Some Structural and Biological Properties of Brucella Endotoxin

INFECTION AND IMMUNITY, Feb. 1970, p. 174-182 Copyright © 1970 American Society for Microbiology

Vol. 1, No. 2 Printed in U.S.A.

Some Structural and Biological Properties of Brucella Endotoxin D. LEONG, R. DIAZ,1 K. MILNER, J. RUDBACH, AND J. B. WILSON Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, and University of Wisconsin, Madison, Wisconsin 53706

Received for publication 21 October 1969 Hot phenol-water extraction of smooth Brucella abortus and B. melitensis cells yielded a toxic fraction which was recovered from the phenol phase (fraction 5). Chemically, fractions 5 from both Brucella species were lipid-carbohydrate-protein-2 keto-3-deoxyoctulosonic acid complexes which were stable to heat and resistant to Pronase digestion. Electron micrographs of the Brucella toxins were morphologically indistinguishable from those of enterobacterial endotoxins. Biologically, Brucella toxins were lethal for mice and immunogenic for rabbits. An intravenous injection of Brucella toxin induced severe leukopenia with subsequent leukocytosis in mice. Cross-tolerance experiments with mice demonstrated that pretreatment with B. abortus toxin lessened the hypoferremia produced by challenge with Escherichia coli endotoxin. Furthermore, fractions 5 from B. abortus and B. melitensis were able to form hybrids with E. coli and Salmonella enteritidis endotoxins and also with each other. Although Brucella toxins possess many structural and biological properties in common with endotoxins from the Enterobacteriaceae, some quantitative differences in their biological potencies were observed. Brucella toxins were relatively innocuous in tests for pyrogenicity in rabbits and lethality for chick embryos. In nonspecific protection tests, Brucella toxin had only 3½,5 the potency of E. coli endotoxin in protecting mice against challenge with virulent S. typhi. However, on the basis of the data presented and on the work done previously, we concluded that the heat-stable toxins of B. abortus and B. melitensis were endotoxins. for 36 hr in shake flasks containing Trypticase Soy Broth (BBL). A strain of Escherichia coli (Rolf) isolated from Swiss Webster mice was grown similarly, but the incubation time was reduced to 18 hr. Salmonella enteritidis strain S-795 and E. coli 0111:B4 (Difco) were grown at 37 C for 24 hr in M9 medium containing 0.5% glucose (24). The cells were harvested by centrifugation and washed repeatedly with cold saline. Preparation of endotoxin. Ether-water (EW) extractions of B. abortus, E. coli (Rolf), and S. enteritidis whole cells were prepared as described previously by Ribi et al. (21). Cell walls were prepared from E. coli 0111 :B4 by cell disruption at 20,000 psi with a Sorvall refrigerated cell fractionator (6). Endotoxin was extracted from the prepared cell walls by the hot phenolwater (PW) method of Westphal et al. (27). Special procedures were utilized for extracting B. abortus and B. melitensis cells by a hot phenol-water method as described in detail by M. S. Redfearn (Ph.D. Thesis, University of Wisconsin, Madison, MATERIALS AND METHODS 1960). Brief descriptions of these procedures and the Cultivation of bacteria. Smooth cultures of Brucella yields of the various fractions are presented, respecabortus 2308 and B. melitensis 16M were grown at 37 C tively, in Fig. 1 and Table 1. Chemical analyses. The analytical methods em1 Present address: University of Navarra Medical School, ployed have been described elsewhere (2, 22). The 2 keto-3-deoxyoctulosonic acid (KDO) values were Pamplona, Spain. 174

Representatives of most families of gram-negative bacteria contain heat-stable toxins. Those extracted from the various species of Enterobacteriaceae have been studied most thoroughly (13). Ordinarily, the heat-stable toxins of enterobacteria may be described as phospholipid-polysaccharidepeptide complexes which elicit a characteristic spectrum of biological reactions (7, 13, 17). They are usually called "endotoxins." Heat-stable toxins from other gram-negative bacteria, in the main, have not received a comparable degree of attention, nor has the relationship of their structure and biological properties to those of enterobacterial endotoxins been thoroughly investigated. It was the purpose of this investigation to study the relationship between heat-stable toxins from two bacterial species in the family Brucellaceae and endotoxins from species of Enterobacteriaceae.

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PROPERTIES OF BRUCELLA ENDOTOXIN

determined by the thiobarbituric acid technique of Weissbach and Hurwitz (26) with equimolar quantities of KDO and 2-deoxy-D-ribose as the standard. Electron microscopy. Fractions 5 from B. abortus and B. melitenisis were negatively stained with potassium phosphotungstate for electron microscopy, by the method described by Ribi et al. (23). Electron micrographs were made with a Siemens Elmiskop I electron microscope. Treatment with Pronase and heat. Fractions 5 of B. abortus and B. melitenisis were dissolved in 0.025 M tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 5 mg/ml. Pronase (B grade, lot 65515, Calbiochem) was added to the solution in a final concentration of 150 ,g/ml, and the mixture was incubated in a 40 C water bath for 15 hr. Two volumes of sodium acetatemethyl alcohol (1 ml of saturated solution of sodium acetate in 99 ml of methyl alcohol) were added to the treated fractions. The precipitates were sedimented by centrifugation at 10,000 X g for 30 min and redissolved in distilled water. These solutions were recentrifuged at 60,000 X g for 12 hr at 5 C, and the resulting pellets were dissolved in distilled water and lyophilized. To test heat stability, fractions 5 of B. abortus and B. melitenisis were suspended in phosphate-buffered saline (pH 7.2) at 5 mg/ml and heated for 15 min in a boiling water bath (96.6 C) in sealed glass tubes, after which the tubes were cooled rapidly to room temperature. Appropriate dilutions were made and injected intravenously into mice for determination of LD3o. Bioassays for endotoxin. Determinations of toxicity (mouse LD/t. X 100 = %70 WBC, where t. was the leukocyte count at a given interval after, and t0 the leukocyte count before, administration of the test dose. The WBC count before injection of fraction 5 was taken as 100%-; % WBC lower than 100% represented leukopenia, and greater than 100% represented leukocytosis. The technique used for measuring serum iron of mice was that of Kampschmidt and Upchurch (11), as modified by Baker and Wilson (3) for bioassay of endotoxin-like activity in cultures of B. abortus and E. coli. Results are expressed as percentage of reduction in the normal level of serum iron (15). This response was also employed for measuring cross-tolerance between B. abortus-EW and

175

E. coli Rolf-EW extracts. For this purpose, each of seven groups of mice (three replicates per group, five mice per replicate) was pretreated with one to seven consecutive daily intraperitoneal inoculations of 10 /ug of B. abortus-EW. A challenging dose of 12.5 ,ug of E. coli Rolf-EW was given intraperitoneally 24 hr after each pretreatment. At 12 hr after the mice were challenged, they were sacrificed, and the depression in serum iron was measured. In another experiment, the same time sequences of pretreatments, challenge, and sacrifice were followed, but the pretreating inoculations were with 10 jig of E. coli RolfEW, and the challenge dose was 100 ,ug of B. abortusEW. The latter challenge produced the same depression of serum iron in normal mice as 12.5 jig of E. coli Rolf-EW. The antigenicity of different fractions obtained from the hot phenol-water extraction of Brucella cells was tested as follows. A 25-,ug amount of each fraction was injected intravenously into rabbits (two rabbits for each fraction). Ten days later the animals were bled (primary bleeding), after which a booster injection of 10 mg was administered. Seven days after the booster injection, the animals were again bled (secondary bleeding). The antibody titers of the sera were measured by the tube agglutination test in which the standard antigen of the U.S. Department of Agriculture was employed at the recommended concentration for diagnosis of bovine brucellosis (4). Formation of endotoxin hybrids. The antigens of B. abortus fraction 5 diffuse only to a limited degree through agar of immunodiffusion plates. Redfearn (Ph.D. Thesis. University of Wisconsin, Madison) had found earlier that the diffusibility of fraction 5 in agar could be increased by mild acid hydrolysis; therefore, conditions were sought which would satisfactorily prepare the antigens for immunodiffusion without destroying their ability to hybridize. The fraction was dissolved at 5 mg/ml in 0.17 M acetic acid and hydrolyzed in a boiling water bath (96.6 C) in screw-cap tubes. Samples were removed at intervals, cooled, and neutralized with 0.2 N Na2CO3. Groups of mice were inoculated intravenously with 0.5 ml of the hydrolysates, and LD5 values were determined (12). After 30 min of hydrolysis, the diffusibility of fraction 5 in agar was enhanced and its toxicity was decreased by only 30% (Table 6). This 30-min hydrolysis product of B. abortus fraction 5, after dialysis against distilled water and lyophilization, was used in the hybridization experiments. Fraction 5 of B. melitenisis diffused readily through agar without prior treatment. However, it was subjected to the same acid hydrolysis and testing in mice for the sake of comparison. Endotoxin hybrids (between B. abortus and B. melitensis, Brucella and E. coli 0111B:4, and Brucella and S. enteritidis) and the corresponding controls for each hybrid pair were prepared as described by Rudbach et al. (25). Specially prepared monospecific antisera against the respective Brucella species, kindly supplied by Lois M. Jones, University of Wisconsin, were required to detect hybridization between B. abortus and B. melitensis fractions 5 (9). Other antisera used for immunodiffusion studies

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LEONG ET AL.

176

TABLE 1. Dry weights of fractions obtained from Brucella abortus and B. melitensis by extraction with hot phenol-water Dry weights

Fraction or cell residue

1 2 3 4 5 6 and 7

Phenol-alcohol phase Total recovered Starting material

B. abortus 2308

B. metitensis

% g 20.13 58.39 2.010 5.829 0.005 0.0145 0.570 1.656 0.884 2.563 4.490 13.022 3.253 9.434

10.60 53.99 0.946 4.819 0.108 0.55 1.037 5.282 0.417 2.124 3.00 15.282 1.742 8.874

31.346 90.91

17.85

34.5 g (dry wt

19.6 g (dry wt

g

90.92

of cells)

of cells)

fractions 5 of both Brucella species. This fraction was precipitated from the phenol phase with sodium acetate-methyl alcohol. The remaining fractions and cell residues obtained from both Brucella species were nontoxic in amounts up to 20 mg per mouse given intraperitoneally. Chemically, fractions S from B. abortus and B. melitensis were protein-lipopolysaccharide-KDO complexes (Table 3). Heat treatment for 15 min in a boiling water bath and Pronase digestion had little effect on the resulting toxicity of fractions 5 from either Brucella species; moreover, treatment with Pronase did not significantly reduce the nitrogen content of the fraction 5 preparations. TABLE 2. Toxicity of fractions obtained by hot phenol-water extractions of Brucella abortus and B. melitensis Mouse LD6o values (mg)"


were obtained after multiple injections of rabbits with heat-killed cells of B. melitensis, B. abortus, S. enteritidis, and E. coli 0111 :B4.

RESULTS

Toxicity and antigenicity of the Brucella fractions. Table 2 presents mouse LD50 values of the different fractions and cell residues obtained by the Redfearn extraction procedure (Fig. 1) from cells of B. abortus and B. melitensis. All of the toxicity, as measured by lethality for mice, was found in

Interphase (fraction 1) Cell residue (fraction 2) Water phase (fraction 3) Water phase (fraction 4) Endotoxin (fraction 5) Phenol phase precipitate (fractions 6 and 7) Phenol-alcohol phase

B. abortus

B. melitensis

>20 >20 >20 >20 0.30 >20

>20 >20 >20 >20 0.30 >20

>20

>20

Fractions injected intraperitoneally.

10 g dried Brucella cells suspended in 340 ml water, 66 C; add equal volume of 88% phenol, 66 C, stirred 20 min, cooled to 5 C, centrifuged

Interphase (fraction 1)

Aqueous phase, dialyzed, pervaporated, 5 vol NaAcMeOH,a centrifuged

Precipitate (fraction 2)

Phenol phase, filtered, NaAc-MeOH (3 vol) centrifuged

Precipitate dissolved in Supernatant fluid, Supernatant fluid di;alyzed, Precipitate extracted water, NaAc-MeOH (4 vol) 5 vol acetone, cen- lyophilized (phenol-alIcohol 3 X with water, cen-

centrifuged

trifuged

phase)

Precipitate lyophilized (fraction 3)

Supernatant fluid (discarded)

Supernatant fluid, di;alyzed, Precipitate dissolved at pervaporated, 3 vol NaAc- pH 11.5, centrifuged MeOH, centrifuged

Precipitate dissolved in water, acetoneMeOHb 10 vol Supernatant fluid

Precipitate,

lyophilized

Supernatant fluid (discarded)

trifuged

Suipernaltant

lyc)philized

fluid

(fracction 6)

Precipitate

lyophilized (fraction 7)

Precipitate

lyophilized

(fraction 4) (discarded) (fraction 5) FIG. 1. Fractionation procedures of Brucella cells by the hot phenol-water method of Redfearn. Footnote a denotes sodium acetate-methanol (I ml of saturated solution of sodium acetate to 99 ml of methanol). Footnote b denotes a 1:1 volume of acetone and sodium acetate-methanol.

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VOL. 1, 1970

TABLE 3. Chemical composition of the fractions obtained from Brucella abortus and B. melitensis cells by extraction with hot phenol-water Total carbo-

Hexose

KDO

Hexosamine FAA + FAE


Na

P

lAb lMd 2A 2M 3A 3M 4A 4M 5A 5M 6A-7A 6M-7M

14.3 14.2 14.1 13.9 10.4 13.5 Trace Trace 6.08 5.88 15.8 16.0

1.23 1.19 0.94 1.21 5.49 7.65 300 0.15

jig

10 0.003

jig

15 0.2

0111:B4

6, and 7, and the phenol-alcohol phase from B. melitensis failed to stimulate the primary antibody response, but secondary responses were elicited after the booster injection was given; fraction 4 was nonantigenic. Leukopenic and leukocytotic effects of B. melitensis fraction 5. Figure 2 shows the leukcpenic and leukocytotic effects of 25- and 100-,ug doses of B. melitensis fraction 5 in mice. Within the first hour after injection of the 100-,ug dose, leukopenia was evident and persisted for 5 to 6 hr. After 6 hr, the WBC counts of the mice increased progessively to a moderate degree of leukocytosis. The 25-,ug dose of the same fraction also induced leukopenia within the first hour after its injection, followed by a marked leukocytosis at

f!| lE1. X | l5

FIG. 3.

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6 hr. At 24 hr, leukocytosis persisted in both groups of mice. Toxicity of E. coli endotoxin compared to that of B. abortus fraction 5. B. abortus fraction 5 was subjected to a variety of quantitative bioassays in parallel with E. coli 0111:B4-PW endotoxin. Table 5 shows that fraction 5 was much less potent than E. coli 01 11: B4-PW endotoxin in pyrogenicity for rabbits (FI4o) and lethality for chick embryos (CELD5o). Fraction 5 had only '15 the potency of E. coli 0111 : B4-PW endotoxin to induce nonspecific resistance to S. typhi challenge in TABLE 6. Effect of acid hydrolysis oni the toxicity oJ Brucella abortiis anid B. nmelitenisis toxinis (J')aciotoll 5) Mouse LD50

Time of hydrolysis"B. abort us

a I

mm

jig

0 15 30 45 60 90

250 325 325 375 325 570

values" B. nmelite nsis jig

250 186 373 430 647 > 1,000

Toxin (5 mg/ml) in 0.17 M acetic acid. Toxin injected intravenously.

..

Electroni micrographls of ntegatively stainted fractionls 5 from B. melitemisis (A) amid B. abortuts

(B).

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mice. Only in mouse lethality were the toxins of approximately equal potency. Electron micrographs of fractions 5 from B. abortus and B. melitensis. Figures 3A and 3B are electron micrographs of negatively stained preparations of fractions 5 from B. melitensis and B. abortus. Both show long and short filamentous structures morphologically indistinguishable from those found in electron micrographs of endotoxins extracted from smooth strains of Salmonella and Escherichia by the hot phenol-water method (23). Effect of acid hydrolysis on fractions 5 of B. abortus and B. melitensis. During the first 15 min of acid hydrolysis of B. melitensis fraction 5, there was an increase in toxicity of the preparation (Table 6). Loss of toxicity began after 15 min of hydrolysis and progressed until little toxicity remained at 90 min. Fraction 5 of B. abortus was more resistant to acid hydrolysis than that of B. melitensis. The LD50 values of B. abortus fraction 5 increased only 30 to 50 % of its original potency up to 60 min of acid hydrolysis. After 90 min, the LD50 of the preparation had doubled. Hybrid formation between endotoxins from the Enterobactericeae and fractions 5 of Brucella. Endotoxins from various species of Enterobacteriaceae can be induced to form hybrids with each other by treating a mixture of endotoxins with sodium deoxycholate and then removing the bile salt (25). A convenient method for detecting hybridization was to examine immunodiffusion plates for a reaction of identity when the endotoxin hybrid was allowed to diffuse against and react simultaneously with both of the specific antisera

FIG. 4. Hybrid formation between toxins. (A) Hybrid formation between B. melitensis (Bm) and E. coli 0111 (Ec) and (B) between B. melitensis and S. enteritidis (Se) toxins. Concentration was I mg/ml; 500 ,gg/ml of each toxin. HYB indicates samples in which the sodium deoxycholate (NaD) was removed from the mixture of the toxins. MIX indicates the controls which were treated separately with NaD; the NaD was removed before the samples were mixed.

179

directed against the individual members making up the hybrid pair. Figures 4A and 4B show, respectively, the precipitation arcs obtained in immunodiffusion plates with hybrids formed between B. melitensis and E. coli 0111 toxins and between B. melitensis and S. enteritidis toxins. The top wells contain the hybrids and the bottom wells the control solutions of the two toxins treated separately with sodium deoxycholate, precipitated with alcohol, and mixed. A single precipitation arc showing continuity can be seen around the top wells containing the hybrid pairs in both plates, and a crossing or a nonfusing of precipitation arcs shows the reaction of nonidentity with the control mixtures in the bottom wells. Similarly, formation of hybrids between B. abortus and E. coli (Fig. 5A), B. abortus and S. enteritidis (Fig. SB), and B. abortus and B. melitensis (Fig. 6) toxins occurred after sodium deoxycholate treatment of the different combinations and removal of the bile salt. Cross-tolerance to toxin-induced hypoferremia. It was reported earlier that an ether-water extract of B. abortus cells caused a reduction in normal serum iron of mice when the extract was injected intraperitoneally (2). A state of tolerance, characterized by a lesser degree of hypoferremia (a serum iron reduction of 25 % or less), could be induced in mice by pretreatment with small doses of B. abortus-EW toxin given prior to a 100-,ug challenge .with the homologous toxin (15). Table 7 shows that a state of cross-tolerance was established in mice pretreated and challenged with two antigenically unrelated toxins. Tolerance to the E. coli Rolf-EW endotoxin was established after the third and subsequent pretreating injections with

FIG. 5. Hybrid formation between toxins. (A) Hybrid formation between B. abortus (Ba) and E. coli (Ec) and (B) between B. abortus and S. enteritidis (Se) toxins. Concentration was I mg/ml; 500 Ag/ml of each toxin. The white arrows point to the precipitation arcs produced with B. abortus toxin and its specific antiserum; also see legend of Fig. 4.

180

LEONG ET AL.

INFEC. IMMUN.

B. abortus-EW toxin. The reduction in serum iron of the tolerant mice was 5 to 25 %, whereas normal mice challenged with the same quantity of E. coli endotoxin had an average reduction in serum iron of 56%. Similarly, tolerance to challenge with B. abortus toxin was observed after the fifth and subsequent pretreating injections with E. coli endotoxin. The tolerant mice responded with 0 to 24% reduction in serum iron levels to the B. abortus challenge, but a 50% reduction in serum iron was observed in the control

TABLE 7. Cross-tolerance between toxins from Brucella abortus and Escherichia coli as measured by the hypoferremic response

mice.

2 3 4 5 6 7 Challenge dose in normal mice

DISCUSSION Generally, when smooth strains of enterobacteria are extracted by the hot phenol-water procedure, the biologically active lipopolysaccharides are isolated from the aqueous phase (27). Recently, however, phenol-soluble endotoxins have been isolated from other gram-negative bacteria such as Citrobacter freundii (18, 19, 20) and Xanthomonas campestris (1, 8, 16). The toxins extracted from smooth B. abortus and B. melitensis cells fit into this latter category. They were recovered from the phenol phase, as originally described by Redfearn (Ph.D. Thesis, University of Wisconsin, Madison, 1960). The toxic fractions

FIG. 6. Hybrid formation between B. abortus (Ba) and B. melitensis (Bm) toxins. Concentration was I mg/ml; 500 ,ug/ml of each toxin. The precipitation arcs of B. abortus and B. melitensis toxins have been reinforced with white ink for contrast. Anti-Bm and anti-Ba were monospecific antisera; also see legend of Fig. 4.

Reduction in serum iron No. of injections

1

B. abortus pretreat-

ment,a E. coli challengec

E. colib pretreatment, B. abortus challenged

51 44 25 19 5 24 20 56

39 38 43 33 24 0 13 50

a B. abortus-EW toxin, 10 ,ug per injection, intraperitoneally (IP). b E. coli Rolf-EW endotoxin, 10,g per injection, IP. c E. coli Rolf-EW endotoxin, 12.5 ,ug, IP. d B. abortus-EW toxin, 100 jug, IP.

from both Brucella species were protein-lipopolysaccharide-KDO complexes, a chemical make-up characteristic of most endotoxins. These Brucella toxins were stable to heat and resistant to Pronase digestion; electron micrographs of the toxins showed forms which were morphologically indistinguishable from those of smooth enterobacterial endotoxins extracted by the phenolwater method. Furthermore, the phenol-soluble toxins from B. abortus and B. melitensis could be induced to form hybrid molecules with E. coli and S. enteritidis endotoxins and also with each other. Previously, it was shown that only endotoxins could be induced to hybridize with endotoxins under the conditions of the experiment (25). We take this to imply some homology between toxins from Brucella and enterobacterial endotoxins. The lipopolysaccharide complex, which contains both the 0-somatic antigens and the endotoxic activity, is found on the cell surfaces of smooth enterobacteria and is diminished or lacking in rough strains. In like manner, rough strains of Brucella are deficient in surface lipopolysaccharides and less toxic for mice (10). Our studies demonstrated that fractions 5 of both Brucella species were potent antigens, were lethal for mice, and could induce leukopenia and leukocytosis. Other work has demonstrated that these antigens of Brucella were capable of inhibiting the aggluti-

VOL. 1, 1970


nation of smooth Brucella cells by homologous 0-antisera (5). Kampschmidt and Upchurch (11) showed that E. coli endotoxin injected intravenously into rats depressed the serum-iron levels of these animals and that the degree of hypoferremia was directly proportional to the quantity of endotoxin injected. Toxins have been extracted from B. abortus which were lethal for mice and could induce a dose-related hypoferremia in these animals (2). Tolerance to the hypoferremic capacity of B. abortus toxin could be induced in mice by pretreatment with small doses of the homologous toxin (15). Our cross-tolerance experiments with mice demonstrated that pretreatment with B. abortus toxin lessened the hypoferremia produced by challenge with E. coli endotoxin. Reciprocal cross-tolerance to the hypoferremic capacity of B. abortus toxin could be established by pretreatment of the mice with E. coli endotoxin. These findings establish further similarities between enterobacterial and brucellar heat-stable toxins. Brucella toxins thus possess many structural and biological properties in common with endotoxins of the Enterobacteriaceae. However, some quantitative differences in their biological potencies were observed. Brucella toxins were relatively innocuous in tests for pyrogenicity in rabbits and lethality for chick embryos. In nonspecific protection tests, Brucella toxin had only 35 the potency of E. coli endotoxin in protecting mice against challenge with virulent S. typhi. Thus, a question must be resolved. Are the toxins of Brucella really endotoxins, deficient in certain chemical structures necessary for the complete spectrum of classical endotoxic activities, or should they be grouped as separate toxic entities, distinguishable from endotoxins? We have concluded on the basis of the data presented and on the work done previously (2, 5, 14, 15) on Brucella toxins that fractions 5 from B. abortus and B. melitensis were endotoxins which possess many immunological, morphological, and toxic characteristics in common with endotoxins extracted from the Enterobacteriaceae. The question of chemical or structural deficiencies which might be responsible for the diminished potency is left for future studies. Alternative possibilities, such as the hypothetical participation of hypersensitivity factors in endotoxic phenomena, are outside the scope of the present work. ACKNOWLEDGMENTS We thank W. D. Bickel, R. Hornung, R. Pfeifer, and R. Reich for their skillful assistance. This investigation was supported by Public Health Service Fellowship 2-F02-AI-34657-03 (to D. L.) from the National Institure of Allergy and Infectious Diseases.

181

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