10% separating gel, MAbs MT1 and MT2 reacted with proteins with molecular masses of 32 and 31 kDa, respectively (Fig. 2). These results indicate that MAbs MT1 and MT2 are directed against two different proteins which comigrate when a 12% separating gel is used. As visualized by immunoelectron microscopy, the epitopes reactive with MAbs MT1 and MT2 are localized at the cell surface of P. multocida strains. These MAbs showed a stronger binding on the non-capsulated strain than on the capsulated strain (Fig. 7A and C), suggesting that the capsule may mask the epitopes which are accessible to antibodies.

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TABLE 1. Serotype and SDS-PAGE analysis of 9 reference and 37 field strains of P. multocida and their reactivities with MAbs MT1 and MT2 in ELISA and Western blots Molecular mass of major OMP in SDS-PAGE (kDa)b

MAb MT1

MAb MT2

Reference strains A (1) B (1) D (type I) (3) D (type II) (2) E (1) F (1)

37 32 32 37 38 36

2 2 1 2 2 2

2 2 1 1 2 2

Field strains A (7) D (type I) (21) D (type II) (9)

37 32 37

2 1 2

2 1 1

Strain and serotype (no.)a

Reactivity in ELISA and Western blot

a

Number of strains examined. SDS-PAGE analysis with whole-cell antigen indicates the approximate molecular mass of the major OMP. On the basis of the migration of the major OMP, two types of OMP patterns, designated types I and II, were found among field and reference strains of P. multocida serotype D. b

The strong binding of MAb MT2 with BC in ELISA and WC in Western blots (Fig. 2B, lane 1) and in immunoelectron microscopy (Fig. 7C) but its weak binding with the OMP preparation (Fig. 2B, lane 2) indicate the insufficient yield of the corresponding protein in the OMP preparation by the method used in the present study. The epitopes reactive with MAb MT1 were partially resistant to treatment with proteinase K in the ELISA, but were completely sensitive in the Western blot (Fig. 5A, lane 2). This may be due to partial masking of the proteinase K-susceptible sites of the major protein with LPS when it is tested in the ELISA (26). The persistence of the reactivity of MAb MT1 with OMP after periodate treatment (Fig. 6) and the absence of its reactivity with LPS (Fig. 2A, lane 4) confirm the protein nature of its corresponding epitopes. The criteria used to characterize pore proteins in Escherichia coli (23), P. multocida (24), and Pseudomonas aeruginosa (26), such as (i) sensitivity to proteinase K (Fig. 5A, lane 2), (ii) resistance to chymotrypsin or trypsin (Fig. 5A, lanes 3 and 4), (iii) inability to be solubilized in sample buffer at 378C (Fig. 3A and B), and (iv) localization on the cell surface (Fig. 7A and B) and OMV (Fig. 2A, lane 3, and Fig. 7B), suggest that MAb MT1 may be directed against the H protein (pore protein) of P. multocida. MAb MT2-binding epitopes were sensitive to treatment with both periodate (Fig. 6) and proteinase K but were partially resistant to treatment with trypsin and chymotrypsin (Fig. 4). In view of the fact that periodate oxidation cleaved the carbohydrate-vicinal hydroxyl groups of the antigen without altering the structure of the polypeptide chains (2), the results obtained in the present study suggest that the immunodominant structure of the epitopes reactive with MAb MT2 may be influenced by a carbohydrate moiety. Perusal of the literature shows that such a glycosidic antigen has not been identified earlier in P. multocida. The detection of multiple bands with MAb MT2 in Western blots (Fig. 2B, lane 1) may be due to variation in the degree of glycosylation of the reactive epitopes mainly because the oligosaccharide components fail to migrate as a single band on the electrophoresis gel (8, 10, 31). Since this protein (i) is resistant to degradation by the chymotrypsin and trypsin (Fig. 4), (ii) is exposed on the cell surface (Fig. 7C and D), and (iii) comigrates with major protein of 32 kDa (Fig. 1, lanes 3 and 4),

FIG. 8. OMP pattern (types I and II) observed among P. multocida serotype D field strains (3001 and 1703) by SDS-PAGE and their reactivities with MAbs MT1 and MT2 by Western blotting. The OMPs were dissolved in sample buffer by boiling and were separated by SDS-PAGE with a 12% separating gel (lanes I and II). One slice was stained with Coomassie brilliant blue (A), whereas the others were transferred to a nitrocellulose membrane and incubated with MAbs MT1 (B) and MT2 (C). Molecular mass markers (in kilodaltons) are indicated on the left.

it is suggested that MAb MT2 may be directed against a W protein of P. multocida whose electrophoretic mobility is indistinguishable from that of H protein (25). The reactivities of P. multocida serotype D strains isolated from a rabbit, a turkey, and a pig (Table 1) with MAb MT2 suggest that the W protein may be conserved among all serotype D strains, regardless of the animal species from which they have been isolated. However, a large-scale study of P. multocida strains from various species and various geographic locations is required to make a definite conclusion. On the basis of the results obtained in the present study (Table 1), it is suggested that MAbs MT1 and MT2 may be used effectively to subclassify serotype D strains. Further studies are in progress to demonstrate the usefulness of MAbs MT1 and MT2 in epidemiological studies of P. multocida infection. ACKNOWLEDGMENTS We gratefully thank G. R. Carter and R. B. Rimler for generous gifts of P. multocida strains. We also thank M. Jacques and Bernadette Foiry for assistance in immunogold labelling of the strains and The ´re`se Bernard-Gendron for formatting the manuscript. REFERENCES 1. Barenkamp, S. J., R. S. Munson, and D. M. Granoff. 1981. Subtyping isolates of Haemophilus influenzae type b by outer membrane protein profiles. J. Infect. Dis. 143:668–676. 2. Bobbitt, J. M. 1956. Periodate oxidation of carbohydrates. Adv. Carbohydr. Chem. Biochem. 11:1–41. 3. 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. 4. Buchana, T. M., and J. F. Hildebrandt. 1981. Antigenic-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein. Infect. Immun. 32:985–994. 5. Carter, G. R. 1955. Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am. J. Vet. Res. 16:481–484. 6. Carter, G. R., and S. W. Rundell. 1975. Identification of type A strains of Pasteurella multocida using staphylococcal hyaluronidase. Vet. Rec. 96:343. 7. Carter, G. R., and P. Subronto. 1973. Identification of type D strains of Pasteurella multocida with acriflavine. Am. J. Vet. Res. 34:293–294.

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VOL. 33, 1995 8. Cloeckaert, A., M. S. Zygmunt, P. de Wergifosse, G. Dubray, and J. N. Limet. 1992. Demonstration of peptidoglycan-associated Brucella outer-membrane proteins by use of monoclonal antibodies. J. Gen. Microbiol. 138:1543–1550. 9. Confer, A. W. 1993. Immunogens of Pasteurella. Vet. Microbiol. 37:353–368. 10. Gill, J., E. Stellwag, and M. Dworkin. 1985. Monoclonal antibodies against cell-surface antigens of developing cells of Myxococcus xanthus. Ann. Inst. Pasteur Microbiol. 136A:11–18. 11. Goding, J. W. 1983. Monoclonal antibodies: principles and practice, p. 85. Academic Press, Inc., New York. 12. Hamel, J., B. R. Brodeur, A. Belmaaza, S. Montplaisir, J. M. Musser, and R. K. Selander. 1987. Identification of Haemophilus influenzae type b by a monoclonal antibody coaaglutination assay. J. Clin. Microbiol. 25:2434– 2436. 13. Hanson, R. S., and J. A. Phillips. 1981. Chemical composition, p. 328–364. In P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 14. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis. 16:925–936. 15. Ireland, L., B. Adler, and A. R. Milner. 1991. Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera. Vet. Microbiol. 27:175– 185. 16. Johnson, R. B., H. J. S. Dawkins, and T. L. Spencer. 1991. Electrophoretic profiles of Pasteurella multocida isolates from animals with hemorrhagic septicemia. Am. J. Vet. Res. 52:1644–1648. 17. Khosraviani, M., T. Nunoya, and M. Matsumoto. 1990. Monoclonal antibodies against surface antigens of Pasteurella multocida strain P.1059. Avian Dis. 34:163–173. 18. Ko ¨hler, G., and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256:495–497. 19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685. 20. Larivie`re, S., L. Leblanc, K. R. Mittal, and G. P. Martineau. 1992. Characterization of Pasteurella multocida from nasal cavities of piglets from farms with or without atrophic rhinitis. J. Clin. Microbiol. 30:1398–1401. 21. Li, Z. S., N. S. Jensen, M. Be´langer, M. C. L’espe´rance, and M. Jacques. 1992. Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9. J. Clin. Microbiol. 30:2941–2947. 22. Lu, Y.-S., L. W. Gerrity, S. P. Pakes, and L. C. Nie. 1991. A monoclonal

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24.

25.

26.

27. 28. 29.

30. 31. 32.

33.

34.

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antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against pasteurellosis. Infect. Immun. 59:172–180. Lugtenberg, B., and L. van Alphen. 1983. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim. Biophys. Acta 737:51–115. Lugtenberg, B., R. van Boxtel, and M. de Jong. 1984. Atrophic rhinitis in swine: correlation of Pasteurella multocida pathogenicity with membrane protein and lipopolysaccharide patterns. Infect. Immun. 46:48–54. Lugtenberg, B., R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik. 1986. Biochemical and immunological characterization of cell surface proteins of Pasteurella multocida strains causing atrophic rhinitis in swine. Infect. Immun. 52:175–182. Mutharia, L. M., and R. E. W. Hancock. 1985. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 31:381–386. Ogden, J. R., and K. Leung. 1988. Purification of murine monoclonal antibodies by caprilic acid. J. Immunol. Methods 111:283–284. Overbeeke, N., and B. Lugtenberg. 1980. Major outer membrane proteins of Escherichia coli strains of human origin. J. Gen. Microbiol. 121:373–380. Rebers, P. A., M. Phillips, R. Rimler, R. A. Boykins, and K. R. Rhodes. 1980. Immunizing properties of Westphal lipopolysaccharide from an avian strain of Pasteurella multocida. Am. J. Vet. Res. 41:1650–1654. Rimler, R. B., and K. R. Rhoades. 1987. Serogroup F, a new capsular serogroup of Pasteurella multocida. J. Clin. Microbiol. 25:615–618. Springer, W. R., and S. H. Barondes. 1983. Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum. J. Biol. Chem. 258:4698–4701. Thomson, M. S., L. H. Lauerman, and G. R. Wilt. 1990. Monoclonal antibody in the identification of Haemophilus somnus. J. Vet. Diagn. Invest. 2:116–119. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc. Natl. Acad. Sci. USA 74:4350–4354. Wijewardana, T. G., C. F. Wilson, N. J. L. Gilmour, and I. R. Poxton. 1990. Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody. J. Med. Microbiol. 33:217–222. Woodward, M. P., W. W. Young, and R. A. Bloodgood. 1985. Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J. Immunol. Methods 78:143–153.