INFECTION AND IMMUNITy, Oct. 1976, p. 990-999 Copyright X) 1976 American Society for Microbiology

Vol. 14, No. 4 Printed in U.S.A.

Immunogenic and Toxic Properties of a Purified Lipopolysaccharide-Protein Complex from Pasteurella multocida DAVID J. GANFIELD,I* PAUL A. REBERS, AND K. L. HEDDLESTON National Animal Disease Center, North Central Region, Agricultural Research Service, Ames, Iowa 50010 Received for publication 23 March 1976

An immunogenic fraction from Pasteurella multocida was found to consist chiefly of a high-molecular-weight protein-polysaccharide complex containing 25 to 27% protein and 10.7% carbohydrate. The starting material was obtained by differential centrifugation at 105,000 x g of a saline extract ofP. multocida cells and further purified by gel filtration on Sepharose 2B. Three peaks were usually obtained after gel filtration. The component in the first peak amounted to about 10% of the starting material and eluted in the void volume. It was predominately carbohydrate, although some protein was present. Two inoculations of 10 to 20 ,ug of the first component induced up to 80% protection in mice against a challenge inoculation with P. multocida that killed 100% of the controls. The second, or major, component amounted to about 75 to 95% of the starting material. This fraction contained 25 to 27% protein and 10.7% carbohydrate. Small amounts, 10 to 20 ,ug, induced active immunity in mice and turkeys, but large amounts could be lethal; the mean lethal dose was 195 ,ug for mice and 5.7 ,ug for 10-day-old chicken embryos. The components in the third peak were primarily proteins that gave reactions of nonidentity with the antigens of peak II in gel diffusion. The components present in the third fraction were definitely less effective in the induction of protective immunity than those present in the first or second. Analyses of the protective antigen(s) by the isoelectric focusing procedure in a pH 3 to 10 gradient showed that all of the precipitinogenic activity was found in the range of pH 3 to 4, with a peak at pH 3.7.

At least 16 serotypes of Pasteurella multocida have been isolated from cases of pasteurellosis (reference 2 mentioils 15 serotypes, but the current number is 16). Each serotype is capable of inducing antiinfection immunity against the homologous serotype, but instances of crossprotection are rare. Cold saline extracts of the cells contain many nonprotective antigens that cross-react in gel diffusion. Heating of the extracts destroys most of the cross-reactivity, whereas the type-specific reactivity is retained, and serves as the basis of the serotyping system (4, 8). Relatively little is known about the chemical nature and biological activities of the type-specific immunizing antigens. However, previous work has shown that 5- to 20-p,g quantities of type-specific antigens that were isolated by ultracentrifugation from saline extracts of P. multocida cells induced active immunity in chickens, turkeys, and mice; larger amounts (200 to 500 ,Ag) were usually toxic or lethal. Their stability to heat, formalin, and preliminary chemical analysis suggested that I Present address: Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA 19107.

lipopolysaccharide-protein complexes were present (9). The differential centrifugation procedure resulted in serologically specific antigens, but electron micrographs and gel precipitin analyses showed that the preparations lacked the required homogeneity for meaningful chemical analyses (9). The purpose of the present investigation was to use additional methods, such as isoelectric focusing and gel filtration, for the purification of the particulate antigens, so that the immunogenicity, toxicity, and serological specificity of each fraction could be compared. This report describes the purification and characterization of a serologically and immunologically specific antigen isolated from serotype 3 P. multocida. This strain, P-1059, is the most common serotype isolated from epiornithics of fowl cholera in the United States (4). Although the isolate from diseased fowl occurs as a virulent, encapsulated, iridescent-colony organism, the antigenic complex described here was prepared from a nonencapsulated, gray-colony, avirulent variant. Despite the loss in virulence, bacterins prepared from the gray mutant are 990

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P. MULTOCIDA LIPOPOLYSACCHARIDE-PROTEIN

capable of inducing protective immunity (9). Because the original P-1059 organisms were isolated from a turkey infection and the turkey is very susceptible to this strain, turkey poults and mice were used for active immunity studies. (Portions of this work have been submitted [D.J.G.] in partial fulfillment of the requirements for the Ph.D. degree, Department of Biochemistry, Iowa State University, Ames, Iowa.) MATERIALS AND METHODS Cultures. P. multocida P-1059 (ATCC 15742) was used throughout these studies. This strain occurs as a virulent encapsulated form, which was used to determine active immunity, designated P-1059F, and also as a nonencapsulated, avirulent form, designated P-1059G, which was used for preparation of the immunogenic complexes (9). Extraction and isolation of the lipopolysaccharide complexes. The procedures used for extraction and isolation by ultracentrifugation are similar to those described previously (9). Briefly, the cells were grown on Difco starch agar containing 0.5% additional agar in Roux bottles at 37°C for 24 h; for harvest, they were washed off the agar with 0.85% NaCl containing 0.3% formalin. A 500-ml suspension of cells from 40 Roux bottles was stirred for 24 to 48 h at 4°C. The suspension was centrifuged for 30 min at 12,000 x g to remove the cells. The suspension was filtered through washed filters, pore size 1.0 ,tm and 0.45 jum (Millipore Corp., Bedford, Mass.) in succession, and the filtrate was centrifuged at 105,000 x g for 2 h in a Spinco no. 40 rotor (Beckman Instruments, Inc., Palo Alto, Calif.). The resulting pellet was gently suspended in 0.85% NaCl containing 0.1% formalin and repelleted three times at 105,000 x g. A slight modification of the previously described ultracentrifugal procedure was used in which the bottom 1 ml of the centrifuged extracts was included with the pellet obtained by ultracentrifugation for 2 h at 105,000 x g. Assay methods. The methods used for measurement of Kjeldahl nitrogen, total phosphorus, and carbohydrate were the same as those described previously; carbohydrate was determined by the phenol-sulfuric acid procedure, with glucose as the standard (9). The total protein content was estimated by the ultraviolet (UV) absorption method of Groves et al. (6) and also by a modified Folin-Lowry method described by Bailey (1), with recrystallized bovine serum albumin as a standard. Ouchterlony plates were prepared with 0.85% NaCl for use with rabbit antisera, and with 8.5% NaCl for use with chicken antisera as previously described (9). Quantitative precipitin reactions were carried out with the fractions from the Sepharose 2B column with rabbit antiserum at 0°C according to the procedure of Heidelberger and Rebers (11; see also reference 13), except that the total protein in the washed precipitates was determined by a modified Lowry procedure with bovine serum albumin as a standard (1). A semiquantitative determination of

991

some of the fractions was also made by a gel diffusion procedure. Since a relationship between the position of the precipitin line and the antigen concentration exists, i.e., at higher antigen concentrations the precipitin line fotms farther from the antigen well, the distances from the antigen wells to the center of the precipitin line were measured accurately. A microcomparator (Gaertner Corp., Chicago, Ill.) was used to magnify the gel diffusion pattern and accurately measure the distances. For a series of fractions, a series of wells was cut parallel to each other. In one row of wells, a constant volume of the antigen fraction was added; to the other, a constant volume of antiserum was added. The height of the bars in the figures is directly proportional to the distances, and is referred to as gel precipitin activity. Immunoelectrophoresis was carried out in 1% agarose in 0.02 M tris(hydroxymethyl)aminomethane-0.02 M citrate buffer at pH 8.6 (8). Source of animals and methods of inoculation. New Zealand rabbits were used for preparation of hyperimmune antisera. Initial inoculations were generally given intradermally in five sites on the shaved back with an adjuvant of Bayol F and Arlacel A as described previously (9). Subsequent inoculations of aqueous solutions were given intravenously into the marginal ear vein at monthly intervals. Beltsville Small White turkey poults obtained from a closed National Animal Disease Center flock free of P. multocida were used for immunity studies. Poults were inoculated intramuscularly as described in Results. Randomized mice (16- to 18-g Swiss Webster females) were inoculated intraperitoneally for use in immunity and toxicity experiments. Nineto ten-day-old New Hampshire chicken embryos used in toxicity studies were inoculated on the chorioallantoic membrane as described by Horsfall and Tamm (12). Challenge of immunity. The immunity of immunized and control turkey poults was routinely challenged by intramuscular inoculation in the thigh with 900 to 1,000 colony-forming units of P. multocida P-1059F. The immunity of mice was challenged with the same number of organisms by the intraperitoneal route. Agarose gel fi'ltration. Agarose gel filtration experiments were performed with Sepharose 2B or 4B (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) in columns 25 mm in diameter and 450 mm long. Two- or three-milliliter samples were applied to the column. A flow rate of 10 to 11 ml/h was maintained by keeping a constant pressure head of approximately 9 cm. Five-milliliter fractions were collected by drop counting. Generally, 250 ml was collected over a 22- to 23-h period; 0.15 M NaCl containing 3% ethyl alcohol was used as the eluant. Ethanol functioned as a preservative that did not interfere with UV measurements or carbohydrate assays (personal commication, B. Bjorklund, State Bacteriological Control Institute, Stockholm, Sweden.) Both ascending and descending gel filtration experiments were performed. The fractions were each examined for UV absorption in a spectrophotometer (Zeiss PMQ II) at four

992

GANFIELD, REBERS, AND HEDDLESTON

wavelengths: 280, 260, 233, and 224 nm. Nitrogen, protein, and carbohydrate contents of each of the fractions were determined as mentioned above. Isoelectric focusing experiments. Electrofocusing equipment (model 8100, LKB-Produckter AB, Stockholm, Sweden) was used to determine the isoelectric point of the P. multocida preparations as described by the LKB Instruction Manual. Both pH 3 to 5 and pH 3 to 10 Ampholine solutions were used. The time of electrofocusing was 20 h with the pH 3 to 10 gradient (300 V) and 72 h with the pH 3 to 5 gradient (450 V). In all experiments, the anode was at the bottom of the column and the cathode was at the top. Sucrose gradients were made manually, and the sample was added to the middle of the gradient. The column was maintained at 2°C, and 3-ml fractions were collected at completion. RESULTS

Isolation of the immunizing complex by ultracentrifugation. Agar-grown cells were extracted and isolated by ultracentrifugation. The washed pellets designated as 40-P preparations were resuspended and stored in formalinized saline at 4°C because they could not be redispersed if they were lyophilized. Immunity studies in turkey poults. The ability of the 40-P preparations to immunize turkey poults and the inoculation dose required were studied. Results are shown in Table 1. In experiment 1, various doses of a 40-P preparation were inoculated. A single intramuscular inoculation of 10 4g protected 70% of the poults against a lethal infection when a standard challenge inoculation of P. multocida was used. A dose-response effect was observed. As shown in experiment 2 of Table 1, the same immunogenic preparation used in experiment 1 was tested for its stability to proteolytic digestion with Pronase (CalBiochem., La Jolla, Calif.), formic acid, alkali, and heat (1210C). Results are given in Table 1. Incubation of the immunogenic preparation with Pronase did not noticeably affect the protection, but both the alkali and heat treatments markedly reduced the protection. The acid treatment slightly reduced the protection. A small precipitate was observed when the preparations were acidified, and this precipitate was included with the neutralized supernatant and injected in a suspended form. The resistance of the protective activity of the 40-P preparation to Pronase digestion agreed with the results of chemical analysis by paper chromatography, which indicated no release of ninhydrin-positive components after Pronase digestion (6 h, 40°C). Amino acids were liberated from the sample by acid hydrolysis (5 N HCl, 24 h, 1000C). Incubation of a mixture of

INFECT. IMMUN.

casein, 40-P preparation, and Pronase resulted in the liberation of free amino acids. Hence, Pronase retained proteolytic activity in the presence of 40-P. On the basis of the above experiments, it was concluded that the immunogenicity is reduced after 1 h at 121°C but is resistant to the broadspectrum proteolytic enzyme Pronase. The immunogenicity of the preparation was more stable in 0.1 N formic acid than in 0.1 N sodium hydroxide. The loss of activity at high pH may indicate either denaturation of protein or possible cleavage of ester linkages. Lethal toxicity of the immunogenic 40-P preparation for mice and chicken embryos. Because experiments reported previously had indicated a toxic effect when similar preparations were injected into mice and chickens, we designed more extensive experiments to quantitate the toxicity of the original 40-P preparations. The toxicity was determined both in mice and in 10-day-old chicken embryos. The results of these experiments are shown in Tables 2 and 3. The mean lethal dose (LDso) value for the mice was 195 ug; for the chicken embryos, the LD4, value was 8.7 ,4g. Gross chemical composition of the 40-P preparations. A large number of 40-P preparations was isolated as described. The analyses of a number of these preparations for total nitrogen (Kjeldahl), carbohydrate, and total phosphorus are summarized in Table 4. Most of these preparations had significant differences in gross chemical composition. Since the differences in the 40-P preparations could arise from either nitrogenous or carbohydrate impurities, or both, the preparations were fractionated by gel filtration. Purification by gel filtration. Attempts were made to fractionate the 40-P preparations by Sephadex G-200 gel filtration. However, no separation was obtained, since essentially all of the material was excluded from Sephadex G200, as determined by the UV absorption at 280 nm and by Ouchterlony gel diffusion. Therefore, agarose columns were used subsequently. Initial experiments with Sepharose 4B were partially successful in separating the major precipitinogens from a less-active fraction obtained in the void volume. However, both the UV absorption and carbohydrate analysis of the fractions indicated poor resolution of the components that were eluted near the void volume. Therefore, the more porous gel Sepharose 2B was used in subsequent experiments. A total of 16 gel filtration experiments was performed with 40-P preparations obtained from 11 lots of microorganisms. All the preparations

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993

P. MULTOCIDA LIPOPOLYSACCHARIDE-PROTEIN

TABLE 1. Active immunization of turkey poults with various doses ofPasteurella multocida P-1059G 40-P preparation and with the 40-P preparation subjected to various stability tests 40-P immunogenr

Expt 1 Untreated 40-P Untreated 40-P Untreated 40-P Controls

Cumulative no. dead by day: 1

2

5

7

14

No. survivors/ no. challenged'

0 3 8 10

1 4 8 10

2 7 9 11

3 7 10 11

3 7 10 11

7/10 3/10 0/10 0/11

Dose (Mg)

10 1.0 0.1 0

Potection 70 30 0 0

M

Expt 2 3 7/10 70 1 1 3 3 Untreated 40-P 10 3 7/10 70 0 1 2 3 Pronase 40-P 10 5 5/10 50 1 4 5 5 Acid 40-Pd 10 9 1/10 10 5 9 9 9 Alkali 40-PI 10 9 1/10 10 6 8 9 9 Heated 40-Pf 10 11 0/11 0 4 10 11 11 Controls 0 a The 40-P preparation (lot 80) contained 4.26% nitrogen, 19.4% carbohydrate, and 2.0% phosphorus. b Two-week-old turkey poults were inoculated intramuscularly with 0.1-ml saline solutions of the untreated or treated 40-P. They were challenge-inoculated 2 weeks later with 900 colony-forming units of P. multocida P-1059F and held for 2 weeks after challenge. A sample of 40-P 134 Mg, in 1.34 ml of phosphate buffer, pH 7.4, ionic strength 0.05, was incubated after addition of 200 Ag of Pronase for 6 h at 40°C. d A sample of 40-P, 134 ug, in 1.34 ml of 0.1 N formic acid, was incubated 4 h at 40°C and neutralized before "

injection.

e A sample of 40-P, 134 ,ug, in 1.34 ml of 0.1 N NaOH, was incubated 4 h at 400C and neutralized before

injection.

' A sample of 40-P, 134 ug, in 1.34 ml of sterile saline, was autoclaved for 1 h at 121°C.

TABLE 2. Lethal toxicity ofPasteurella multocida P1059G 40-P preparation in mice Amt inoculated" (Lg)

No. dead at 48 h/ no. inoculated

Deaths (%

500W (lot 249)

94 15/16 250 56 9/16 38 162 6/16 12 83 2/16 6 41 1/16 I Each of the 16-18g mice was inoculated intraperitoneally with the toxin in 0.15 M NaCl solution containing 0.3% formalin and observed for 10 days. All, deaths occurred in 2 days. b Values for the LD5, and lower and upper 95% confidence limits (195 Mg [148 to 265 Mg]) were determined by the probit method of analysis.

had a common major component that had a peak elution volume of between 120 and 130 ml. The void volume (55 ml) was determined with blue dextran and, the internal volume (200 ml) was determined with formaldehyde. Each 5-ml fraction was examined for its absorption at 224, 233, 260, and 280 nm. Readings were made at 224 and 233 nm so that protein could be estimated by the procedure of Groves, at 260 nm for estimation of nucleic acids, and at 280 nm, the usual wavelength for estimation of proteins. A typical elution profile is shown

TABLE 3. Toxicity of P. multocida P-1059G 40-P preparation in chicken embryos before and after fractionation on Sepharose 2B Sample

Original 40-P (lot 116)P

Peak I (lot 116) Peak II (lot 116)d

Amt inocu-

No. dead at 48 hino. inoculated

Deaths (

22 11 5.5 2.8 or 38.0

8/10 7/11

4/12

80 63 42 0 0 33

22.2 11.1 8.5 5.5 2.8

10/12 9/12 8/12 7/12 3/12 0/10

83 75 67 58 30 0

lateo lated((cg) pg)

Ob

5/12 0/10 0/10

a One-tenth milliliter samples were inoculated onto the chorioallantoic membrane of 10-day-old embryos, and eggs were candled daily for 5 days after inoculation. All deaths occurred in 2 days. b LDN for original 40-P is 8.7 MAg (5.8 to 14.0 ug [upper and lower confidence limits, by the probit method]). c Formalinized saline: 0.85% NaCl containing 0.1% formalin; the diluent alone was used as a control. d LD50 for peak II is 5.2 ,Mg (2.0 to 8.0 MAg [upper and lower confidence limits, by the probit method]).

994

INFECT. IMMUN.

GANFIELD, REBERS, AND HEDDLESTON

in Fig. 1, the readings at 224 nm were plotted as the most sensitive indication of protein; those at 260 nm were plotted to indicate nucleic acids. The ratio of the absorbancy (A) at 260 nm to A224 nm suggested that the components of peak II contained more protein than those of peak I, and that the components of peak I might contain nucleic acids. The components of peak I failed to give any reaction in the Ouchterlony gel diffusion test, but strong reactions were obtained with those of peak II, and moderate reactions were obtained with those of peak III. Reactions of nonidentity were observed between the components of peaks II and III. The peak II component gave a single line at TABLE 4. Gross chemical composition of P. multocida P-1059G 40-P preparations % Nitro- % Carbo- % Phosphorug' gena (wt/ hydrate° Prepn (wtlwt) wt) (wt/wt) 2.1 6.2 15.5 No. 1 (lot 30) 5.7 13.5 No. 2 (lot 59) 2.0 19.4 4.26 No. 3 (lot 80) 13.0 5.0 No. 4 (lot 88) 15.9 No. 5 (lot 109) 2.75 17.8 4.36 No. 6 (lot 116) 2.41 16.0 5.1 No. 7 (lot 196) 2.39 14.1 5.6 No. 8 (lot 197) 2.56 13.0 8.4 No. 9 (lot 198) 3.67 22.9 6.35 No. 10 (lot 199) 2.84 17.5 7.1 No. 11 (lot 200) a Total nitrogen values were determiined by micro-Kjeldahl procedure. b Total carbohydrate expressed as gluc ose equivalents in the phenol-sulfuric acid proceduxre. ' Total phosphorus. PEAK

I

PEAK

11

PEAK it

I l

0.5 to 1.0 mg/ml, but gave two lines of equal intensity after concentration to 6 mg/ml. Perhaps concentration induced some aggregation. The Sepharose 2B fractions were also analyzed for their total protein and total carbohydrate content. Two methods were used to estimate the total protein content of the fractions, a differential UV absorption method (A224-A2.3) (6) and the Lowry method (1). Agreement between the two methods was good. The total protein, as well as the total carbohydrate content of the Sepharose 2B fractions of another 40P preparation, are shown in Fig. 2. Several of the 40-P preparations, as examined by Sepharose 2B filtration, did not appear to contain the lower-molecular-weight component designated peak III in Fig. 1. The major component in peak II had approximately the same ratio of protein to carbohydrate (2.6:1 to 2.8:1) in all the preparations. The excluded material, peak I, had a significantly higher carbohydrate content (Fig. 2). In a recycling experiment, the peak II fractions from a Sepharose 2B column were divided into three equal parts based on their protein content and were concentrated by ultrafiltration. The part representing the concentrated trailing fraction was reapplied to the same column and eluted. The elution profile was essentially the same as that of peak II; the elution peak occurred in the same position. The ratio of protein to carbohydrate was unchanged. Serological analysis of the three concentrated subfractions of peak II in Ouchterlony gel diffusion plates indicated that these three parts of peak II were serologically identical. The recoveries of the 40-P preparations from the Sepharose 2B column were essentially

quantitative; recoveries, based on

total solids,

90 to 100%. To>- tal solids were determined by dialysis against , distilled water, followed by lyophilization of a t portion into a tared bottle. A typical 40-P preparation (preparation 6 on Table 4) that contained z 21.6 mg of solids gave 2.1 mg in peak I and 19.0 , mg in peak II, for a recovery of 93%. The amounts in peak II in various other 40-P prepaI 0 rations varied from 75 to 95% of the total weight. The results of the quantitative precipitin 50 175 75 150 l00 125 analyses of the fractions from a Sepharose 2B MO0 ELUTION VOLUME (ml) column are shown in Fig. 3, along with the FIG. 1. Sepharose 2B gel filtration e lution pat- total protein content of the fractions used as terns of Pasteurella multocida P-1059G 41O-P preparation. A sample, 42 mg, was applied ti a column antigens. These results show that the peak II component is more active than peak I in precip (45 by 2.5 cm) and eluted with 0.15 M Na C ing 3% ethyl alcohol. Bars represent the presence of itating antibody. In addition to the chemical analysis of the precipitin lines in gel diffusion plates, and their height is proportional to the distance of th e line from individual fractions, the fractions representing the edge of the antigen well. peak I and peak II were pooled, concentrated,

protein,

or

carbohydrate,

were

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P. MULTOCIDA LIPOPOLYSACCHARIDE-PROTEIN

120

-a Z

0

80

< 60 z

Q 40 O 20

75

100

125

150

175

EWTION VOLUME (ml)

FIG. 2. Sepharose 2B gel filtration elution pattern of P. multocida P-1059G 40-P preparation in which each fraction was analyzed for its protein and carbohydrate content.

75

125

ELUTION \VOLUME (ml)

FIG. 3. Sepharose B fel filtration of a P. multocida P-1059G 40-P preparation (lot 109). Lower curve, protein concentration, in micrograms per milliliter, as determined by a modified Lowry method with bovine serum albumin as a standard. Upper curve, quantitative precipitin reaction of the fractions, 0.50-ml fraction, 0.05 ml of hyperimmune P1059G rabbit antiserum (no. 71), 2 weeks at 0°C, total protein concentration of antibody and antigen determined per milliliter of the Sepharose 2B fraction by the Lowry method with bovine serum albumin as a standard.

and analyzed for their protein and carbohydrate content. Peak I contained 61% carbohydrate and 24.3% protein based on the Groves UV method (6). Peak II contained 10.7% carbohydrate and 25.6% protein based on a microKjeldahl nitrogen content of 4.1% N, or 27.8% protein based on the UV method. The original 40-P preparation contained 17.8% carbohydrate and 27.3% protein, based on a micro-Kjeldahl nitrogen content of 4.36%. Immunity studies with the Sepharose 2B

995

fractions. The fractions representing the peaks from the column were separately pooled, concentrated, and tested for their ability to immunize either mice or turkey poults against lethal infections with P. multocida. The results are shown in Table 5. The major fraction, from peak II, was just as effective as the original 40P preparation in providing immunity when inoculated into either mice or turkeys. The excluded substance, from peak I, was also effective in immunizing mice and, to a lesser extent, turkey poults. The peak I fraction, as stated previously, failed to react in gel diffusion. The peak III fraction, which was found only in some preparations and consisted of lower-molecularweight protein antigens, was not as effective as that from peak II in immunizing mice. Lethal toxicity of the Sepharose 2B fractions. The fractions representing the peaks were also tested for their lethal toxicity for chicken embryos as shown in Table 3. The peak II fraction appeared to be more toxic than the unfractionated 40-P preparation. The peak I fraction was less toxic. However, only the peak II fraction was isolated in large enough amounts to study the dose-response effect.The LDs,, of 5.2 ,ug for the peak II fraction was less than the LD?,, of 8.7 ,ug for the unfractionated 40-P preparation. Gel diffusion patterns. The gel diffusion patterns of the 40-P preparations before and after fractionation on Sepharose 2B are shown in Fig. 4A and B, respectively, with 12 serotypes of avian antisera. Purification on Sepharose 2B improves the specificity of the reaction; a strong homologous reaction is obtained with type 3 antiserum, and the strongest cross-reaction is observed with type 2. For comparison, the reactions of Westphaltype lipopolysaccharide from P. multocida P1059G are shown with the same antisera in Fig. 4C. A striking similarity is readily seen between the patterns in Fig. 4B and C. To illustrate that the antigenic determinants of the 40-P and lipopolysaccharide are not identical, these antigens were added to the adjacent outer wells of the pattern shown in Fig. 5, and hyperimmune antiserum was added to the center well. The lines of reaction of the 40-P preparation or of the Sepharose 2B purified antigen do not fuse with those of the lipopolysaccharide. In a previous publication (15), definite spurs were obtained with the lipopolysaccharide and 40-P antigens of P. multocida serotype 1. Immunoelectrophoresis of the 40-P preparation resulted in the formation of two lines the same distance from the well in which the antigen was added, differing only in their distance from the trough to

996

INFECT. IMMUN.

GANFIELD, REBERS, AND HEDDLESTON

TABLE 5. Induction of active immunity in mice and turkeys with Sepharose 2B fractions ofP. multocida P1059G 40-P preparations Dose (mg) Cumulative dead by day no.: No. survivors/ Protection Immunogen no. chal( First Second 1 2 4 11 lenged Expt 1 (mice)P 10 10 0 1 Peak 1 (lot 200) 2 2 10/12 83 24 0 0 0 0 12/12 24 Peak II (lot 200) 100 3 7 7 7 5/12 50 50 Peak III (lot 42 226) 20 0 1 20 1 2 10/12 Unfractionated 83 40-P (lot 200) 10 11 11 11 0/11 Controls 0 Expt 2 (mice)a Peak I (lot 116) Peak II (lot 116) Unfractionated 40-P (lot 116) Controls

20 20 20

20 20 20

0 0 0

0 0 1

0 1 4

2 4 4

10/12 10/14 10/14

83 71 71

13

13

13

13

0/13

0

Expt 3 (turkeys)" 6 7 7 6 20 2/9 Peak I (lot 109) 22 4 3 5 5 20 Peak II (lot 109) 4/9 44 2 20 4 5 6 4/10 Unfractionated 40 40-P (lot 109) 9 10 10 10 Controls 0/10 0 a Each mouse was inoculated intraperitoneally with two equal doses of antigen. The second was given 26 days after the first. The challenge inoculation was given intraperitoneally 3 weeks after the second dose of antigen and contained 1,000 colony-forming units of P. multocida P-1059F. b Each turkey was given a single dose of antigen intramuscularly in the thigh, and the challenge inoculation was given 3 weeks later by the same route with the 1,000 colony-forming units of P. multocida P1059F.

which the rabbit antiserum was added. Such a of avirulent nonencapsulated forms of the orgapattern might be produced if the main compo- nism contained substances that effectively imnent could exist in two different states of aggre- munized chickens, mice, and rabbits (9). For gation that had the same electrophoretic mobil- this reason and because we believed that saline extracts of encapsulated virulent P. multocida ity but different rates of diffusion. Isoelectric focusing. Preparations of 40-P might contain a more complex array of antiwere applied to an isoelectric focusing column gens, the extracts of avirulent nonencapsulated with a pH gradient of 3-10. After electrofocus- organisms were chosen for investigation. ing for 20 h, fractions were collected and anaThe protection of 70% of the turkeys with a lyzed by the Ouchterlony procedure (Fig. 6). single intramuscular inoculation of 10 ,ug of the The precipitinogenic fractions were all in the 40-P preparation against a challenge inoculapH range of 3 to 4, with a maximum activity at tion with 900 colony-forming units of P. multopH 3.7. The isoelectric focusing procedure gave cida is considered significant, since this chalonly one precipitating region. When the time of lenge inoculation killed 32 out of 32 controls in electrofocusing was longer than 20 h or when a these experiments, and previous studies have gradient of pH 3 to 5 was used, considerable shown that 25, 50, 100, 150, and 200 colonyamounts of precipitation occurred in the isoe- forming units killed 91, 92, 94, and 96% of conlectric focusing column. trol groups of turkeys (3) (Table 1). The degree of protection of turkeys described in Table 5 was DISCUSSION less, only 40 to 45%, but a different 40-P prepaThe primary purpose of purification of the ration was used, one that was lower in carbohyisotonic saline extractable antigens ofP. multo- drate and higher in phosphorus content. Even cida was to enable a better characterization of this content of protection was considered imthe antigen(s) capable of inducing antiinfec- portant because after 1 day, 90% of the controls tious immunity. Previous experiments from were dead, whereas only 20, 33, and 66% of the this laboratory have shown that saline extracts vaccinated turkeys had died.

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P. MULTOCIDA LIPOPOLYSACCHARIDE-PROTEIN

997

The active component of the 40-P preparation is believed to be a lipopolysaccharide-protein complex (17; D. Ganfield, Ph.D. thesis, Iowa State Univ. Ames, 1971). Despite the relatively high protein content of the 40-P preparation, its immunogenicity was retained and no amino acids were liberated by treatment with the proteolytic enzyme Pronase. The resistance of the protein to proteolytic digestion may mean that the protein is protected by the lipopolysaccharides or a phospholipid. Protection of protein of a lipopolysaccharide-protein complex against attack by Pronase has been described by Rothfield and Pearlman-Kothencz (17). Since the

FIG. 5. Wells labeled A, B, and C contain the antigens described in Fig. 4. Center well contains serum from a rabbit inoculated eight times over a 4month period with a 40-P preparation.

FIG. 4. Ouchterlony gel diffusion patterns of P. multocida P-1059G antigens and 12 serotypes of avian antisera. The avian antisera were placed in the outer wells labeled 1 through 12 of (A) and by the same pattern in (B) and (C). (A) P. multocida P1059G 40-P antigen was placed in the center wells; (B) the Sepharose peak II fraction of the 40-P preparation was placed in the center wells; (C) a Westphaltype lipopolysaccharide from P. multocida P-1059G cells was placed in the center wells. Note the strong homologous reaction with the serotype 3 antiserum and the cross-reaction with serotype 2 antiserum with all of the antigens; the degree of cross-reactivity is least in (C) and greatest in (A).

immunogenicity of the 40-P preparation was not reduced by treatment with Pronase, it may be inferred that the immunogenicity is not due to a free or unprotected protein. If protein is required, it is present in a protected form. Since the pelleting procedure at 105,000 x g of the 40-P preparation would not be expected to produce a pure antigen, it was anticipated that further purification could be obtained by gel filtration. Sepharose 2B gel filtration of the 40P preparations resulted in as many as three peaks. The first peak contained a fraction with the highest ratio of carbohydrate to protein and appeared to be a large aggregate, because it came out in the void volume and would not give lines in the Ouchterlony gel diffusion test. The second fraction, which represented the major component, contained a lower ratio of carbohydrate to protein, was a smaller complex, and gave a gel precipitin line with antiserum. Both these fractions had some similar antigenic determinants when tested by the quantitative precipitin reaction and induced active immunity in mice and turkeys. Active immunity was induced in mice and turkeys with the antigens from peaks I and II to about the same extent as was obtained with the original 40-P preparation. Better protection was obtained in mice that had received two doses of vaccine than in the turkeys that had received only one. However, the purpose of giving the turkeys only one dose was to attempt to adjust the schedule so that only partial protection would be obtained, since it was believed

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40-P antigen and the type 2 antiserum used to prepare the pattern shown in Fig. 4. Other type 2 antisera do not show this cross-reaction. It should be pointed out that bacterins prepared with type 3 P. multocida do not induce immunity against the type 2 organism (10, 14). Typespecific protective immunity has been demonstrated with P. multocida types 1, 2, 3, and 4. Although the Westphal lipopolysaccharide appears to have similar antigenic determination ~~~~~~z to those of the 40-P antigen, it should be mentioned that it is easy to induce active immunity 5.0 in mice with the 40-P preparations, but the Westphal lipopolysaccharide has previously 4.0 failed to do so (2, 16). This evidence, along with (LJ the patterns shown in Fig. 5, suggests that 3.0 --J 3.0~~~~~ important differences exist between the 40-P preparation and the Westphal lipopolysaccha2.0 ride. Despite the well-known heterogeneity of gram-negative lipopolysaccharides as shown by Nowotny et al. (15), as well as the heterogeneI i.. . ity of lipopolysaccharide-protein complexes (20), 0 15 30 45 60 75 90 the P. multocida lipopolysaccharide-protein ELUTION VOLUME (ml) complexes purified by gel filtration on SephaFIG. 6. Isoelectric focusing experiment with a P. rose 2B appeared homogeneous because the multocida P-1059G 40-P preparation in a pH 3 to 10 peak II fraction had a relatively constant ratio gradient. The preparation (10 mg) was electrofo- of protein to carbohydrate from three different cused for 20 h at 300 V and 2C. Solid line represents the pH gradient of the eluted fractions. Solid bars preparations of 40-P. Furthermore, rechromarepresent the relative concentration of serological tography of the peak II fraction on Sepharose reactivity of the fractions as estimated by measuring 2B gave no evidence of further fractionation. The variability in the composition of the 40-P distance of precipitin lines from the antigen well in Ouchterlony plates. preparations may simply reflect different amounts of the components found in peaks I, IH, that it would be easier to detect differences in and III. The relative toxicity of the peak II fraction immunogenicity of the fractions under these conditions. The challenge inoculation given the was greater than that of the peak I fraction in mice was strong enough to kill 24 of 24 control the chicken embryo test. The greater toxicity of mice. A previous experiment showed that 100 the second may be the result of its smaller size colony-forming units of this strain was a strong or of the presence of more protein facilitating enough dose to kill 32 out of 32 mice (7). The its transport across the chorioallantoic memchallenge was strong enough in any case so brane. The LD50 of 5.7 ,tg for embryos and 195 that the partial protection obtained is meaning- ,ug for mice with the second fraction is as low as ful. The third fraction that was found with that generally reported for Westphal lipopolyseveral of the 40-P preparations did not contain saccharides (5, 19). These figures indicate a detectable carbohydrate, and gave lines of noni- very toxic complex in spite of the presence of dentity with the peak II fraction in Ouchterlony greater than 50% noncarbohydrate compotests. Some protection was observed with the nents. peak III fraction, and this could mean that the LITERATURE CITED soluble cross-reactive proteins of peak III are capable of inducing immunity. However, the 1. Bailey, J. L. 1967. Techniques in protein chemistry, 2nd ed. Elsevier Publishing Co., New York. peak III fraction may have derived its activity R. V. S., and K. W. Knox. 1961. The antigens of from traces of the peak II component as a result 2. Bain, Pasteurella multocida type 1. II. Lipopolysaccharides. of incomplete separation on the column. Immunology 4:122-129. The gel diffusion studies reported in this pa- 3. Bairey, M. H. 1975. Immune response to fowl cholera antigens. Am. J. Vet. Res. 36:575-578. per and previously (9) indicate that protective B. O., K. L. Heddleston, and C. J. Pfow. antibody may be detected with the 40-P anti- 4. Blackburn, 1975. Pasteurella multocida serotyping results 1971gen. However, this is not always the case. A 1973. Avian Dis. 19:353-356. cross-reaction was obtained between the type 3 5. DaMassa, A. J., H. E. Adler, and C. E. Franti. 1975. A 10.0

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quantitative study of the effect of endotoxin on embryonating chicken eggs. Avian Dis. 19:1-5. 6. Groves, W. E., F. C. Davis, Jr., and B. H. Sells. 1968. Spectrophotometric determination of microgram quantities of protein without nucleic acid interference. Anal. Biochem. 22:195-210. 7. Heddleston, K. L., and P. A. Rebers. 1974. Fowl cholera bacterins: host specific cross-immunity induced in turkeys with Pasteurella multocida propagated in embryonating turkey eggs. Avian Dis. 18:213-219. 8. 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. 9. Heddleston, K. L., P. A. Rebers, and A. E. Ritchie. 1966. Immunizing and toxic properties of particulate antigens from two immunogenic types of Pasteurella multocida of avian origin. J. Immunol. 96:124-133. 10. Heddleston, K. L., K. R. Rhoades, and P. A. Rebers. 1967. Experimental pasteurellosis: comparative studies on Pasteurelia multocida from Asia, Africa and North America. Am. J. Vet. Res. 28:1003-1012. 11. Heidelberger, M., and P. A. Rebers. 1958. Cross reactions of polyglucoses in antipneumococcal sera. VI. Precipitation of type VIII and type III antisera by ,glucans. J. Am. Chem. Soc. 80:116-118. 12. Horsfall, F. L., Jr., and I. Tamm (ed.). 1965. Viral and richettsial infections of man, 4th ed. J. B. Lippincott Co., Philadelphia, PA. 13. Kabat, E., and M. Mayer (ed.). 1968. Immunochemistry, 2nd ed. Charles C Thomas Publisher, Springfield, Ill.

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14. Little, P. A., and B. M. Lyon. 1963. Demonstration of serological types within the nonhemolytic Pasteurella. Am. J. Vet. Res. 4:110-112. 15. Nowotny, A., K. R. Cundy, N. L. Neale, A. M. Nowotny, R. Radvany, S. P. Thomas, and D. J. Tripodi. 1966. Relation of structure to function in bacterial 0antigens. IV. Fractionation of the components. Ann. N.Y. Acad. Sci. 133:586-W603. 16. Rebers, P. A., and K. L. Heddleston. 1974. Immunological comparison of Westphal-type lipopolysaccharides and free endotoxins from an encapsulated and a nonencapsulated avian strain of Pasteurella multocida. Am. J. Vet. Res. 35:555-560. 17. Rebers, P. A., K. L. Heddleston, and K. R. Rhoades. 1967. Isolation from Pasteurella multocida of a lipopolysaccharide antigen with immunizing and toxic properties. J. Bacteriol. 93:7-14. 18. Rothfield, L., and M. Pearlman-Kothencz. 1969. Synthesis and assembly of bacterial membrane components. A lipopolysaccharide-phospholipid-protein complex excreted by living bacteria. J. Mol. Biol. 44:477-492. 19. Tate, W. J., III, H. Douglas, A. I. Braude, and W. W. Wells. 1966. Protection against lethality of E. coli endotoxin with "O" antiserum. Ann. N.Y. Acad. Sci. 133:746-762. 20. Work, E. 1971. Production, chemistry, and properties of bacterial pyrogens and endotoxins, p. 2346. In G. E. Wolstenholme and J. Birch (ed.), Pyrogens and fever, Ciba Foundation Symposium. Churchill Livingstone, Edinburgh.