LEPTOSPIRES. Washington, D. C. preparations since they contained insoluble materials.

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1 INFRARED SPECTROPHOTOMETRY OF CELLULAR CONSTITUENTS OF LEPTOSPIRES MORRIS D. SCHNEIDER AND JOSEPH McLAUGHLIN, JR. Third Army Area Medical Laboratory, Fort McPherson, Georgia, and the Division of Biochemistry, Army Medical Service Graduate School, Walter Reed Army Medical Center, Washington, D. C. Received for publication December 30, 1954 Infrared spectrophotometry has proved useful in studies of whole bacteria (Stevenson and Boldaun, 1952; Levine et al., 1953a) and typespecific and glycogen-like bacterial polysaccharides (Levine et al., 1953 a, b, c, d). An investigation of the immunochemistry of certain serogroups of leptospires has been undertaken and data pertaining to this subject reported (Schneider, 1953, 1954a, b). The purpose of this communication is to describe reproducible differences in infrared spectroscopic findings of complement-fixing fractions of selected leptospires which may (a) shed light on the number of complement-fixing antigen systems, (b) serve to identify the fractions, and (c) provide a rapid supplement to the classical serological methods for distinguishing members of the Leptospira group. MATERIALS AND METHODS Complement-fixing fractions. Strains of the leptospires are from the Leptospira Type Collection of the Veterinary Division, Army Medical Service Graduate School, Walter Reed Army Medical Center, Washington 12, D. C. Cell-free fractions were isolated from LeptoVira icterohaemorrhagiae AB (Wijnberg), Leptospira canicola, and Leptospira bataviae. A flow chart of their preparation, chemical composition, and serological reactivity may be found in the previously cited references. Briefly, the cellular constituents are nondialyzable materials, contain pentose, and react very well in the complement fixation test. Distinctive polysaccharide containing substances, represented by Fractions 1 and 2, comprise approximately 90 per cent of the complement-fixing principle of the leptospire. Approximately 50 per cent of the nitrogen and virtually all of the protein comprise the preparation designated Fraction 3. Physical methods were not suitable for appraising the purity of the preparations since they contained insoluble materials. Infrared pectra. Approximately 1 to 10 mg of each fraction, representing 1 to 10 thousand test doses of the complement-fixing principle, were suspended in water, spread on a silver chloride disk, and allowed to dry. Spectra were determined in the 2 to 15 microns (ju) region by means of a recording Perkin-Elmer Infrared spectrophotometer, Model 21. Complement fixaion test. Preparations in this study were tested against hyperimmune rabbit serums by means of a semiquantitative complement fixation procedure (Kolmer et ai., 1951). RESUILTS Infrared spectroscopy. Intense absorption regions of the complement-fixing antigens were observed at 2.8 to 3.1, 3.45, 5.7 to 6.95, and 9.0 to 9.75,p. Fraction 1. Aqueous cell-free extracts of L. icterohaemorrhagiae and L. bataviae show absorption bands at: 3.0, 3.45, 6.25, 6.90, 8.10, and 9.35,. The band at 3.0,p is a composite of NH, OH, and CH hydrogen stretching. The NH and OH may be in associated or unassociated form. The absorption at 3.45ju has been attributed to CH stretching and the band at 6.25 p to a carboxylate structure and zwitterion type. The band at 6.9,p has been attributed to carbon-hydrogen deformation such as CH2 and CH3. The 9.35 pu band is of a group of absorptions which occur between 9.0 and 9.75 p and appears to be related to polysaccharide linkages (Whistler and House, 1953). Fraction 2. The absorption of the alcoholic cell-free extract has become much stronger in the 3.45,p region. A carboxyl peak is present at 5.7 to 5.8 p. Note its absence in Fraction 1. The peaks at 6.0 to 6.75 p are more numerous, suggesting basic similarities to Fraction 1. The band at the 9.0 to 9.75,u region shows several smaller peaks, indicating that a 87

88 M. D. SCHNEIDER AND JOSEPH McLAUGHLIN, JR. [VOL. 70 LEPTOSPAEIR ACAE LEPTOSPI# F>CI 00 FRACT I FRACTION 2 FRMTIOFE 2 2 50 I FRA~~~CTION 3 5?1 RACTIO10a 2t.5 5.5 8.

2 88 M. D. SCHNEIDER AND JOSEPH McLAUGHLIN, JR. [VOL. 70 LEPTOSPAEIR ACAE LEPTOSPI# F>CI 00 FRACT I FRACTION 2 FRMTIOFE I FRA~~~CTION 3 5?1 RACTIO10a 2t T52X S LE"osPIRA CANICOLA FRACTION I S kveleg6th, IN MICRONS Figure 1. Infrared spectroscopy of leptospiral preparations. Dissimilarities in absorption spectra of Fraction 1 of each of Leptospira icterohaemorrhagiae, Leptospira canicola, and Leptospira bataviae are illustrated in curves 533, 530, and 560. Distinctive characteristics of Fraction 2 of each of L. icterohaemorrhagiae and L. bataviae are shown in the spectral bands identified by numbers 511 and 572. Recognizable contributions of polysaccharide materials are indicated by strong absorptivities of Fractions 1 and 2 in the 9.35 microns region. Spectra of the protein containing Fraction 3 of each of L. icterohaemorrhagiae and L. bataviae (curves 507 and 574) are nearly identical. See the text for discussion of the absorption bands. more complex saccharide is possibly present in Fraction 2. Fraction S. The preparations illustrate spectra typical of proteins. They show no new points of interest. The spectral bands occur at 3.0, 6.1, and 6.5 I in each of the preparations of L. icterohaemwrrhagiae and L. bataviae. Fraction 1 of L. canicola. A complete evaluation of the spectral data of L. canicola cannot, unfortunately, be made. A single preparation of Fraction 1 which was available, however, has been studied. It possesses a peak at 3.0,u and is attributed to NH and OH stretching vibrations; the band at 5.8 A is derived from a carboxyl group. The latter band may be seen in the spectra of Fraction 2, but a weaker band at 3.45 u is the same as in TABLE 1 Serological relationship of cell-free extracts of Leptospira canicola as tested with hyperimmune serums prepared against members of the Leptospira group Hyperimmune Serums Antigen Fraction 1 I Fraction 2 Density (ug) per complement fixation tube Complement fixation end points L. andaman L. semeranga L. hyos L. bataviae L. pomona L. djasmin L. sentot L. autumnalis strain FBF L. australis strain A L. ballum L. canicola L. icterohaemorrhagiae L. australis strain B... L. pyrogenes L. grippotyphosa L. medanensis L. hebdomadis L. sejro... 4 L. saxkoeking L. bovis-palestine. 4 8 L. biflexa * Figures are the reciprocals of the highest dilution of antiserum reacting with the density (jzg) of antigen indicated. Hyperimmune serums were diluted 1:4, 8, 32, 128, and 512. Fraction 1 of each of L. bataviae and L. icterohaemorrhagiae. The 9.3,u band of Fraction 1 of L. canicola is well developed. Since the absorbance at 12.3 I is weak, it is possible that Fraction 1 of L. canicola contains a polysaccharide structure intermediate between those found in the polysaccharide containing fractions of the other serogroups studied. The distinctive spectra are shown in figure 1. Complement fixation reactivity. The carbohydrate containing preparations of L. canicola reacted in complement fixation tests preferentially with homologous hyperimmune rabbit serum. Group reactivity of Fraction 1 of L. canicola (table 1) is similar to that reported

1955] INFRARED SPECTROPHOTOMETRY OF LEPTOSPIRES 89 TABLE 2 Fraction 2 antigen of Leptospira canicola reacts poorly in the complement fixation test with hyperimmune serums against heterologous

3 1955] INFRARED SPECTROPHOTOMETRY OF LEPTOSPIRES 89 TABLE 2 Fraction 2 antigen of Leptospira canicola reacts poorly in the complement fixation test with hyperimmune serums against heterologous serogroups of leptospires Hyperimmune Serums Antigen Diluted 1: L. icwokasmor- L. canicola L. bataviae rhagias~ ~ ~ ~.baaea Complement fixation end points 2.5 < < <15 10 < <15 20 < <15 40 < <15 80 < < < < <15 15 <15 * See table 1 for explanation. (Schneider, 1954b) for L. icterohaemorrhagiae. The ability of Fraction 2 to distinguish group reactive antibodies was poor (tables 1 and 2). It is apparent that a genus-specific principle is asociated with Fraction 1 although it illustrated a preference for reacting with homologous hyperimmune serum. The complement fixation antigen system in Fraction 2 appears to be speciesspecific. DISCUSSION Comparatively small infrared spectral differences are found in whole bacteria of the Enterobacteriaceae group (Levine et al., 1953a). The infrared spectra of eighteen strains of Salmonella, for example, illustrated a constant pattern of bands. In this study a unique opportunity has been afforded to apply infrared spectroscopy to arbitrary cell-free constituents rather than the whole leptospire. This discussion is restricted to an analysis of the spectral bands of the selected serotypes investigated. Similarly prepared chemically complex cellular constituents of L. bataviae and L. icterohaemorrhgiae exhibit well characterized, significantly dissimilar and reproducible absorption bands, the full interpretation of which will depend on future research. Chemical functional groupings may be distinguished by infrared spectrophotometry. Proteins, for example, illustrate strong bands at 3.0, 6.1, and 6.5 p. Monosaccharides and monosaccharide derivatives and glycogen absorb in the 9.0 to 9.75 region (Whistler and House, 1953; Levine et al., 1953a; Schwartz et al., 1954). Fractions 1 and 2 of three serogroups of leptospires contain carbohydrate materials which are distinguished by strong absorbances in the 9.0 to 9.75 I region. Fraction 3 is low in carbohydrate but comprises 40 to 50 per cent of the cell's nitrogen content. The spectral bands are typical of protein. Likenesses in absorptivities of similarly prepared Fraction 3 materials of L. bataviae and L. icterohaemorrhagiae suggest that the protein moiety is a common denominator constituent. Nucleic acid absorption of enteric bacteria may be largely responsible for the 8.0 to 8.1 p band, according to Levine et al. (1953a). The cell-free aqueous extracts of each of L. bataviae, L. canicola, and L. icterohaemorrhagiae absorb very well in the 8.0 to 8.1Up region. Since desoxypentosenucleic acid comprises about 8 per cent of the density of Fraction 1, the spectral characteristics of this material appear to be consistent with the chemical data. The 8.0 to 8.10,p band is apparently attenuated or mising in each of Fraction 2 and 3 preparations of the leptospires studied. The polysaccharide containing preparations of the strains of leptospires investigated clearly illustrate uniquely characteristic infrared spectra in the 2 to 15 p range. The infrared spectral characteristics of the cell-free extracts supplemented the classical serological methods for distinguishing the serogroups, but the protein containing constituent appears not to have proved useful for this purpose. The distinctive bands of the polysaccharide containing fractions appear to be strong enough to be of practical value in exploring further the question of characterizing serogroups of leptospires by infrared spectroscopy. Criteria are not yet available for ascertaining the purity of selected leptospiral fractions. Their absence does not lessen the value of these observations. The characteristic infrared absorptivities indicate that substantial amounts of chemically complex materials comprise the leptospiral organism. The possibility that the propagation medium might have contributed artifacts to the spectra is precluded since the cells had been adequately washed prior to fractionation. Although chemical reagents are involved in disruption of the cells, preparations have been dialyzed and lyophilized and should contribute little to nonspecific spectral bands.

$9o M. D. SCHNEIDER AND JOSEPH McLAUGHLIN, JR. [VOL. 70 Since the yield of complementfixing principle of the combined fractions is 2- to 4-fold greater than Randall et al'8.$

4 9o M. D. SCHNEIDER AND JOSEPH McLAUGHLIN, JR. [VOL. 70 Since the yield of complementfixing principle of the combined fractions is 2- to 4-fold greater than Randall et al'8. (1949) whole cel antigen (Schneider, 1954a), there is no justification to suspect that the chemical reagents modified the infrared spectra but for unexplained reasons failed to degrade the complement-fixing principle. The infrared spectral bands have provided these facts: (1) three components of a leptospire imustrate spectra which are dissimilar and reproducible in duplicate preparations, and (2) protein containing constituents, although derived from distinct serotypes, are characterized by spectral similarities which are reproducible in duplicate preparations. It must be a remote concurrence for unspecific factors to contribute to spectral dissimilarities and likenesses in the same and different serogroups of leptospires. By comparing the infrared spectral characteristics of the leptospires with those of certain type-specific polysaccharides of pneumococci (Levine et al., 1953b) we have been afforded a possible criterion of purity. It is a coincidence that the soluble specific substances of certain of the pneumococcal types and the cell-free, polysaccharide containing extracts of the leptospires studied illustrate spectral resemblances. The significance of this observation is not understood. The infrared spectral data, however, suggest that the homogeneity attained in the leptospiral fractions compares reasonably well with the purity of pneumococcal polysaccharides by a different fractionation method (Brown, 1939). Finally, a limited investigation of antigenic potency in vivo in rabbits together with serological tests has disclosed that aqueous (Fraction 1) and alcoholic (Fraction 2) extracts of L. bataviae and L. icterohaemorrhagiae comprise immunologically distinct antigens (Schneider, 1955). The evidence provided by the chemical composition, immunological properties, and infrared spectral characteristics clearly distinguishes the cellular constituents of L. bataviae and L. icterohaemorrhagiae from each other. The obvious conclusion to be drawn is that the leptospiral fractions studied comprise independent antigen systems and that infrared spectroscopy has not only served to identify the components but also supplemented the serological typing in differentiating the serogroups. An unusual opportunity exists for extending the study of the spectral bands of members of the Leptospira group. A unified picture of the spectral characteristics of the cell-free, polysaccharide containing components may possibly aid in resolving serological differences and relationships among these spirochetes. ACKNOWLEDGMENTS The authors' thanks are due to the Departments of Veterinary Bacteriology and Veterinary Clinical Pathology for supplying suspensions of leptospires, hyperimmune serums, and performing complement fixation tests. SUMMARY Cellular constituents of Leptospira icterohaemorrhagiae strain AB (Wijnberg), Leptoepira batavie, and Leptospira canicola illustrated distinctive infrared spectra. Although this investigation has been restricted to a selected number of serotypes, the technique of infrared spectroscopy appears to be of potential value as a rapid supplement to classical serological methods for distinguishing and classifying members of the genus Lepto8pira. Extension of this type of study of the spectral bands of the cell-free, polysaccharide containing constituents may aid in resolving both serological differences and relationship among this group of spirochetes. REFERENCES BROWN, R Chemical and immunological studies of the pneumococcus. V. The soluble specific substances of Types I-XXXII. J. Immunol., 37, KOLMER, J. A., SPAULDING, E. H., AND ROBINSON, H. W Approved laboratory technic. 5th Ed. New York, Appleton-Century-Croft, Inc., p LEVINE, S., STEVENSON, H. J. R., CHAMBERS, L. A., AND KENNER, B. A. 1953a Infrared spectrophotometry of enteric bacteria. J. Bacteriol., 65, LEVINE, S., STEVENSON, H. J. R., AND KABLER, P. W. 1953b Qualitative studies of pneumococcal polysaccharides by infrared spectrophotometry. Arch. Biochem. and Biophys., 45, LEVINE, S., STEVENSON, H. J. R., TABER, E. C., BORDNER, R. H., AND CHAMBERS, L. A. 1953c Glycogen of enteric bacteria. J. Bacteriol., 66,

1955] INFRARED SPECTROPHOTOMETRY OF LEPTOSPIRES 91 LEVINE, S., STEVENSON, H. J. R., AND BORDNER, R. H. 1953d Identification of glycogen in whole bacterial cells by infrared spectrophotometry.

5 1955] INFRARED SPECTROPHOTOMETRY OF LEPTOSPIRES 91 LEVINE, S., STEVENSON, H. J. R., AND BORDNER, R. H. 1953d Identification of glycogen in whole bacterial cells by infrared spectrophotometry. Science, 118, RANDALL, R., WETmoRE, P. W., AND WARNER, A. R Sonic-vibrated leptospirae as antigens in the complement-fixation test for the diagnosis of leptospirosis. J. Lab. Clin. Med., 34, SCHNEIDER, M. D Properties of a serologically active substance from Leptospira icterohaemorrhagiae AB (Wijnberg). Proc. Soc. Exptl. Biol. Med., 82, SCHNEIDER, M. D. 1954a Isolation and chemical composition of complement-fixing antigens from leptospires. Proc. Soc. Exptl. Biol. Med., 85, SCHNEIDER, M. D. 1954b Polysaccharide antigens of leptospires. J. Infectious Diseases, 94, SCHNEIDER, M. D Antigenic potency of cell-free extracts of leptospires. Exptl. Parasitol., 4, SCHWARTZ, H. P., RIGGS, H. E., GLICK, C., Mc- GRATH, J., CHILDS, R., BEW, E., JR., AND STONE, F Infrared spectroscopy of liver glycogen in normal and irradiated adult and fetal rats. Proc. Soc. Exptl. Biol. Med., 85, STEVENSON, H. J. R., AND BOLDAUN, 0. E. A Infrared spectrophotometry as a means of identification of bacteria. Science, 116, WHISTLER, R. L., AND HousIE, L. R Infrared spectra of sugar anomers. Anal. Chem., 25, Downloaded from on October 3, 2018 by guest

Received for publication May 9, Since the absolute transmission values at any. disk, the film thickness, scattering of light and

Received for publication May 9, Since the absolute transmission values at any. disk, the film thickness, scattering of light and INFRARED SPECTROPHOTOMETRY OF ENTERIC BACTERIA SEYMOUR LEVINE, HEBER J. R. STEVENSON, LESLIE A. CHAMBERS, AND BERNARD A. KENNER Environmental Health Center, United States Public Health Service, Cincinnati,