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INFECTION AND IMMUNITY, Sept. 1986, p. 460-463 0019-9567/86/090460-04$02.00/0 Copyright ©D 1986, American Society for Microbiology Pasteurellosis in Laboratory Rabbits: Characterization of Lipopolysaccharides of Pasteurella multocida by Polyacrylamide Gel Electrophoresis, Immunoblot Techniques, and Enzyme-Linked Immunosorbent Assay PATRICK J. MANNING,* MARK A. NAASZ, DAVID DELONG, AND STEVEN L. LEARY Division of Comparative Medicine, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received 10 March 1986/Accepted 21 May 1986 The lipopolysaccharides (LPSs) of five isolates of Pasteurella multocida from rabbits were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblots, and enzyme-linked immunosorbent assay. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of purified unaggre- gated LPSs resembled those of semirough strains of gram-negative enterobacteria and consisted of one or two bands that migrated within an interval just ahead or slightly behind the migration of the Ra chemotype of "Salmonella minnesota," which has a molecular size of 4.3 kilodaltons. Polyclonal rabbit antisera to P. multocida whole cells used in Western blots and enzyme-linked immunosorbent assays of unabsorbed and LPS-absorbed antisera revealed that the LPS of these isolates of P. multocida contained at least two types of antigens: a nonserospecific antigen and a serospecific antigen. The LPSs of four isolates each had a different serospecific antigen. The nonserospecific antigen was expressed in two isolates and was the only demonstrable LPS antigen in one other isolate. Mucopurulent rhinitis or "snuffles" in laboratory rabbits is the major clinical manifestation of chronic pasteurellosis caused by Pasteurella multocida. The stresses of experimen- tation on infected rabbits commonly contribute to more serious forms of pasteurellosis, including pneumonia, pul- monary abscesses, and otitis media. These and other mani- festations of pasteurellosis in rabbits are refractory to anti- biotic therapy. Prolonged clinical infection either kills the animal or prompts euthanasia and thereby limits the useful- ness of rabbits as laboratory animals, particularly in long- term studies (9). Although several capsular and somatic serotypes of P. multocida are involved in pasteurellosis of domestic farm animals and birds (3, 10), there appear to be relatively few serotypes that are pathogenic for rabbits (1, 5, 7, 13). In a previous report (15), we evaluated the lipopolysaccharides (LPSs) of 10 rabbit isolates of P. multocida by enzyme- linked immunosorbent assay (ELISA) and concluded that there was considerable antigenic homology among their LPSs, even though results of gel diffusion precipitin tests of cell wall extracts suggested much antigenic heterogeneity. We have extended these observations by characterizing the LPSs of five of these isolates of P. multocida by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), immunoblots, and ELISA with LPS-absorbed anti- sera. MATERIALS AND METHODS Bacterial isolates. Several characteristics of the five rabbit isolates of P. multocida are shown in Table 1. Purified LPS was prepared by inoculating each isolate into 30 liters of brain heart infusion broth. The broth was incubated at 37°C overnight, and the culture was killed by addition of 0.03 volume of 37% formaldehyde. The organisms were packed * Corresponding author. by centrifugation and washed three times in phosphate- buffered saline (PBS; 0.02 M Na2PO4 in 0.85% NaCl, pH 7.2). Each batch of organisms was dried by acetone, and LPSs were extracted either as described previously (15) or as described by Darveau and Hancock (6) with the substitu- tion of sodium deoxycholate for SDS. Each method yielded a comparable amount of LPS (1 to 2% of dry cell weight) that contained less than 1.5% protein (15) and had indistinguish- able silver-stained SDS-PAGE profiles. Purified LPS and the Ra chemotype of "Salmonella minnesota" were purchased from List Biologics (Campbell, Calif.). SDS-PAGE and silver staining. SDS-PAGE was performed with the Laemmli buffer system (12) on 5-,ul samples con- taining 1 to 3 jig of LPS in Laemmli buffer. The 5% stacking gel and 15% separating gel contained SDS; the separating gel also contained 4 M urea to aid in disaggregation of LPS (16). Electrophoresis (equipment from LKB Sverige AB, Brom- ma, Sweden) was done at 50 mA constant current for 30 min longer than required for the bromophenol blue dye front to leave the gel (about 5 h) in Tris-glycine (pH 8.3) buffer containing 0.1% SDS. The reservoir was cooled with flowing tap water. In some gels, we also applied a low-molecular- weight protein marker (Bio-Rad Laboratories, Richmond, Calif.) and "S. minnesota" LPS to compare with the band- ing patterns and mobilities of P. multocida LPSs. Also, an estimate of the molecular weights of P. multocida LPSs was obtained by including the Ra chemotype of "S. minnesota" in some gels. The gels were fixed and stained for LPS by the silver technique described by Dubray and Bezard (8). Immunoblotting. After SDS-PAGE, LPSs were electro- phoretically transferred (Western blot) from the gel onto nitrocellulose sheets (Transphor Unit; Hoefer Scientific In- struments, San Francisco, Calif.) in the presence of 150 mM glycine-20 mM Tris (pH 8.3) in 20% methanol at 0.6 A for 18 h in a reservoir cooled with flowing tap water. After electrotransfer, the nitrocellulose sheets were immersed in a 460 Vol. 53, No. 3 on March 7, 2019 by guest http://iai.asm.org/ Downloaded from
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Page 1: Pasteurellosis in Laboratory Rabbits: … forms of pasteurellosis, including pneumonia, pul-monaryabscesses, and otitis media. These and other mani-festations ofpasteurellosis in rabbits

INFECTION AND IMMUNITY, Sept. 1986, p. 460-4630019-9567/86/090460-04$02.00/0Copyright ©D 1986, American Society for Microbiology

Pasteurellosis in Laboratory Rabbits: Characterization ofLipopolysaccharides of Pasteurella multocida by PolyacrylamideGel Electrophoresis, Immunoblot Techniques, and Enzyme-Linked

Immunosorbent AssayPATRICK J. MANNING,* MARK A. NAASZ, DAVID DELONG, AND STEVEN L. LEARY

Division of Comparative Medicine, Department of Laboratory Medicine and Pathology, University of Minnesota MedicalSchool, Minneapolis, Minnesota 55455

Received 10 March 1986/Accepted 21 May 1986

The lipopolysaccharides (LPSs) of five isolates of Pasteurella multocida from rabbits were characterized bysodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblots, and enzyme-linked immunosorbentassay. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of purified unaggre-gated LPSs resembled those of semirough strains of gram-negative enterobacteria and consisted of one or twobands that migrated within an interval just ahead or slightly behind the migration of the Ra chemotype of"Salmonella minnesota," which has a molecular size of 4.3 kilodaltons. Polyclonal rabbit antisera to P.multocida whole cells used in Western blots and enzyme-linked immunosorbent assays of unabsorbed andLPS-absorbed antisera revealed that the LPS of these isolates of P. multocida contained at least two types ofantigens: a nonserospecific antigen and a serospecific antigen. The LPSs of four isolates each had a differentserospecific antigen. The nonserospecific antigen was expressed in two isolates and was the only demonstrableLPS antigen in one other isolate.

Mucopurulent rhinitis or "snuffles" in laboratory rabbitsis the major clinical manifestation of chronic pasteurellosiscaused by Pasteurella multocida. The stresses of experimen-tation on infected rabbits commonly contribute to moreserious forms of pasteurellosis, including pneumonia, pul-monary abscesses, and otitis media. These and other mani-festations of pasteurellosis in rabbits are refractory to anti-biotic therapy. Prolonged clinical infection either kills theanimal or prompts euthanasia and thereby limits the useful-ness of rabbits as laboratory animals, particularly in long-term studies (9).Although several capsular and somatic serotypes of P.

multocida are involved in pasteurellosis of domestic farmanimals and birds (3, 10), there appear to be relatively fewserotypes that are pathogenic for rabbits (1, 5, 7, 13). In aprevious report (15), we evaluated the lipopolysaccharides(LPSs) of 10 rabbit isolates of P. multocida by enzyme-linked immunosorbent assay (ELISA) and concluded thatthere was considerable antigenic homology among theirLPSs, even though results of gel diffusion precipitin tests ofcell wall extracts suggested much antigenic heterogeneity.We have extended these observations by characterizing theLPSs of five of these isolates of P. multocida by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblots, and ELISA with LPS-absorbed anti-sera.

MATERIALS AND METHODS

Bacterial isolates. Several characteristics of the five rabbitisolates of P. multocida are shown in Table 1. Purified LPSwas prepared by inoculating each isolate into 30 liters ofbrain heart infusion broth. The broth was incubated at 37°Covernight, and the culture was killed by addition of 0.03volume of 37% formaldehyde. The organisms were packed

* Corresponding author.

by centrifugation and washed three times in phosphate-buffered saline (PBS; 0.02 M Na2PO4 in 0.85% NaCl, pH7.2). Each batch of organisms was dried by acetone, andLPSs were extracted either as described previously (15) oras described by Darveau and Hancock (6) with the substitu-tion of sodium deoxycholate for SDS. Each method yieldeda comparable amount of LPS (1 to 2% of dry cell weight) thatcontained less than 1.5% protein (15) and had indistinguish-able silver-stained SDS-PAGE profiles. Purified LPS and theRa chemotype of "Salmonella minnesota" were purchasedfrom List Biologics (Campbell, Calif.).SDS-PAGE and silver staining. SDS-PAGE was performed

with the Laemmli buffer system (12) on 5-,ul samples con-taining 1 to 3 jig of LPS in Laemmli buffer. The 5% stackinggel and 15% separating gel contained SDS; the separating gelalso contained 4 M urea to aid in disaggregation of LPS (16).Electrophoresis (equipment from LKB Sverige AB, Brom-ma, Sweden) was done at 50 mA constant current for 30 minlonger than required for the bromophenol blue dye front toleave the gel (about 5 h) in Tris-glycine (pH 8.3) buffercontaining 0.1% SDS. The reservoir was cooled with flowingtap water. In some gels, we also applied a low-molecular-weight protein marker (Bio-Rad Laboratories, Richmond,Calif.) and "S. minnesota" LPS to compare with the band-ing patterns and mobilities of P. multocida LPSs. Also, anestimate of the molecular weights of P. multocida LPSs wasobtained by including the Ra chemotype of "S. minnesota"in some gels. The gels were fixed and stained for LPS by thesilver technique described by Dubray and Bezard (8).

Immunoblotting. After SDS-PAGE, LPSs were electro-phoretically transferred (Western blot) from the gel ontonitrocellulose sheets (Transphor Unit; Hoefer Scientific In-struments, San Francisco, Calif.) in the presence of 150 mMglycine-20 mM Tris (pH 8.3) in 20% methanol at 0.6 A for 18h in a reservoir cooled with flowing tap water. Afterelectrotransfer, the nitrocellulose sheets were immersed in a

460

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LPS OF P. MULTOCIDA 461

TABLE 1. Capsular and somatic types of P. multocida isolatesfrom rabbits

Isolate Capsular Somatic type"no. Source typeaPflO. type ~~~~NADC Lab LPS

1 Skin abscess A 3 (4) 4 (7, 12, 3) 12 (4, 3, 7)2 Scrotal D 3 (4) 3 (12, 4) 3

abscess6 Mesenteric D 4 12 (4, 5, 7) 12

abscess10c Pennsylvania D 1 1 ld

cottontail (3, 4, 5)llc Rabbit from A 15 Untypable (15)

Brazila Type A isolates produce a hyaluronic acid capsule, the production of

which is inhibited by hyaluronidase; type D isolates aggregate in 0.1% solutionof acriflavine (15).

b Minor (faint) or delayed (faint and occurring after 24 h) precipitin lines areincluded within parentheses. Somatic typing was done in a gel diffusionprecipitin test in which type-specific chicken antisera (kindly provided by theNational Animal Disease Center, Ames, Iowa) was reacted against a Forma-lin-saline heat-stable extract of the isolate either at the Ames Laboratory(NADC) or in our laboratory (Lab) with chicken antisera kindly provided byBilly Blackburn of the NADC (10). In the column labeled LPS, purified LPSwas used as antigen, and the tests were done in our laboratory.

c Kindly provided by the Microbiology Laboratory, National Animal Dis-ease Center, Ames, Iowa.

d LPS of this isolate was present mainly in the phenol layer when cells wereextracted by the hot phenol-H20 method of Westphal and Jann (21). LPSsfrom other isolates were within the aqueous phase.

blocking buffer (1% bovine serum albumin, 0.85% NaCl, 20mM NaHPO4 [pH 7.2], 0.01% thimerosal) for 3 h and then inimmune serum diluted 1:100 in blocking buffer for 1 h atroom temperature. The sheets were washed three times for10 min in PBS and immersed for 1 h at room temperature ina solution of protein-A-conjugated horseradish peroxidase(Sigma Chemical Co., St. Louis, Mo.) diluted to 2 ,ug/ml inblocking buffer. After three 10-min washes in PBS, substratesolution consisting of 0.05% (wt/vol) 3,3'-diaminobenzidine,0.01% (vol/vol) hydrogen peroxide, 0.05 M citric acid, and0.05 M sodium citrate in distilled water was added. Thebrown reaction product was allowed to form for 10 to 40 s.The sheets were rinsed in distilled water and air dried.Immune sera. Immune sera to killed whole cells were

obtained from pasteurella-free New Zealand White rabbitsmaintained and immunized as described previously (15).ELISA. Lyophilized LPS was diluted in carbonate buffer

(0.05 M carbonate, pH 9.6) to 100 ,ug/ml and sonicated for 1min at a 35% setting (Fisher Scientific Co., Pittsburgh, Pa.).Sonicated LPS solution (0.1 ml; 10 ,ug of LPS) was put intowells of polystyrene plates (Falcon 3070; Becton DickinsonLabware, Oxnard, Calif.), which were then covered andkept at 37°C overnight. The wells were washed three timeswith PBS. Blocking buffer (PBS with 10% fetal bovineserum, 1% bovine serum albumin, and 0.3% gelatin) wasadded to fill each well, and the plates were kept at roomtemperature for 3 h. The wells were washed three times withPBS. Antisera (0.1 ml) diluted 1:1,000 in diluting buffer (PBScontaining 1% bovine serum albumin and 0.3% gelatin) wasadded; the plates were kept at room temperature for 20 minand were washed five times with PBS. Protein-A-conjugatedhorseradish peroxidase (0.1 ml, original concentration, 1mg/ml; Sigma), diluted 1:500 in diluting buffer was added;the plates were kept at room temperature for 20 min andwere again washed five times with PBS. Substrate, consist-ing of 0.05% O-phenylenediamine (Sigma) in a buffer com-posed of 0.05 M citric acid, 0.05 M sodium citrate, and 0.01%hydrogen peroxide, was prepared just before use, and 0.1 ml

was added to each well. After 1 h at room temperature, thereaction was stopped by the addition of 0.1 ml of 1.6 Nsulfuric acid to each well. Optical densities were read at 492nm (Titertek Multiscan; Flow Laboratories, Inc., McLean,Va.). Control wells lacked only LPS, and all determinationswere done in duplicate.Serum absorption. Antiserum, diluted 1:500 in diluting

buffer, was added to an equal volume of diluting buffercontaining 20 p.g of LPS per ml. The sera were absorbed for1 h at room temperature with constant rocking. Unabsorbedantisera at a dilution of 1:1,000 served as a control.

RESULTS

SDS-PAGE profiles of silver-stained LPSs are shown inFig. 1. None of the LPS profiles of the five isolates demon-strated the ladderlike profile characteristic of semiroughEscherichia or Salmonella strains including "S. minnesota"(11). Rather, the LPS profiles of P. multocida contained oneor two (isolate 6 occasionally had a third trailing band)argentophilic bands which migrated somewhat faster than aprotein standard of 14 kilodaltons. All the bands were withinan interval slightly ahead to slightly behind the Rachemotype of "S. minnesota." The Ra chemotype has anestimated molecular weight of 4,311 (16) and consists of lipidA and core polysaccharide but lacks repeating oligosaccha-ride units (11).Immunoblots and ELISA of absorbed antisera. The possi-

ble antigenic relatedness among the LPSs of these fiveisolates of P. multocida was evaluated by use of im-munoblots and ELISAs. The results of several Western blotsand ELISAs are shown in Fig. 2 and 3. Antiserum to isolate1 reacted strongly to moderately with LPSs of isolates 1, 2,and 6 and weakly with isolate 11 LPS (Fig. 2a). The strongcross-reactivity of isolate 1 antisera to LPSs of isolates 2 and6 was confirmed by ELISAs on immune sera and LPS-absorbed immune sera (Fig. 3A). Nearly all the antibody toLPS in isolate 1 antisera was removed by absorption withLPS of isolates 2 and 6. Conversely, LPSs of isolates 10 and11 had negligible absorptive potency on isolate 1 antisera.The reactivity of antisera to isolates 2 and 6 (Fig. 2b and c)

was very similar to that of isolate 1 antiserum in im-munoblots, except that isolate 6 antiserum reacted veryweakly with isolate 2 LPS (Fig. 2c). Based upon the activityof isolate 6 antiserum to isolate 2 LPS in the ELISA (Fig.3C), a stronger reaction was expected in the immunoblot.The faint band shown in Fig. 2c, lane 2, is a minor band ofisolate 2 that was also seen occasionally in silver-stainedSDS-polyacrylamide gels. This band, though not seen in Fig.1, migrated just ahead of the band shown in Fig. 1, lane 2,and coincided with the band of isolate 1 (Fig. 1, lane 1). Itwould appear from these observations that the LPS of isolate2 includes traces of incomplete LPS molecules composedmainly of common (nonserospecific) antigen and that theminor disparity between the ELISAs and immunoblots isinfluenced by the amount of nonserospecific LPS antigenwithin a given preparation of LPS. In this instance, the batchof LPS from isolate 2 used for the ELISA contained morenonserospecific LPS than did the batch of LPS used in theimmunoblot. ELISAs on LPS-absorbed antisera to isolate 2and isolate 6 revealed that each isolate possessed a distinctserospecific LPS antigen not possessed by the other isolates(Fig. 3B and C), in addition to the nonserospecific LPSantigen shared by isolates 1, 2, and 6.

Finally, immunoblots of these LPSs reacted with antiserato isolates 10 and 11 are shown in Fig. 2d and e. Each

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462 MANNING ET AL.

Antisera to Whole Cells of Isolate 1 vs. LPS of Isolates 1, 2, 6, 10 & 11

IA.

0

"~~~~~~~~~~~

I_

mlb_

1 2 6 16 11

FIG. 1. Silver-stained profiles in SDS-PAGE of LPS of P.multocida. Lane numbers correspond to the isolates as numbered inTable 1. Each lane contains 5 ,ul (2 ,ug) of LPS.

antiserum reacted strongly with homologous LPS andweakly or not at all with LPSs of the other isolates. ELISAof absorbed and unabsorbed antisera to isolates 10 and 11demonstrated clearly that the major antigenic characteristicsof LPSs from these isolates were serospecific (Fig. 3D andE).

DISCUSSIONThe semirough characteristics of P. multocida LPSs re-

semble the SDS-PAGE profiles of LPSs from several otherbacterial genera, including Neisseria gonorrhoeae (16),Campylobacterjejuni (18), Neisseria meningitidis (19), andBacteroides fragilis (20). Moreover, LPS profiles of thesefive isolates of P. multocida are very similar to those of P.multocida isolated from the nasal cavities of swine (14),

a c

'I.

1 2 t IS 111 2 6 10 1

b

1 2 6 10 11 1 2 6 11

1 2 I le I1I

FIG. 2. Western blots of LPS transferred from SDS-PAGE onto

nitrocellulose and reacted with polyclonal antisera to whole cells.

The lane numbers correspond to the isolates as numbered in Table

1. The antisera used were to isolate 1 (a), isolate 2 (b), isolate 6 (c),

isolate 10 (d), and isolate 11 (e).

1.0 1.0

0.5

Abso,bu None 1 2 6 10 1

Antisera to Whole Cells of Isolate 6 vs. LPS of Isolates 1, 2. 6. 10 & 11

C.

ab.e None 10 2 6 1 1

10iertWhl10 l fIoae1 s LSo slts1 ,61 10

00.5 0.5

A Lhe None 1 2 6 10 11

Antisera to Whole Cells of Isolate 11 vs. LPS of Isolates 1, 2, 6, 10 & 111.0rbu None 10 2 10to1 1 0US

0

FIG. 3. ELISAs of absorbed and unabsorbed antisera to wholecells of isolates 1, 2, 6, 10, and 11. Antiserum to each of theseisolates was mixed with LPS of isolates 1, 2, 6, 10, and 11; theabsorbed antiserum was reacted with various LPSs from isolateswhose number appears directly above the bar. 0D492, Opticaldensity at 492 nm.

except that LPS PAGE profiles of our isolates had fewerbands. In our initial studies on P. multocida, we also noticedup to seven variably spaced LPS bands within an estimatedmolecular-size range of 15 to 26 kilodaltons in silver-stainedgels. These LPS bands appear to be the result of clustering ofLPS molecules to form aggregates with various molecularweights as has been described for the LPSs of N. gonor-rhoeae (16). Aggregation is inhibited or diminished either byincorporation of 4 M urea into the separating gel or by mildalkaline hydrolysis of LPS before SDS-PAGE (16). Eitherprocedure proved satisfactory for our studies and resulted invery similar SDS-PAGE LPS profile banding patterns (Fig.1).Although the LPSs of these P. multocida isolates lack the

several repeating oligosaccharide units responsible forserospecificity among several genera of the Enterobacteria-ceae family, these LPSs contain, within a relatively smallmolecule that is similar in size to "S. minnesota" chemotype

Antisera to Whole Cells of Isolate 2 vs. LPS of Isolates 1, 2. 6,10 & 11B.

2 22

2 2

a

I I 6 1011 2 6 101 I 0 'OilI 6 to I 6

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LPS OF P. MULTOCIDA 463

Ra (4.3 kilodaltons), both serospecific and nonserospecificantigens. Isolates 2, 6, 10, and 11 each had a differentserospecific LPS antigen. The serospecificity of LPSs ofisolates 10 and 11 confirms an earlier report by Cary et al.(4), who evaluated the LPSs of these two isolates by ELISAinhibition tests.The nature of the nonserospecific antigen(s) is not yet

clear, but the occurrence of common antigens in the LPSs ofP. multocida has also been suggested by other investigators(4, 17). If this antigen is common to the LPSs of all fiveisolates, it appears to be sterically shielded in isolates 10 and11. Alternatively, there may be different core antigensamong these LPSs of P. multocida, or there may be avariable LPS antigen, as has been demonstrated for theLPSs of N. gonorrhoeae (16). The nonserospecific antigenon our isolates appears to be common to many rabbit isolatesof P. multocida because Western blots of LPS of five otherisolates (15) reacted strongly with antisera of isolates 1 and 6(unpublished observations). Moreover, this nonserospecificantigen is probably the LPS antigen responsible for broadcross-reactivity demonstrated by extracts of isolates 1, 2,and 6 with several typing sera in the gel diffusion precipitintests (2, 4, 10) shown in Table 1 (column 5).

In summary, the LPSs of five rabbit isolates of P.multocida were purified and evaluated by SDS-PAGE,ELISA, and Western blots. The SDS-PAGE profiles weresimilar to semirough LPSs of enterobacteriaceae. The LPSscontained a nonserospecific antigen, a serospecific antigen,or both. Four of the isolates each had a different serospecificLPS antigen. The nonserospecific antigen was expressed onthe LPS preparation of two isolates (strongly in isolate 6 andweakly in isolate 2) and was the only demonstrable LPSantigen in one other isolate.

ACKNOWLEDGMENTS

We thank Betty Werner for her expert technical assistance andPatt Mercer Sass for typing the manuscript.

This research was supported by Public Health Service grantRR01234 from the Division of Research Resources, National Insti-tutes of Health.

LITERATURE CITED1. Brogden, K. A. 1980. Physiological and serological characteris-

tics of 48 Pasteurella multocida cultures from rabbits. J. Clin.Microbiol. 11:646-649.

2. Brogden, K. A., and P. A. Rebers. 1978. Serologic examinationof the Westphal-type lipopolysaccharides of Pasteurellamultocida. Am. J. Vet. Res. 39:1680-1682.

3. Carter, G. R. 1955. Studies on Pasteurella multocida. I. Ahemagglutination test for the identification of serological types.Am. J. Vet. Res. 16:481-486.

4. Cary, C. J., G. K. Peter, C. E. Chrisp, and D. F. Keren. 1984.Serological analysis of five serotypes of Pasteurella multocidaof rabbit origin by use of an enzyme-linked immunosorbent

assay with lipopolysaccharide as antigen. J. Clin. Microbiol.20:191-194.

5. Changappa, M. M., R. C. Meyers, and G. R. Carter. 1982.Capsular and somatic types of Pasteurella multocida fromrabbits. Can. J. Comp. Med. 46:437-439.

6. Darveau, R. P., and R. E. W. Hancock. 1983. Procedure forisolation of bacterial lipopolysaccharides from both smooth andrough Pseudomonas aeruginosa and Salmonella typhimuriumstrains. J. Bacteriol. 155:831-838.

7. DiGiacomo, R. F., L. E. Garlinghouse, Jr., and G. L. VanHoosier, Jr. 1983. Natural history of infection with Pasteurellamultocida in rabbits. J. Am. Vet. Med. Assoc. 183:1172-1175.

8. Dubray, G., and G. Bezard. 1982. A highly sensitive periodicacid-silver stain for 1,2-diol groups of glycoproteins andpolysaccharides in polyacrylamide gels. Anal. Biochem.119:325-329.

9. Flatt, R. E. 1974. Bacterial diseases, p. 193-205. In S. W.Weisbroth, R. E. Flatt, and A. L. Kraus (ed.), The biology ofthe laboratory rabbit. Academic Press, Inc., New York.

10. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Geldiffusion precipitin test for serotyping Pasteurella multocidafrom avian species. Avian Dis. 16:925-936.

11. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heter-ogeneity among Salmonella lipopolysaccharide chemotypes insilver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.

12. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

13. Lu, Y.-S., S. P. Pakes, and C. Stefanu. 1983. Capsular andsomatic serotypes of Pasteurella multocida isolates recoveredfrom healthy and diseased rabbits in Texas. J. Clin. Microbiol.18:292-295.

14. Lugtenberg, B., R. van Boxtel, and M. de Jong. 1984. Atrophicrhinitis in swine: correlation of Pasteurella multocida pathoge-nicity with membrane protein and lipopolysaccharide patterns.Infect. Immun. 46:48-54.

15. Manning, P. J. 1984. Naturally occurring pasteurellosis inlaboratory rabbits: chemical and serological studies of wholecells and lipopolysaccharides of Pasteurella multocida. Infect.Immun. 44:502-507.

16. Mintz, C. S., M. A. ApicelHa, and S. A. Morse. 1984. Electro-phoretic and serological characterization of the lipopolysaccha-ride produced by Neisseria gonorrhoeae. J. Infect. Dis.149:544-552.

17. Namioka, S. 1978. Taxonomy and nomenclature of Pasteurellamultocida, p. 273-292. In T. Bergan and J. Norris (ed.),Methods in microbiology, vol. 10. Academic Press, Inc., NewYork.

18. Perez, G. I. P., J. A. Hopkins, and M. J. Blaser. 1985. Antigenicheterogeneity of lipopolysaccharides from Campylobacterjejuni and Campylobacter fetus. Infect. Immun. 48:528-533.

19. Tsai, C.-M., R. Boykins, and C. E. Frasch. 1983. Heterogeneityand variation among Neisseria meningitidis lipopolysaccha-rides. J. Bacteriol. 155:498-504.

20. Weintraub, A., B. E. Larsson, and A. A. Lindberg. 1985. Chem-ical and immunochemical analyses of Bacteroides fragilis lipo-polysaccharides. Infect. Immun. 49:197-201.

21. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide.Methods Carbohydr. Chem. 5:83-91.

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