the antigens of pasteurella multocida

Immunology, 196I, 4, 122.

The Antigens of Pasteurella multocida Type I*

II. LIPOPOLYSACCHARIDES

R. V. S. BAIN

Department of Veterinary Pathology and Bacteriology, University of Sydney

AND

K. W. KNOXt

Department of Biochemistry, University of Sydney

(Received 23rd May, I 960)

Summary. Asian strains of Pasteurella multocida Type I were extracted with salineto remove superficial material and then with phenol-water to extract the lipopoly-saccharides. This product was recovered from the aqueous phase by centrifuging atI05,000 g for 2 hours. The component sugars are galactose, glucose and glucosa-mine and a heptose sugar, possibly D-glycero-L-mannoheptose. The preparationwas toxic for rabbits and was absorbed on human erythrocytes.An Australian strain of P. multocida, which by conventional mouse protection

and haemagglutination tests was identified as Type I yielded a lipopolysaccharidewith different properties. The disease of cattle, haemorrhagic septicaemia, which iscaused by P. multocida Type I in Asia is unknown in Australia.

INTRODUCTION

The studies of Davies and co-workers have shown that type-specific lipopolysaccharidesmay be isolated from members of the genus Pasteurella after removal of surface proteinmaterial with saline; these preparations have been obtained from P. pestis (Davies, I956),P. septica (P. multocida) (MacLennan and Rondle, I957) and P. pseudotuberculosis (Davies,1958; Crumpton, Davies and Hutchinson, I958). The lipopolysaccharides contain analdoheptose sugar and conform generally to the behaviour of this class of compounds inbeing pyrogenic, toxic and antigenic. In our fractionation of P. multocida, Type I (Roberts'classification) a similar substance has been isolated and studied.

MATERIALS AND METHODS

Strains, Cultivation, Serological Methods, Materials, Methods of Estimation of Nitrogen,Phosphorus and Carbohydrates and Chromotography, see Knox and Bain (i960).ULTRACENTRIFUGAL ANALYSIS. Examination of material in the Spinco Model E was

kindly performed by Mr. M. Smith, Division of Food Preservation and Transport,C.S.I.R.O. Because of the poor solubility of lipopolysaccharides in the presence of salt

* Aided by a grant from the Research Fund of the University of Sydney.t Present address: Institute of Dental Research, United Dental Hospital, Sydney, N.S.W., Australia.

I22

The Antigens of Pasteurella multocida Type I

(cf. Davies, I956) a portion of the solution which had not been freeze-dried was examinedat a concentration of 0.2 per cent (w/v) in water. Photographs were taken 4 minutesafter reaching maximum speed of 42,040 rev./min. (I28,430 g).

INFRA-RED SPECTRUM. An examination of products was kindly made by Dr. R. I.Cox, University of Sydney.

RESULTS

PHENOL EXTRACTION

The lipopolysaccharide has been extracted by the method of Westphal, Luderitz andBister (I952). The saline-extracted cells were suspended in water to give approximatelyio per cent (w/v) concentration warmed to 65-680 and an equal volume of go per cent(w/v) aqueous phenol at the same temperature added. The mixture was stirred for 30minutes at 65-680, cooled under running water, left at 20 overnight and then centrifugedat 20. The opalescent aqueous phase was removed and the lipopolysaccharide present wasseparated from contaminating nucleic acid by the two procedures described below. Thedenatured protein in the phenol phase has not been further investigated. Heptose wasfound to be a characteristic component of the lipopolysaccharide and the relative amountsof lipopolysaccharide in different fractions has been followed by determining this com-ponent (as D-glycero-D-galaheptose). Fructose, a characteristic component of the cap-sular polysaccharide (Knox and Bain, I960) was absent from lipopolysaccharide fractions.

In early experiments lipopolysaccharide was prepared by ethanol fractionation at o0,the material insoluble at I7 per cent concentration being removed by centrifugating atIO,OOO g for 30 minutes. In a typical experiment on 40 g. cells this fraction (I5/WS/I)weighed I90 mg. and contained I9.4 per cent heptose, I.7 per cent N and I.8 per cent P.The supernatant from the above precipitation yielded a further precipitate at 50 per centethanol concentration (i5/WS/2) containing 5.6 per cent heptose, I2.5 per cent N and3.9 per cent P. Examination of the ultra-violet absorption spectrum of this preparationrevealed the presence of nucleic acid, whereas I5/WS/I was essentially free from con-tamination by nucleic acid.

Preparations of lipopolysaccharide obtained in this way were poorly soluble and sub-sequently milder procedures of isolation were followed. The opalescent aqueous phase(from the phenol extraction) was dialysed for a week with frequent changes of distilledwater. During this period, the opalescence gradually disappeared, and a clear viscoussolution was obtained. The solution was centrifuged at IO5,000 g for 2 hours (SpincoModel L preparative ultra-centrifuge) and the supernatant removed (2I/WS/2); theresidue was washed twice by re-suspending and again centrifuging under the same condi-tions. Portions of the solution were freeze-dried (2I/WS/i) and the remaining solutionadjusted to I per cent (w/v) on the basis of the weight of the dried material. As evidencedby decreased viscosity and the opalescence of the solution freeze-drying altered thesolubility ofthe material. Chemical analysis was carried out on the dried material after re-constitution to give o.s per cent suspension, and biological tests on the remaining solution.The separation of nucleic acid from lipopolysaccharide was deemed to be satisfactory,

since nucleic acid could not be detected in the lipopolysaccharide preparation 2I/WS/I(examination of the ultra-violet absorption spectrum) and the nucleic-acid fraction21 /WS/2 contained only a trace of heptose. The yield of lipopolysaccharide was 630 mg.from 70 g. of cells.

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R. V. S. Bain and K. W. Knox

PROPERTIES OF THE LIPOPOLYSACCHARIDECompositionFrom the results of Westphal and Davies it was expected that the product obtained by

phenol extraction would be a lipopolysaccharide (Westphal et al., I952; Davies, I956).This conclusion was supported by the nitrogen and phosphorus contents, the presence ofaldoheptose, the poor solubility ofpreparations and also by the results ofmild acid hydroly-sis; heating at io00 for 4 hours in O.0I N HCI or I per cent (w/v) acetic acid yielded anacid-insoluble fraction which was soluble in chloroform and pyridine, but insoluble inether. A lipid material with similar properties has been obtained from various species ofEnterobacteriaceae by Westphal and Luderitz (I954) and from members of the genusPasteurella by Davies (I956, I958).

Hydrolysis of the preparation of 2I/WS/I in 0.5 N HCI for i6 hours at I00° released8 per cent hexosamine and 30 per cent reducing sugar (as glucose); as indicated above thisproduct also contained 1.7 per cent N, 3.0 per cent P and I I.4 per cent heptose. Thisheptose content was typical for preparations obtained by ultracentrifugation, thoughpreparations obtained by ethanol precipitation had a higher heptose content, e.g. I9.4per cent for I5/WS/I . The nitrogen content of these two preparations was the same (I.7per cent) and nucleic acid was not detectable. However, the preparation 2i/WS/i had ahigher P content (increase from I.8 to 3.0 per cent) and lower reducing sugar content(decrease from 35 to 30 per cent). The anomalous results could be due to a higher phos-pholipid content, for the preparations obtained by ultracentrifugation and examination ofthis product (2I/WS/ I) in the Spinco Model E analytical ultracentrifuge revealed a majorcomponent and a slower moving minor component.

ChromatographyPaper chromatograms of hydrolysates were run in butanol-pyridine-water and sprayed

for possible components. Three spots were obtained and these corresponded to the positionsof galactose, glucose and glucosamine. No additional spot corresponding to the heptosecomponent could be detected in this or other solvent systems.To detect the position of the heptose component, the hydrolysis products were applied

as a band at an appropriate position on a sheet of Whatman No. I paper with markerspots at each side. The chromatogram was run in butanol-pyridine-water and the sidestrips containing the markers sprayed. The portion of the chromatograms correspondingto the galactose and glucose components was cut into five strips I cm. wide. The stripswere eluted with water and examined for the presence of heptose by the H2S04-cysteinereaction (Dische, I953). The highest heptose value was found in the strip correspondingto the middle of the glucose spot. The shapes of the absorption curves between 380 m, and420 mi produced additional evidence for the conclusion that galactose and glucose werepresent (Dische, 1953).

Davies (I957) has examined the positions of the heptose in a number ofchromatographysolvents and related the rates of movement to that of more readily available carbohydrates.The position occupied by the unknown heptose in the lipopolysaccharide corresponds tothat of D-glycero-L-mannoheptose in butanol-pyridine-water. Further evidence insupport of this conclusion was obtained by running a chromatogram in propan-i-ol-benzyl alcohol-formic acid-water (50: 72: 20: 20). In this system, the heptose movedwith the same RF as galactose, the position to be expected of D-glycero-L-mannoheptose(or the optical enantiomorph).

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Heptose has been measured as D-glycero-D-galaheptose and II.4 per cent of thisproduct would correspond to I9.4 per cent of D-glycero-L-mannoheptose (Dr. M. J.Crumpton, private communication).

BIOLOGICAL PROPERTIES

HaemagglutinationHaemagglutination of group 0 human erythrocytes plated with crude Pasteurella

extracts and type-specific antisera has been described by Carter (I955). This has beenconfirmed in our laboratory. Various fractions, crude and comparatively purified, obtainedduring fractionation of Type I cells, were tested to determine which were absorbed on thered cells and subsequently agglutinated by sera against whole bacteria. Results are shownin Table I. Lipopolysaccharide or fractions containing this product (as judged bytoxicity and pyrogenicity for rabbits and a positive heptose test) were readily absorbedby human red cells without signs of direct haemagglutin4tion. Small additions of homo-logous type antisera produced strong haemagglutination but sera against other types did

TABLE 1

THE ABILITY OF FRACTIONS TO BE ABSORBED ON GROUP 0 HUMAN ERYTHROCYTES

Haemagglutination in antisera to whole bacteriaFraction onred cells Type I Type II Type III Type IV

Crudesaline extract + + + + + +Trypsinizedpolysaccharide _ - _Protein (heptose -) _ _ _Protein (heptose +) + + + + ±Lipopolysaccharide + + + +

not. The ketose-containing capsular polysaccharide fraction when heptose-free gaveentirely negative tests on all occasions.

ToxicityThis has been observed mainly in adult rabbits. Guinea pigs and mice are insusceptible

to doses below 500 ,ug. Crude iso-electric precipitates from saline extracts of whole cellswere found by Bain (I955) to be erratically toxic for rabbits. We now know that this is duesubstantially to the presence of lipopolysaccharide. However, even purified lipopoly-saccharide produces erratic results in rabbits as noted by Landy and Johnstone (I955)using lipopolysaccharide from Salmonella typhosa. Our preparations always killed rabbitsin a dose of soo ,ug. intravenously, but the effects of lower doses were inconsistent, con-sequently the determination of an L.D. 50 was virtually impossible with moderate numbersof animals. Irrespective of sex, weight, strain or age (above 3 months) some rabbitsreceiving 20-50 pg. intravenously died in 3-4 hours, a few of those receiving doses be-tween so ,ug. and 500 p'g. died in from 2-36 hours whilst others survived. When sufficienttime elapsed for the development of lesions, toxic changes were seen in the liver cells,kidney tubules and heart muscle. Haemorrhages were seen at times in the adrenals andinvariably in the spleen; indeed splenic haemorrhage came to be regarded as an indicator

B IMMUNOL.

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of lipopolysaccharide toxicity (in contradistinction to a possible protein toxicity which isbeing investigated at present). Lipopolysaccharide preparations from eleven batches ofcells were tested in fifty-four rabbits with results as outlined above. Affected rabbitssurviving for more than 12 hours commonly showed diarrhoea; a few recovered afterbeing in a state of collapse for I-2 hours.Two week-old calves collapsed 2 hours after receiving intravenously 300 ,ug. and 700 pg.

respectively of lipopolysaccharide contained in crude iso-electric precipitate, but theyrecovered subsequently. The blood of cattle dying from haemorrhagic septicaemia yieldedfrom 20 to I00 mg. dry weight of pasteurellas per litre. These, if similar to cells grown invitro, would contain about o. per cent of lipopolysaccharide. An adult infected ox there-fore might have a gramme or more of pasteurellas in its blood and over a milligramme oflipopolysaccharide. Three adult buffaloes receiving intravenously I.0, 1.5 and 2 mg.

respectively of purified lipopolysaccharide showed some malaise, diarrhoea and elevatedtemperatures in the subsequent 24 hours but at no stage appeared seriously ill.

In common with other bacterial endotoxins, pasteurella lipopolysaccharide wheninjected intravenously into rabbits elicited a sharp febrile response in from 2-4 hours.Temperatures of the rabbits described above rose to I05-Io6° F. and remained elevatedwith some fluctuation from 2-I2 hours. Six rabbits immune to challenge with live cultureand possessing abundant mouse-protective antibody were injected with 50 pg. lipopoly-saccharide. Five showed the usual febrile response but no other symptom. In mice whichreact to the intravenous injection of lipopolysaccharide the temperature drops to as lowas 920 F.The sample was submitted to acid hydrolysis (4 hoursI00° inO.OI N HCI). A waxy

material separated and was removed. The residual 'polysaccharide' in a doseof 400pg.,

the usual certain killing dose, failed to kill two rabbits but caused their temperatures torise toI06° F.

AntigernicityThe lipopolysaccharide was antigenic and produced in rabbits precipitins which could

be identified on Ouchterlony plates. In common with anti-endotoxin sera generally our

sera did not protect rabbits against lethal dosesof500 pg. of lipopolysaccharide.In repeated tests purified lipopolysaccharide from twelve different batches, in doses of

from 20-200,ig., protected on the average about 20 per cent of mice against challenge withI00

L.D.50 bacteria intraperitoneally 3 weeks after injection. Experiments with multiplesof the standard dose (40pg. intraperitoneally) did not produce a graded response as was

expected but merely erratic survivals not exceeding20 per cent in any group of twenty.All of the above preparations removed some but not all of the mouse protective property

of rabbit and cattle sera. Mice in groups of six receiving5 M.P.D. of absorbed serum alldied on challenge and those receiving20 M.P.D. all survived.

In Table 2 an experiment is presented to show whether Pasteurella lipopolysaccharidehas the capacity to protect mice non-specifically against challenge a short time afterinoculation. The mice, 24 hours after challenge, showed enhanced resistance as comparedwith the controls but this vanished in the ensuing I 2 hours. This effect held for72 hoursafter inoculation and was demonstrated with four different preparations from the 'Insein'strain. The challenge was ioo L.D.50 intramuscularly and the lipopolysaccharide was

administered intraperitoneally.

I26


LIPOPOLYSACCHARIDES IN DIFFERENT STRAINS OF Pasteurella multocida TYPE I

As discussed elsewhere (Knox and Bain, I960) a colonial ('blue') variant of the Type Iorganism differed in some respects from the parent strain. However, no difference eitherchemical or biological could be detected between the corresponding lipopolysaccharides.

Lipopolysaccharide preparations from a number of Type I strains were examined bythe H2SO4-cysteine reaction (Dische, I953). Under suitable conditions the shape of the

TABLE 2

PROTECTION AFFORDED TO MICE* BY 40 Pg. OF LIPOPOLYSACCHARIDEAT VARIOUS TIMES OF CHALLENGE (IOO L.D. 50)

Time between Survivors at 24 hours Survivors at 36 hoursinoculation

and challenge Controls Experimental Controls Experimental

12 hours 0 4 0 024 hours 0 7 0 036 hourS I 4 O 072 hours 0 I I 0 014 days 0 IO 0 3

* Twelve mice in each group.

absorption spectrum provides information on the amounts of the non-hexosamine com-ponents present. As shown in Fig. I the spectrum for the classical 'Insein' strain showedmaxima at 390 mll. (glucose) 410 m". (galactose) 5I0 m". (aldoheptose) and a minorpeak at 6oo m". associated with the presence of hexose.

FIG. I. Examination of lipopolysaccharides from 'Insein' strain(0-0) and Strain 989 (*-) by the sulphuric acid-cysteine re-action. The optical density was read 22 hours after adding cysteine.

Only one of the strains examined, 989, yielded a lipopolysaccharide with an absorptionspectrum which differed significantly from that of the 'Insein' strain (see Fig. I). Whencompared with the 'Insein' lipopolysaccharides this product contains less heptose (4 percent as D-glycero-D-galaheptose) and apparently more glucose (from the greater extinc-tion at 4IO mP.).The lipopolysaccharide from Strain 989 contained the same carbohydrate constituents

as that from the 'Insein' strain with, in addition, a component of very high mobility (inbutanol-pyridine-water). The Rglucose was 2.10 which corresponds closely with that

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found for dideoxyhexoses (Davies, I955). It was not possible to identify this componentfurther.

Strain 989 was of particular interest as by mouse protection and haemagglutinationtyping it was Type I, which causes haemorrhagic septicaemia of cattle in Asia. The strainwas isolated in Australia where that disease has never been seen clinically. In routine testsfor identity, it differed from the classical Asian Type I strains by failing to ferment xyloseand in being lethal to chickens. The polysaccharide and protein fractions of Strain 989could not be differentiated chemically or serologically from those of the 'Insein' strain orother Type I Asian strain. The lipopolysaccharide, however, showed the followingdifferences in addition to those given in Fig. i. It produced direct haemagglutination ofhuman 0 erythrocytes. It was toxic for mice by intravenous injection, i mg. killing 8o percent of the mice inoculated and smaller doses proportionately fewer; not one of 200 micereceiving up to a mg. of lipopolysaccharide from Asian Type I strain showed more thanhypothermia and brief malaise followed by recovery. On Ouchterlony plates the 989fraction reacted clearly with 989 serum but not with Asian Type I serum. It failed com-pletely to protect mice against homologous challenge in the standard dose of 40 pg.intraperitoneally, and still failed when the dose was based on heptose content and increasedthreefold to make it comparable to the 'Insein' lipopolysaccharide. The 989 and 'Insein'lipopolysaccharides produced indistinguishable effects on rabbits and chickens and infra-red spectrograms of the two substances showed only minor differences of doubtful signi-ficance. A second strain, I036 of the same type as 989, was isolated recently in Australiafrom cattle and its lipopolysaccharide proved to be identical with 989.

DISCUSSION

Carter's technique for typing strains of P. multocida by a haemagglutination test usescrude extracts of the bacteria obtained by warming them at 560. These extracts do notproduce direct haemagglutination even when obtained from Strain 989. As shown inTable i, of the 'purified' fractions only the lipopolysaccharide is able to link with the sur-face of the erythrocytes under simple direct conditions. Where crude extracts are used, thelipopolysaccharide, bound to other antigens, acts as 'cement' linking the antigens to theerythrocytes and thus hiding its own specificity. The importance of this is that it is epide-miologically meaningful. Bovine strains which could be called 'Type I' are rare inAustralia. The only two which have come to hand (989 and I036) did not cause haemor-rhagic septicaemia in the cattle from which they were isolated. Each had similar lipopoly-saccharide antigen which differed from that of Asian Type I. This shows that the currenttechniques for typing, i.e. mouse-protection tests and haemagglutination tests do not gofar enough.MacLennan and Rondle (I957) found type-specific lipopolysaccharides in Types I,

IV and V. Hitherto the type-specific antigens have been assumed, without any very goodevidence, to be polysaccharides. The numerous cross reactions experienced in all attemptsat typing Pasteurella by diverse methods have always underlined the antigenic complexityof the group. The mouse-protection test and the haemagglutination test agree generallyand subdivide the species into serotypes which have some significance with regard to hostrange and the types of disease produced. Closer examination of purified lipopolysaccha-rides might give more precise information on the true status of the serotypes.

Since the only demonstrable chemical difference between Strains 989 and 'Insein' was

128

The Antigens of Pasteurella multocida Type I I29

the nature of the lipopolysaccharide, it was tempting to speculate that this might accountfor the pathogenicity for chickens of 989 and the non-pathogenicity of 'Insein' and otherAsian Type I strains for this host. However, limited inoculation experiments using four-week-old chicks failed to show any difference in toxicity in the lipopolysaccharide fractions,both groups of twelve showing malaise and depression but no deaths after receiving I mg.of lipopolysaccharide intravenously.The lipopolysaccharide preparations isolated from P. multocida Type I are similar in

composition and properties to those which have been isolated from other members of thespecies (MacLennan and Rondle, I957), from P. pestis (Davies, I956) and from P. pseudo-tuberculosis (Davies, 1958; Crumpton, Davies and Hutchinson, I 958) . In particular, chroma-tographic evidence suggests that in each case D-glycero-L-mannoheptose (or its opticalenantiomorph) is present (MacLennan and Rondle, 1957; Forster, Davies and Crumpton,I958; Davies, I958). Additional carbohydrate components always present are one ormore hexoses and hexosamines; in the preparations we have examined, galactose, glucose,glucosamine were present. The only additional components found by MacLennan andRondle (I957) in strains of Types I, IV and V were galactose and glucosamine, thesame as reported for P. pestis by Davies (I957). However, the studies of Davies (1958) oncarbohydrate components of various strains of P. pseudotuberculosis suggest that widevariations in composition can occur.The possible presence of a dideoxyhexose in the strain of Type I isolated in Queensland

reveals a similarity to preparations from some of the strains of P. pseudotuberculosis in whichsugars of this group were sometimes found.

ACKNOWLEDGMENTSWe acknowledge with thanks the valuable information made available by Dr. D. A. L. Davies

and Dr. M. J. Crumpton, Microbiological Research Establishment, Porton, England, and theample material produced by Mr. R. F. Jones of the Department of Veterinary Bacteriology,University of Sydney, by the techniques of mass-cultivation.

REFERENCES

CARTER, G. R. (I955) Studies on Pasteurella multocida.I. A haemagglutination test for the identification ofserological types.' Amer. J. vet. Res., I6, 481-4.

CRUMPTON, M. J., DAVIES, D. A. L. and HUTCHINSON,A. M. (1958). 'The serological specificities ofPasteurella pseudotuberculosis somatic antigens.' J. gen.Microbiol., I8, I29-39.

DAVIES, D. A. L. (I955). 'The specific polysaccharideof some gram-negative bacteria.' Biochem. j., 59,696-704-

DAVIES, D. A. L. (1956). 'A specific polysaccharide ofPasteurella pestis.' Biochem. J., 63, 105- I 6.

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DISCHE, Z. (I953). 'Qualitative and quantitativecolorimetric determination of heptoses.' J. biol.Chem., 204, 983-97.

FORESTER, A. B., DAVIES, D. A. L. and CRUMPTON,M. J. (I958). 'Periodate on some polysaccharidescontaining aldoheptose sugars.' Nature, Lond., I8I,412-13.

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LANDY, M. and JOHNSTONE, A. G. (I 955). 'Studies onthe 0 antigen of Salmonella typhosa: IV. Endotoxicproperties of the purified antigen'. Proc. Soc. exp.Biol., 90, 57-62.

McKENZIE, H. A. and WALLACE, HEATHER S. (I954).'The Kjeldahl determination of nitrogen: a criticalstudy of digestion conditions -temperature, cat-alyst and oxidizing agent.' Aust. J. Chem., 7, 55-70.

MAcLENNAN, A. P. and RONDLE, C. J. M. (I957).'Antigens of Pasteurella septica.' j. gen. Microbiol.,67, Vii.

ROBERTS, R. S. (I947). 'An immunological study ofPasteurella septica.' j. comp. Path., 57, 261-78.

WESTPHAL, D. and LUDERITZ, D. (I954). 'ChemischeErforschung von Lipopolysacchariden Gram-negativBakterien.' Angew. Chem., 66, 407-I7.

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the antigens of pasteurella multocida

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