JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1990, p. 654-6590095-1137/90/040654-06$02.00/0Copyright C 1990, American Society for Microbiology

Comparisons of Pasteurella multocida Lipopolysaccharides bySodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis ToDetermine Relationship between Group B and E HemorrhagicSepticemia Strains and Serologically Related Group A Strains

RICHARD B. RIMLER

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70,Ames, Iowa 50010

Received 11 October 1989/Accepted 18 December 1989

Lipopolysaccharides (LPSs) purified from 16 reference somatic serotypes of Pasteurella multocida were

examined and compared by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolu-tion of LPS patterns in a gel was optimum when sample wells were cast separately from the stacking gel andthe running gel consisted of 15% T (total monomer) polyacrylamide and 4 M deionized urea. Band patterns ofP. multocida LPSs in a gel differed from control Salmonella minnesota wild-type and core mutant LPSs.Although the band patterns and mobilities of LPSs from some P. multocida reference serotypes were similar,none were identical. Evidence for O antigens similar to those produced by enterobacteria was not observed.Proteinase K digestion of whole P. multocida cells resulted in LPS band patterns similar to those of purifiedLPS. The presence or absence of a capsule on a strain had no major influence on band patterns in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. Comparisons of LPS patterns of group B and E hemor-rhagic septicemia strains with those of serologically related group A strains of P. multocida indicated that theywere similar. Typing antisera made with purified serotype 2 or 5 LPS reacted with electroblots of all thesestrains. However, the reactions did not distinguish strains as being serotype 2 or 5.

Pasteurella multocida is a pathogen in diseases of avariety of domestic and feral mammals and birds. Sixteensomatic serotypes of P. multocida (numbered 1 through 16)have been described, and these serotypes occur in any offive distinct capsular groups (lettered A, B, D, E, and F).Somatic serotypes of P. multocida are determined by a geldiffusion precipitin test (GDPT), which utilizes heat-stableantigens extracted from formalinized saline suspensions ofthe bacterium. The serotype specificity of the test is due tothe lipopolysaccharide (LPS) component of the heat-stableantigens. Lipopolysaccharide has been purified from each ofthe different somatic serotypes and, although their biochem-ical structures have not been determined, constituents of P.multocida LPSs are like those found in typical gram-negativebacteria (15).

Hitchcock and Brown (4) have shown that biochemicaldifferences of purified LPS or LPS in proteinase K digests ofwild-type and mutant strains of Salmonella sp. were re-flected in unique patterns in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). Examinationof the LPSs from other bacteria with this technique hasresulted in similar findings (3, 10). Manning et al. (7) utilizedSDS-PAGE to examine LPSs from four serotypes of P.multocida isolated from rabbits. Their findings indicated thatpatterns produced by P. multocida LPSs were similar to thatproduced by a Ra mutant of Salmonella minnesota; Ramutants form rough LPSs which lack O antigens. Similarly,in a study of 25 swine isolates of P. multocida (serotypesunknown) by SDS-PAGE, Lugtenberg et al. (6) observedthat proteinase K digests produced LPS patterns character-istic of an LPS that lacks O antigens. Comparisons ofSDS-PAGE patterns of LPSs from all of the 16 differentsomatic serotypes of P. multocida have not been done.Hemorrhagic septicemia in cattle, bison, and buffalo is a

specific disease produced by certain strains of P. multocida.These strains are found in capsule group B or E and areenzootic to certain geographical areas. For example, in Asia,southern Europe, and, rarely, North America, hemorrhagicsepticemia is produced by strains with group B capsules. InAfrica, the disease is produced by strains with group B or Ecapsules. Hemorrhagic septicemia-producing strains werepreviously assigned to somatic serotype 2 on the basis offindings in the GDPT. However, recent research with typingantisera made in chickens by using purified LPS has shownthat cross-reactions occur between somatic serotype 2 andsomatic serotype 5 antigens in serologic and immune protec-tion tests (14). The validity of assigning strains of P. multo-cida to somatic serotype 2 or 5 is now questionable. Becausethe biochemical constituents of their LPSs are markedlysimilar (14), SDS-PAGE analysis might be used to resolvewhether there are any real differences between somaticserotypes 2 and 5.

In this study, purified LPS and LPS in proteinase Kdigests from all the 16 somatic serotypes of P. multocidawere examined by SDS-PAGE. Different concentrations ofacrylamide, the presence of urea, storage of treated samples,and use of capsulated and noncapsulated strains were testedto determine their influence on the LPS pattern in SDS-PAGE. P. multocida strains which serotype as both 2 and 5were analyzed by SDS-PAGE and immunoblot methods todetermine whether their LPSs differed.

MATERIALS AND METHODS

Bacteria and LPSs. The capsule group, somatic serotypeas determined by the GDPT, and geographical and animalsources of 33 strains of P. multocida used are listed in Table1. The LPSs from reference strains of somatic serotypes 1

654

Vol. 28, No. 4

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

P. MULTOCIDA LIPOPOLYSACCHARIDES 655

TABLE 1. Capsule group, somatic serotype, and geographic andhost sources of P. multocida

Capsule group SourceStrain and somatic

serotype Geographic Host

X-73 A: 1 Maryland ChickenP-4383 A:1 California Mallard duckP-4795 A: 1 California MuskratP-1059 A:3 West Virginia TurkeyP-1662 A:4 South Carolina TurkeyP-1702b A:2,5c Virginia TurkeyP-3201 A:2,5 Texas TurkeyP-3203 A:2,5 Texas TurkeyP-5178 A:2,5 Washington Bighorn sheepP-2192 - :6d Texas TurkeyP-1997 -:7 New York Herring gullP-1581 -:8 Massachusetts Pine siskinP-2095 A:9 Minnesota TurkeyP-2100 A: 10 Indiana TurkeyP-903 -:11 Maryland SwineP-1573 A: 12 Iowa HumanP-1591 - :13 Iowa HumanP-2225 A: 14 Iowa BovineP-2237 - :15 Iowa TurkeyP-2723 A: 16 Indiana TurkeyP-3269 B:1 California SwanM_1404e B:2,5 Wyoming BisonP-5146 B:2,5 India SwineP-4653 B:2,5 India BuffaloP-4656 B:2,5 India DeerP-3705 B:2,5 Sri Lanka BuffaloP-4866 B:2,5 Malaysia BuffaloP-4258 B:2,5 China YakP-1235 E:2,5 Africa BovineP-4120 E:2,5 Cameroon BovineP-4121 E:2,5 Senegal BovineP-4123 E:2,5 Mali BovineP-4110 E:2,5 Sudan Bovine

'Boldface means reference strain of somatic serotype.b Somatic serotype 5 reference strain.' Reacts as both serotype 2 and serotype 5 in the GDPT.-, Absence of letter for capsule group, denoting a noncapsulated strain.

e Somatic serotype 2 reference strain.

through 16 were extracted either by the phenol-chloroform-petroleum ether method (1) or the phenol-water method (19),purified as described previously (14), and lyophilized. Thesepurified LPSs are referred to as T-1 through T-16 in the textand figures. Control LPSs were from wild-type and Ra andRe mutants of S. minnesota (List Biochemical Co., Camp-bell, Calif.).Sample treatment. Lyophilized LPS was dissolved in

distilled water and heated at 56°C for 0.5 h. The LPS solutionwas mixed with an equal volume of 2x treatment buffer(0.125 M Tris, 4.0% SDS, 20% glycerol, and 10% 2-mercap-toethanol; pH 6.8) and heated at 100°C for 5 min.For proteinase K treatment, bacterial cells grown 18 h on

dextrose starch agar plates were suspended in 0.05 M Trisbuffer (pH 7.3). The suspension was adjusted to 20 times aMcFarland number 1 turbidity standard with the samebuffer, and 1.0 ml of the adjusted suspension was centrifugedat 31,000 x g for 15 min. The supernatant was removed byaspiration and discarded. One milliliter of lx treatmentbuffer was added to suspend the pellet, and the suspensionwas heated at 100°C for 10 min. After cooling to roomtemperature, 100 ,ul of proteinase K solution (3 mg ofproteinase K [EM Science Co., Cherry Hill, N.J.] dissolvedin 1.0 ml of 1 x treatment buffer) was added, and theresulting solution was incubated at 60°C for 1 h.

To determine the influence of storage, treated sampleswere electrophoresed within 20 min of preparation or frozenat -20°C until required for electrophoresis.SDS-PAGE. SDS-PAGE was done with slab gels by using

the buffer system described by Laemmli (5). Running gels(0.15 by 12 by 14 cm) were used, and acrylamide concentra-tions were 10% T (total monomer)-2.7% C (cross-linker),12.5% T-2.7% C, or 15% T-2.7% C. Running gels contained4 M deionized urea. Stacking gels (0.15 by 12 by 2 cm) andsample wells were cast separately as recommended byNeuhoff et al. (8) and were made without urea; acrylamideconcentrations were 4% T-2.7% C. Electrophoresis wasperformed with a constant current of 30 mA per slab at 15°Cfor 2.5. 3.5, or 4 h. Prestained electrophoresis standards(Diversified Biotech, Newton Center, Mass.) were used toestimate molecular weight. Gels were silver stained by themethod of Tsai and Frasch (18). Unless stated otherwise,sample wells were loaded with 10 ,ug of purified LPS or 20 ,ulof proteinase K digest.

Electroblots. LPS was electrophoretically transferred fromacrylamide gel slabs to nitrocellulose membrane sheets(BA85; Schleicher & Schuell, Inc., Keene, N.H.) by themethod of Towbin et al. (17). Unreacted sites on the mem-branes were blocked with a solution (PBSGT) that consistedof phosphate-buffered saline containing 1% fish gelatin(Hipure; Norland Products, Inc., New Brunswick, N.J.)and 0.05% Tween 80. Blocked membranes were incubatedfor 1 to 2 h with a 1:200 dilution of adsorbed or unad-sorbed chicken anti-LPS serum and washed three times withPBSGT. Membranes were incubated for 1 to 2 h with a1:1,000 dilution of peroxidase-conjugated goat anti-chickenimmunoglobulin G (Kirkegaard and Perry Laboratories,Inc., Gaithersburg, Md.) and washed twice with PBSGT andtwice with saline. Bound immunoglobulin G was visualizedwith a 4-chloro-1-naphthol solution (Kirkegaard and Perry).

Anti-LPS sera. Antisera were made against purified LPSfrom strain M-1404 (reference serotype 2) and strain P-1702(reference serotype 5) in groups of 30 chickens as describedpreviously (11, 14) and pooled according to strain.Immunoadsorbents. Cells from 18-h cultures of strains

M-1404 and P-1702 were lysed as described previously (12).For preparation of immunoadsorbents, the lysates weredialyzed against saline and polymerized with bovine serumalbumin by the gluteraldehyde method of Ternynck andAvrameas (16). Anti-LPS sera diluted 1:3 with PBS wereadsorbed exhaustively against the heterologous immunoad-sorbent and concentrated to the original volume by ultrafil-tration over a YM-30 membrane (Amicon Corp., Danvers,Mass.).

Gel diffusion precipitation tests. Adsorbed and unadsorbedanti-LPS sera were reacted with purified LPSs and heatedformalinized-saline extracts of reference strains M-1404 andP-1702 in the GDPT. The GDPT was done as described byHeddleston et al. (2).

RESULTS

Influence of gel casting and acrylamide concentration. Theusual SDS-PAGE method of casting a stacking gel in con-junction with sample wells resulted in poor definition andlateral streaking of LPS bands. Streaking was eliminated,and enhanced LPS band definition resulted when gel castingwas done by the method of Neuhoff et al. (8) and 4 M ureawas incorporated into the running gel. Examination of mo-bilities and resolutions of silver-stained band patterns ofcontrol LPSs and LPS of each of the different P. multocida

VOL. 28, 1990

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

J. CLIN. MICROBIOL.

_ -_ _~~~~m

_~~~~~gum111..; m . ICa

g. Mies

'_b~I c

.I

_ ~f

,~~~~~~~~~~~~~~~~~~~~~~~q,,5>..le f'b8'

=_"ss--s~t

FIG. 1. Silver-stained 15% T polyacrylamide gels comparingmigration and band patterns of LPSs purified from 16 referenceserotype strains of P. multocida. Gels were electrophoresed 2.5 h.Sal W, S. minnesota wild-type LPS; Sal Ra, S. minnesota Ra mutantstrain LPS; Sal Re, S. minnesota Re mutant strain LPS; T-1 throughT-16, purified P. multocida LPSs.

reference serotypes in running gels of 10% T, 12.5% T, and15% T acrylamide indicated that optimum results were

obtained with the 15% T acrylamide concentration. Thisconcentration was used exclusively in running gels for therest of the study.Comparisons of serotype 1 through 16 LPSs by SDS-PAGE.

Comparisons were made in silver-stained gels between LPSspurified from each of the 16 reference somatic serotypes ofP. multocida (T-1 to T-16) and control S. minnesota LPSs(Fig. 1). Control LPSs produced typical band patterns withcharacteristic mobilities. The LPSs from P. multocida couldbe divided into two groups on the basis of leading bandmobility. Group 1 (T-1 to T-6, T-8, T-9, T-12, T-13, and T-16)had leading bands with mobilities between those of theleading bands of S. minnesota wild-type and Ra LPSs.Group 2 (T-7, T-10, T-11, T-14, and T-15) had leading bandswith mobilities between those of the leading bands of Ra andRe LPSs.The P. multocida LPS band patterns consisted of a short

series of repeating units whose number varied with theparticular LPS. Both heavy and light silver-stained bandsoccurred within a repeating unit. Although the band patternsof P. multocida LPSs representing the different serotypeswere not identical, similarities between patterns occurred(Fig. 1, T-1, T-2, T-4, and T-5). Also, band patterns of P.multocida LPSs were unlike those of control S. minnesotaLPSs.

T K iCi P K'i P-2 C 9 P-4 8J 4

FIG. 2. Silver-stained band patterns of purified LPS from capsu-lated (T-1C) and noncapsulated (T-1) P. multocida X-73 (referenceserotype 1), LPS in proteinase K digests of capsulated (PK1C) andnoncapsulated (PK1) strain X-73, and LPS in proteinase K digests ofother serotype 1 strains (P-3269, P-4383, and P-4795). Samples wereelectrophoresed 3.5 h in a 15% T polyacrylamide gel. Arrowsindicate bands not seen in proteinase K digests.

Freezing of P. multocida LPSs after heating to 100°C intreatment buffer resulted in patterns with an increase in thenumber of repeating units in the upper portions of silver-stained gels. These patterns, a result of LPS aggregation,mimicked S. minnesota wild-type LPS. The mobilities ofleading bands in a preparation that had been frozen did notdiffer from the mobilities of leading bands in a freshly treatedLPS preparation. Reheating of a thawed preparation to100°C for 5 min prior to SDS-PAGE restored the LPSpatterns to the original number of repeating units, and thepatterns appeared like those of the freshly treated LPSsshown in Fig. 1.Comparison of purified LPS with LPS in proteinase K

digests. The LPS patterns of proteinase K digests of each ofthe 16 reference serotype strains of P. multocida were verysimilar to the patterns of their respective purified LPSs.However, with some strains, certain bands seen in a purifiedLPS preparation were less intense or absent in the respectiveproteinase K digest (Fig. 2, arrows). The positions of thesebands generally occurred in the fast-migrating, lower portionof the pattern. Increasing the sample load did not result inthe appearance of missing bands. All bands seen in aproteinase K digest had corresponding bands in the respec-tive purified LPS pattern.

Influence of capsulation on LPS patterns. Purified LPS andLPS in proteinase K digests of a capsulated and noncapsu-lated variant of reference serotype 1 (strain X-73) werecompared (Fig. 2). Except for the fact that LPS purified fromthe capsulated bacteria (T-1C) produced band patterns withmore repeating units than LPS purified from the noncapsu-lated bacteria (T-1), the mobilities and positions of purifiedLPS bands were similar. Unlike purified LPS, bands seen inproteinase K digests of capsulated (PKiC) and noncapsu-lated (PK1) strain X-73 occurred in the 4- to 15.5-kilodaltonrange and were nearly identical. All bands in the proteinaseK digests had corresponding bands in the purified LPSpatterns.LPS band patterns of proteinase K digests of capsulated

and noncapsulated strain X-73 (PKiC and PK1) and othercapsulated serotype 1 strains (P-3269, P-4383, and P-4795)

656 RIMLER

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

P. MULTOCIDA LIPOPOLYSACCHARIDES 657

m.w.kDamw kD.,

4ætii~~~~~;;t~_-r

FIG. 3. Silver-stained band patterns of LPSs in proteinase Kdigests of capsule group B (M-1404, P-5146, and P-4258) and capsulegroup E (P-1235, P-4120, P-4121, and P-4123) hemorrhagic septice-mia P. multocida strains from various hosts and geographic origins.Samples were electrophoresed for 3.5 h in a 15% T polyacrylamidegel. Arrows indicate bands not seen in LPSs of capsule group Estrains.

were similar (Fig. 2). Certain bands in PK1C and PK1 weremore intensely stained. However, increasing the sampleconcentration of the other serotype 1 strains did not signif-icantly improve the intensity or resolution of weakly stainedbands to a comparable degree. Except for differences in stainintensity, band patterns of proteinase K digests of strainswith a group A capsule which had serotype 1 LPS (strainsX-73, P-4383, and P-4795) were similar to that of a strain witha group B capsule that had serotype 1 LPS (strain P-3269).Comparisons between hemorrhagic septicemia strains from

different locations. Slight differences occurred among LPSsin proteinase K digests of hemorrhagic septicemia P. multo-cida isolated from various animal species and geographicorigins. For example, the LPSs of capsule group B strains(M-1404, P-5146, and P-4258) differed with regard to mobilityand the presence or absence of light-stained bands (Fig. 3).No differences were seen among LPSs of capsule group Estrains (P-1235, P-4120, P-4121, and P-4123). Except for thepresence of two light-stained bands of strain M-1404 thatwere detected in the 8.1- to 14.4-kilodalton range (Fig. 3,arrows), the pattern and mobilities of bands seen with strainM-1404 were like those of capsule group E strains.

Relationship between strains designated serotypes 2 and 5.Proteinase K digests of strains belonging to different capsulegroups which serotype as both 2 and 5 were compared (Fig.4). Light-stained bands in the 8.1- to 14.4-kilodalton range ofstrain M-1404 (reference serotype 2) and light-stained lead-ing bands of strain P-5178 were not seen or were poorlystained in other proteinase K digests (Fig. 4, arrows). Exceptfor the absence of these light-stained bands, patterns of LPSsin proteinase K digests of capsule group A (P-3203, P-3201,P-5178, and P-1702 [reference serotype 5]), capsule group B(M-1404), and capsule group E (P-1235) were similar.

Figure 5 compares silver-stained LPS patterns of anSDS-PAGE gel with electroblots of a companion gel that wasreacted with unabsorbed antiserum made against T-2 (puri-fied LPS of reference serotype 2). As observed previously,LPS in the proteinase K digest of strain M-1404 (referenceserotype 2) showed a mobility that was slightly differentfrom that of the LPS of the other capsule group B hemor-rhagic septicemia strains. Antibodies directed against T-2recognized antigens in T-5 (purified serotype 5 LPS) andLPSs in proteinase K digests of all the capsule group B andE strains. The antibody reactions with LPSs in proteinase K

.-r

-O(___ _

rc- 3 2 C. 3 R77O 4t-i4 ; P- 2 3

FIG. 4. Comparison of silver-stained band patterns of purifiedLPSs from reference serotype 2 (T-2) and serotype 5 (T-5), LPSs inproteinase K digests of capsule group A strains (P-3203, P-3201,P-5178, and P-1702), and LPSs in proteinase K digests of capsulegroup B and E hemorrhagic septicemia strains (M-1404 and P-1235).Samples were electrophoresed 3.5 h in a 15% T polyacrylamide gel.Arrows indicate bands not seen or poorly stained in other proteinaseK digests.

digests of the different capsule group B strains were similar,except for the slight variation in intensity with strains P-4656and P-4653. The strength of these antibody reactions did notseem to correlate with the concentration of silver-stainingmaterial in the bands. The reactions of antibodies with LPSsof capsule group E strains (P-4110 and P-1235) appeared tobe different from reactions with those of capsule group Bstrains. Only certain bands reacted, and these reactions didnot appear to be dependent on the concentration of silver-stained material in a given band.

After antiserum made against T-2 was treated with strain

A

E!_ E;U _- -

B_--"s

FIG. 5. (A) Silver-stained band patterns of purified LPSs ofreference serotype 5 (T-5) and serotype 2 (T-2) and LPSs inproteinase K digests of capsule group E (P-4110 and P-1235) andcapsule group B (P-4866, P-3705, P-4656, P-4653, P-5146, andM-1404) hemorrhagic septicemia P. multocida strains. Sampleswere electrophoresed 3.5 h in a 15% T polyacrylamide gel. (B)Immunoblot of companion gel developed with anti-serotype 2 LPS.

VOL. 28, 1990

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

J. CLIN. MICROBIOL.

P

~4 V P

b

~ F4 rI-14C P -2 35

FIG. 6. (a) Silver-stained band patterns of purified LPSs from

reference serotype 2 (T-2) and serotype 5 (T-5), LPSs in proteinaseK digests of capsule group B (M-1404 and P-5146) and capsule group

E (P-1235 and P-4110) hemorrhagic septicemia P. multocida strains,

and LPSs in proteinase K digests of capsule group A (P-3203,

P-5178, and P-1702) strains. Samples were electrophoresed 3.5 h in

a 15% T polyacrylamide gel. (b) Immunoblot of companion gel

developed with anti-serotype 5 LPS.

P-1702 (reference serotype 5) immunoadsorbent, no anti-

body reaction occurred with any of the above LPS prepara-

tions electroblotted onto membranes. Reprobing membranes

with the unadsorbed antiserum resulted in specific antibodyreactions (Fig. 5).

Figure 6 compares silver-stained LPS patterns of an

SDS-PAGE gel with electroblots of a companion gel that was

reacted with unadsorbed antiserum made against T-e, thepurified LPS of reference serotype 5 (strain P-1702). Anti-

bodies recognized antigens in T-2 (purified serotype 2 LPS)

and LPSs in proteinase K digests of capsule group A strains

(P-3203, P-5178, and P-1702), group B strains (M-1404 and

P-5146), and group E strains (P-1235 and P-4110). The

strength of these antibodyreactions did not seem to corre-

late with the concentration of silver-staining material in the

bands. No differences were seen among reactions with LPSs

of the capsule group E strains. Antibody reactions among

LPSs ofthe three capsule group A strains differed. The LPSs

of strains P-3203 and P-1702 reacted similarly. However, a

6.5-kilodalton band that reacted with these LPSs dod not

react with a similarly positioned band in the LPS of P-5178.

After antiserum made against reference serotype 5 LPS

was treated with strain M-1404 (reference serotype 2) immu-noadsorbent, no antibody reaction occurred with any of the

above LPS preparations electroblotted onto membranes.

Reprobing membranes with the unadsorbed antiserum re-

sulted in specific reactions (Fig. 6).

GDPT. No differences between the specîficities ofantiseraagainst M-1404 LPS and P-1702 LPS could be detected in the

GDPT. Lines of identity were produced from reactions of

M-1404 LPS antiserum with purified LPS and heated ex-tracts of strains M-1404 and P-1702 and from reactions ofP-1702 LPS antiserum with the same antigens (data notshown). Reciprocal adsorption of M-1404 or P-1702 LPSantiserum with immunoadsorbent of the heterologous straincompletely removed all antibodies; no reaction in the GDPTwas detected between adsorbed antiserum and LPS orheated extract of the homologous or heterologous strain.

DISCUSSION

LPSs of gram-negative bacteria are heterogeneous com-pounds, and their heterogeneity can be demonstrated bySDS-PAGE. Silver stains of purified P. multocida LPSssubjected to SDS-PAGE revealed patterns quite differentfrom those of wild-type and core mutant S. minnesota LPSs.The LPS patterns consisted of a short series of repeatingunits whose number varied with the particular LPS. Theladderlike pattern produced by wild-type S. minnesota LPSis characteristic of a high-molecular-weight LPS that con-tains long polysaccharide O antigens. This pattern wasmimicked by P. multocida LPS when samples were frozenbefore testing. The mimicry was a result of aggregation andhas been similarly observed with Brucella abortus LPS thathas been stored frozen at -20°C (M. Phillips, personalcommunication). Caution is therefore warranted before pre-suming that ladderlike patterns in a gel are indicative of anLPS with long polysaccharide O antigens.Hitchcock and Brown (4) demonstrated that the mobilities

of the fastest-migrating LPS bands of S. minnesota core-defective mutants (Ra to Re) were related to the size of theoligosaccharide core; shortening of the core oligosaccharideresulted in a concomitant increase in electrophoretic mobil-ity. The LPSs of P. multocida could be divided into twogroups on the basis of mobilities of the leading bands. Themobilities of leading bands of some group 1 LPSs weresimilar to that of S. minnesota Ra LPS (molecular weight,4,585). This similarity indicated that the molecular weightsof the basic structures of these P. multocida LPSs also maybe similar. The mobilities of the leading bands of LPSs fromgroup 2 were intermediate between those of S. minnesota RaLPS and S. minnesota Re LPS (molecular weight, 2,685).This finding indicated that the molecular weights of theirrespective basic structures may occur between 2,685 and4,585.The LPSs of different P. multocida somatic serotypes

produced different patterns in SDS-PAGE. Comparison of apurified LPS with LPS in a proteinase K digest of therespective parent cells showed patterns and mobilities thatwere markedly similar. This similarity indicated that protein-ase K treatment could offer a simple alternative to LPSpurification when comparisons of LPSs from a variety ofstrains were required. Because capsule polysaccharides areheterogeneous and produce ladderlike patterns in PAGE (9),it was important to determine whether the capsulation of astrain influenced the mobility and pattern of its LPS. Cap-sulation was not a significant influence because no majordifferences were observed between the band patterns andrelative mobilities of LPSs purified from a capsulated strainand its noncapsulated variant. Also, the presence or absenceof a capsule did not influence band patterns and relativemobilities of LPSs in proteinase K digests of P. multocida.Four slightly different LPS patterns were seen among the

hemorrhagic septicemia strains, and these patterns weresimilar to those of capsule group A strains P-3203, P-1702,and P-5178. None of the variations that occurred in any of

658 RIMLER

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

P. MULTOCIDA LIPOPOLYSACCHARIDES 659

the hemorrhagic septicemia strain patterns was related to itsorigin in a particular animal host. Except for slight differ-ences of stain intensity, the LPS patterns among group Estrains were identical. This finding makes it tempting tospeculate that LPSs from all group E hemorrhagic septice-mia strains will produce the same pattern because group Estrains are indigenous to Africa.The close antigenic relationship between strains which

react with serotype 2 and 5 antisera was reflected in thesimilarity of their LPS patterns in SDS-PAGE. Electroblotsof LPSs from all the hemorrhagic septicemia strains andserologically related group A strains reacted with antiseramade against both purified serotype 2 and 5 LPSs. Therelationship between the intensity of the silver stain reactionof bands in SDS-PAGE and the strength of the correspond-ing reaction with either antiserum in electroblots was tenu-ous. Some bands that were intensely silver stained did notreact or reacted weakly with antisera, while others that wereweakly silver stained reacted strongly with antisera. Therewas some disparity between the two antisera in the way thatthey recognized certain bands of an LPS pattern. However,this disparity between antiserum reactions is not sufficient todistinguish strains as being serotype 2 or 5.

P. multocida LPSs behave as haptens, and an immuno-logic carrier is necessary to induce a good serologic response

against them (11, 13). Immunization of chickens with immu-nologic carrier-associated LPSs has improved specificity ofserotyping of P. multocida by the GDPT (15). The antiseraused in this study were prepared by that method, which isalso the method now used by my laboratory and the NationalVeterinary Services Laboratory, Ames, Iowa, to prepare

reference antisera. Seemingly, this method of antiserumproduction has resulted in recognition of the major antigenicdeterminants of P. multocida LPS. No differences in theGDPT were seen between the specificities of antisera madeagainst serotype 2 and serotype 5 LPSs, indicating that themajor antigenic determinants of these LPSs are the same.

The fact that reciprocal adsorptions removed all antibodyreactivity for the GDPT and electroblots suggests that des-ignation of 2 and 5 as distinct serotypes is unwarranted.However, the findings that some bands in the electroblotsreacted poorly or not at all with antisera indicate thatunrecognized antigenic determinants may be present. Thesedeterminants may constitute minor differences between thedifferent LPSs. Possibly, these determinants could be rec-

ognized with antisera prepared by other means and therebyprovide a basis for subtyping of strains. Regardless ofwhether antiserum can be developed to distinguish minorantigenic determinants, it is probably appropriate to con-sider the abolition of either serotype 2 or 5.

LITERATURE CITED1. Galanos, C., O. Luderitz, and O. Westphal. 1969. A new method

for extraction of R-lipopolysaccharides. Eur. J. Biochem. 9:245-249.

2. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Fowlcholera: gel diffusion precipitin test for serotyping Pasteurella

multocida from avian species. Avian Dis. 16:925-936.3. Hitchcock, P. J. 1984. Analyses of gonococcal lipopolysaccha-

ride in whole-cell lysates by sodium dodecyl sulfate-polyacry-lamide gel electrophoresis: stable association of lipopolysaccha-ride with the major outer membrane protein (protein I) ofNeisseria gonorrhoeae. Infect. Immun. 46:202-212.

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

5. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:422-427.

6. 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.

7. Manning, P. J., M. A. Naasz, D. DeLong, and S. L. Leary. 1986.Pasteurellosis in laboratory rabbits: characterization of lipo-polysaccharides of Pasteurella multocida by polyacrylamide gelelectrophoresis, immunoblot techniques, and enzyme-linkedimmunosorbent assay. Infect. Immun. 53:460-463.

8. Neuhoff, V., K.-S. Choeng-Kim, and K. Altland. 1986. Improveddesign of one-dimensional vertical polyacrylamide slab gel elec-trophoresis avoids lateral streaking. Electrophoresis 7:56-57.

9. Pelkonen, S., J. Hayrinen, and J. Finne. 1988. Polyacrylamidegel electrophoresis of the capsular polysaccharides of Esche-richia coli Ki and other bacteria. J. Bacteriol. 170:2646-2653.

10. Peppler, M. S. 1984. Two physically and serologically distinctlipopolysaccharide profiles in strains of Bordetella pertussis andtheir phenotype variants. Infect. Immun. 43:224-232.

11. Rimler, R. B., R. D. Angus, and M. Phillips. 1989. Evaluation ofthe specificity of Pasteurella multocida somatic antigen-typingantisera prepared in chickens, using ribosome-lipopolysaccha-ride complexes as inocula. Am. J. Vet. Res. 50:29-31.

12. Rimler, R. B., and K. A. Brogden. 1986. Pasteurella multocidaisolated from rabbits and swine: serologic types and toxinproduction. Am. J. Vet. Res. 47:730-737.

13. Rimler, R. B., and M. Phillips. 1986. Fowl cholera: protectionagainst Pasteurella multocida by ribosome-lipolysaccharidevaccine. Avian Dis. 30:409-415.

14. Rimler, R. B., P. A. Rebers, and M. Phillips. 1984. Lipopoly-saccharides of the Heddleston serotypes of Pasteurella multo-cida. Am. J. Vet. Res. 45:759-763.

15. Rimler, R. B., and K. R. Rhoades. 1989. Pasteurella multocida,p. 37-73. In C. Adlam and J. M. Rutter (ed.), Pasteurella andpasteurellosis. Academic Press Inc., San Diego, Calif.

16. Ternynck, T., and S. Avrameas. 1976. Polymerization andimmobilization of proteins using ethylchloroformate and glutar-aldehyde, p. 29-35. In E. Ruoslahti (ed.), Immunadsorbents inprotein purification. University Park Press, Baltimore.

17. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

18. Tsai, C., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharide in polyacrylamide gels. Anal. Bio-chem. 119:115-119.

19. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides,p. 83-91. In R. L. Whistler and M. L. Wolfrom (ed.), Methodsin carbohydrate chemistry, vol. 5. Academic Press, Inc., NewYork.

VOL. 28, 1990

on August 23, 2020 by guest

http://jcm.asm

.org/D

ownloaded from