1W Hl‘ ‘ mum THS 3773333355 cm 'E‘fiE i‘EXOTGXEC $AC‘?OR$ OF zfimgsma §>C>MQMA 3N WTRG AND :3: CVO ":ifi v. WI‘*“OW‘ and. mum “a. gem Thesis For fire Eegros a? M. S. MtCHEGAN SYATE UNEVER‘SWY Rabat“? Bruce Lacey, $11. 1963 THESIS LIBRARY Michigan State University . STUDIES ON THE EXOTOXIC FACTORS OF LEPTOSPIRA POMONA m VITRO AND 31 ovo BY Robert Bruce Lacey, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1963 ACKNOWLEDGEMENTS I wish to thank Dr. Ralph C. Belding, Associate Professor of Microbiology and Public Health, and Dr. Stuart D. Sleight, Assistant Professor of Veterinary Pathology, for their en- couragement and guidance throughout this investigation. My sincere appreciation to Miss Pauline Schiop, Grace Hospital, Detroit, Michigan, who first inspired my interest in pathogenic bacteriology, and to Mrs. Athalie Lundberg for her mental encouragement. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4 Hemolysin . . . . . . . . . . . . . . . . . . . 4 Electrophoresis . . . . . . . . . . . . . . . . 7 Gel filtration . . . . . . . . . . . . . . . . 8 Tissue culture technics . . . . . . . . . . . 9 Embryonating egg technics . . . . . . . . . . . ll Leptospires and the embryonating egg technic . . 12 MATERIALS AND METHODS . . . . . . . . . . . . . . . 13 Hemolysin Studies . . . . . . . . . . . . . . . 13 Organisms 13 Preparation of hemolysin 13 Alternative method of hemolysin preparation 14 Hemolytic activity assay method 15 Electrophoresis 16 Gel filtration 17 Electrophoretic analysis of the normal chicken serum fractions 20 Effect of pH on stability 21 Effect of storage on stability 21 Lipase analyses 21 Studies on Other Toxic Factors . . . . . . . . . 23 Organisms 23 Fractionation prOcedures 23 Tissue culture experimentation 24 Chicken embryo experimentation 26 Heat lability studies 27 Electrophoresis 27 Sterility controls 28 Treatment of the 60-80 per cent fractions with Sephadex G-50 ' 28 Lipase analyses 29 RESULTS . . . . . . . . . . . . . . . . . . . . . . 30 iii Page DISCUSSION . . . . . . . . . . . . . . . . . . . . . 51 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 60 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 62 iv LIST OF TABLES Table Page 1. Comparison of hemolytic activity of the euglobulin precipitate and supernatant fluid from the 0-40 per cent ammonium sulfate fraction of strain LW culture filtrate . . . . . . . . . . . . . . . . . 31 2. Hemolytic activity of ammonium sulfate fractions from strain LW, strain J and uninoculated Stuart's medium . . . . . . . . 37 3. Embryo lethality following yolk sac in— oculation of 56 C treated toxic ammonium sulfate fractions . . . . . . . . . . . . . 39 4. Embryo lethality following yolk sac in- oculation of culture filtrate ammonium sulfate fractions. (Trial #1) . . . . . . . 41 5. Embryo lethality following yolk sac in— oculation of culture filtrate ammonium sulfate fractions. (Trial #2) . . . . . . . 42 6. Embryo lethality following intravenous inoculation of culture filtrate ammonium sulfate fractions . . . . . . . . . . . . . 43 7. Lipase activity of various ammonium sulfate fractions . . . . . . . . . . . . . 44 Figure LIST OF FIGURES Page Protein analysis of the fractions of normal chicken serum after passage through.Sephadex G-200 . . . . . . . . . . . 45 Protein analysis of the fractions of crude hemolysin (strain LW 0—40 per cent ammonium sulfate fraction) after passage through Sephadex G-200 . . . . . . . . . . . . . . 46 Storage stability of hemolysin at 4 C and at -20 C . . . . . . . . . . . . . . . . 47 Measurement of hemolytic activity of the hemolysin in vitro with varying pH . . . . . 48 Electrophoretic migration patterns of the 60-80 per cent ammonium sulfate fractions in barbitone acetate buffer . . . . . . . . 49 Electrophoretic migration patterns of the 60-80 per cent ammonium sulfate fractions in Tris-E.D.T.A.-Boric Acid buffer . . . . . 50 Schematic representation of the structure of a lysolecithin . . . . . . . . . . . . . 54 vi INTRODUCTI ON Disease is defined as a definite state of ill health having a characteristic train of symptoms, involving the whole body or any of its parts, and with the etiology, pa— thology and prognosis either known or unknown. Microor- ganisms which incite disease within the host are called pa- thogens, and the ability of any pathogen to produce disease is called its pathogenicity. In order for an organism to cause infection it must first overcome the natural defenses of the host and must establish itself on or within the host and multiply (Raffel, 1961). The host presents a wide array of defenses against such in- vasions by microorganisms; likewise, the invading microbe may possess one or several aggressive systems to aid in gaining a foothold within the host. Hyaluronidase, fibrin- olysin and leukocidin are prominent examples. Toxins produced by microorganisms, either in the form of exotoxins or endo- toxins, kill or injure tissues and constitute perhaps the greatest threat to the health and well-being of the host. Classic examples of toxin producing microorganisms are Corynebacterium diphtheriae and Clostridium botulinum. Much has been learned about the pathogenicity and viru- lence of many microorganisms. The leptospires, however, still remain available for much investigation in this respect, for very little is known regarding their disease—producing characteristics. This statement has added significance when one considers that over fifty leptospiral serotypes have been described, three-fourths of which have been associated with disease in man (Alston and Broom, 1958). The first notable step in solving the key to leptospiral infections occurred in 1956 when a "hemolysin" was demon- strated in certain pathogenic serotypes. Injection of this hemolytic component into susceptible animals produced a syndrome comparable to the actual symptoms of the disease itself (Sleight and Langham, 1962). Other serotypes of pathogenic leptospires, however, do not produce any demonstrable ig_vitro hemolytic activity. Perhaps, then, the hemolytic factor is not solely operative in the course of the disease and an additional factor, or factors, may be present. The object of this study, therefore, was two-fold. First, to further characterize the "hemolysin" produced by certain leptospires, primarily through electrophoretic and gel filtration technics. Second, it was hoped that the presence or absence of toxic substances other than "hemolysin" could be demonstrated. LITERATURE REVIEW Leptospirosis Spirochetal fever was first described in 1886 as a febrile disease associated with jaundice and characterized by involve- ment of the spleen and kidney (Dubos, 1958). In 1915 Inada _§_§1, (from Alston and Broom, 1958) described Leptospira icterohemorrhagiae as the causative agent of Weil's Disease in man. Since Inada's first descriptions on the mode of infection, etiology, and specific therapy of "spirochaetosis icterohaemorrhagica" many workers have investigated the leptospires in an attempt to unravel the mysteries of their pathogenicity. Fukushima _£_§1, (from Van Theil, 1948) demonstrated the effects of a leptospiral toxin on guinea pigs. When introduced into the animal this toxin produced hyperemia and fever. Stavitsky (1948) could not find any trace of this toxin, nor could he find any detectible amounts of hyaluronidase, fibrinolysin, leukocidin or coagulase. A significant break-through occurred when Alexander g£_ a1, (1956) described the presence of a hemolysin in the supernatant fluids of certain leptospiral cultures. This hemolysin was water-soluble, non-dialyzable, thermolabile and oxygen—stable (Russell, 1956; Alexander gt a1., 1956). Levels of hemolysin were highest one to three days following optimal growth (Alexander §§_a1., 1956). These workers theorized the hemolytic activity was due to a toxin pro- duced by the organism. Russell (1956) noted that most hemolytic activity occurred at 37 C and that the hemolysin was heat—labile but unaltered by repeated freezing and thawing. This hemolysin easily lysed the erythrocytes of cattle, sheep and goats, but had no effect on guinea pig erythrocytes (Bauer and Morse, 1958). Dozsa §t_§;, (1960) determined the hemolytic activity of leptospiral hemolysin against the erythrocytes of 44 wild and domesticated animal species. Only the erythrocytes of the Pecora (red deer, roe deer, black buck, Mongolian gazelle, mouflon, Barbary sheep, goat, Alpine ibex, cattle, buffalo and zebu cattle) were susceptible. The effect of hemolysin on lambs was investigated by Kemenes (1958). A single intravenous injection of 150 to 200 m1 of the supernatant fluid of Leptospira pomona cul- tures, or repeated injections of smaller amounts, resulted in anemia. Bauer _§_§l, (1961), using concentrated and partially purified hemolysin, produced a hemolytic anemia in lambs evidenced by icterus and hemoglobinuria. Bauer §L_§L, (1961) also studied the l vivo hemolytic process following infection with washed L, pomona cells. This study indicated that hemolysin was associated with a leptospiremia, and hemolysis continued up to 2 days in the presence of agglutinins, suggesting that the hemolytic proc- ess was irreversibly established before antibody was produced. Rogols _L_§L, (1959) suggested that the hemolysin might be a phospholipase since phospholipids acted as inhibitors of hemolysin. Bertok and Kemenes (1960) demonstrated that leptospiral lipase activity is present in non—pathogenic and pathogenic strains. This enzyme had different properties than hemolysin. Lipase was more heat stable than hemolysin and appeared earlier and persisted longer in cultures. Like hemolysin, it could not be dialyzed. Lipase, specifically tributyrinase, activity was present in virulent serotypes, e.g., L, ictero- hemorrhaqiae, L, canicola, L, pomona, and others. Wolff (1954) found that pathogenic leptospires decreased in virulence upon repeated transfer in culture media. Bauer and Morse (1958) could find no correlation between hemolysin production and virulence. Bertok and Kemenes (1960) stressed that lipase and hemolysin production, p§£_§§, cannot be as- sociated with virulence. Most of the hemolytic activity associated with the growth of leptospires is found in the culture supernatant fluid (Russell, 1956). Imamura gL_§L, (1957), however, subjected L, icterohemorrhaqiae to sonic vibration and in- jected the cell-free portion of the sonic extract intra— cutaneously into rabbits and guinea pigs. Large, erythematous areas approximately 20—30 mm in diameter developed. The non-protein nature of the substance producing this erythema was apparent due to it its positive Molisch and Bial's orcinol reaction and its negative biuret reaction. Electrophoresis Electrophoresis is the movement of charged particles suspended in a liquid medium, under the influence of an ap- plied electric field (Overbreek and Lijklema, 1959) . A variety of supporting media is available to the investigator including starch gel, starch block, filter paper, agar gel and sponge rubber (Smith, 1960). Kohn (1957) introduced cellulose acetate as a supporting medium for electrophoresis. Cellulose acetate is advantageous in electrophoretic analysis since the time of the electrophoretic run is greatly de— creased from that of conventional filter paper strips. Also, adsorption is minimal, thus resulting in practically no "tailing." Fractions which do not separate well on filter paper may be demonstrated on cellulose acetate. Of partic— ular biological significance is the separation of serum proteins by this method (Putnam, 1960). Flynn and DeMayo (1951) and Owen (1956) have pioneered much work in the electrophoretic studies of serum protein. Aaronson and Gronwal (1957) identified twelve separate serum fractions using cellulose acetate electrophoresis. Gel filtration While electrophoresis is based on migration of particles in an electrical field, gel filtration is based entirely on the separation of substances according to their molecular dimensions. Sephadex (Pharmacia), composed of small grains of hydrophilic, insoluble cross-linked dextran, represents a great advance in molecular investigations (Pharmacia, 1959). Treatment of the sample undergoing gel filtration is excep- tionally mild since the only fractionating process occurring is one of filtration. Sephadex possesses the added advantage of automatic regeneration after the sample has passed through the column (Pharmacia, 1959). This is easily accomplished by washing the column thoroughly With an eluent, such as water or a suitable buffer. Sephadex is also useful in effecting a concentration of a given substance (Flodin, gL_§L,, 1960). Due to the hydrophilic nature of the gel, water is easily removed and the activity of the substance undergoing concentration remains unaltered. The use of Sephadex also eliminates lengthy dialysis procedures such as those inherent in the preparation of fluorescent antibody reagents (Killander, _L_§L,, 1961). Sephadex has been used successfully for the purification of a variety of substances which include virus suspensions (Matheka and Wittmann, 1961), fractionation of human sera (Epstein and Tan, 1961), enzyme preparations (Gelotte and Krantz, 1959), and rattlesnake venom (Bjork and Porath, 1959). Tissue culture technics Tissue culture, pg£_§g, is the growth and maintenance of cells L§_vitro on a suitable supporting menstrum (Cunningham, 1960). As early as 1913 Steinhardt §L_§L, (from Dubos, 1958) demonstrated that vaccinia virus survived several weeks in a tissue suspension made from corneas of rabbits and guinea pigs. Rous and Jones (1916) devised a method for the prepara- tion of suspensions of tissue cells by treatmentwith trypsin. 10 This treatment facilitated the growth of monolayer cultures and introduced uniformity into tissue culture preparations by insuring an approximately equal number of cells in each culture flask. Youngner (1954) observed better results in preparing tissue cultures by subjecting cells undergoing trypsinization to mechanical agitation. Dosser (1961) stressed the importance of the inoculation of the optimum number of cells for a given amount of medium as a strict prerequisite for obtaining suitable monolayer cultures. Fischer eL_§L, (1948) noted that embryo extract alone could not furnish all substances necessary for normal cell growth. Ebeling (1921) used a medium containing fibrinogen, serum, and tissue juice and noted that the cells grew as well in this mixture as in plasma and embryo extract. Baker (1929) suggested that a definite oxidation-reduction potential in the medium might be as important for the con- tinuous proliferation of fibroblasts as a given hydrogen ion concentration or osmotic pressure. Baker and Carrel (1925) have shown that the growth- inhibiting action of serum was due largely to serum lipids. Swim and Parker (1955) reported storage of HeLa cells at 4 C for six weeks without replacement of nutrient during stor- age and even several days after the cells were returned to 37 C. ll Antibiotics have had great importance in tissue culture investigations. When present in the growth and maintenance media, they help prevent bacterial contamination and allow greater availability of number and size of tissue cultures (Goldberg, 1959). Penicillin and streptomycin are still the antibiotics of choice and are commonly added in concentrations of 100,000 units per liter and 1,000 mg per liter, respective- ly (Goldberg, 1959; Cunningham, 1960). Embryonating_egq technics The history of embryonating egg technics dates back to nearly the same time as the first pioneering efforts in tis- sue culture. Rous and Murphy (1911) used embryonating avian eggs to study chicken sarcomas. Gay and Thompson (1929) noted an increase in vaccinia virus introduced into the yolk sac of a developing embryo. Woodruff and Goodpasture (1931) successfully infected the chorio-allantoic membrane with fowl- pox virus. Goodpasture and Buddingh (1933) and Stevenson and Butler (1933) nearly simultaneously reported the production of a vaccine against smallpox which was prepared from ex- tracts of infected chorio-allantoic membrane. 12 Leptospires and the embryonating egg technic The leptospires and embryonating egg technics have only recently been united into a feasible research tool. Most of the research has been limited to studies on newly hatched t 2;, (1955) experimented with chicks. However, Byrne embryonating chicks by injecting live leptospiral cultures into the yolk sac of l7-day—old embryos. Upon hatching, the leptospires could be recovered in blood or urine cultures. These chicks died four to five days after hatching. Injec- tion of 1—day-old chicks with L, canicola produced a lepto- spiremia of approximately 28 days duration. During this time, no other evidence of leptospirosis was observed. Ringen and Okazaki (1956) studied 2-day-old chicks to deter- mine their susceptibility to infection. Hoag §L_§L, (1953) reported that a leptospiremia persisted in all chicks ex— amined through the ninth day following inoculation and often as late as the twenty-first day. MATERIALS AND METHODS A. Hemolysin Studies Organisms Two strains of Leptospira pomona obtained from stock laboratory cultures were used in these experiments. The Lethal Wickard strain (LW), originally isolated in 1956, was employed in the production of hemolysin. Johnson (J), a non- hemolysin-producing strain, served as a control culture. Large volumes of culture supernatant fluid were obtained by inoculating 5 ml of a 7-10 day culture into 450 ml Stuart's medium (Difco) containing 50 m1 sterile, inactivated rabbit serum. The cultures were incubated at 29 C for approximately 14 days, or 1-2 days after maximum turbidity was observed. The organisms were then removed by positive pressure through a Seitz-type filter. Preparation Q§_hemolysin Hemolysin was precipitated from the culture filtrate by 40 per cent saturation with ammonium sulfate at 4 C for 12 hours (Bauer_g§_§L., 1961; Carter, 1957). The precipitate was harvested by centrifugation in the cold at 10,000 rpm for 30 minutes in an I. E. C. Model HR-l refrigerated centrifuge l3 14 with an 858 angle head. The precipitate was resuspended to 5 per cent of the original culture volume in 0.15 M phosphate buffered saline (PBS), pH 7.2, ionic strength 0.15, and dialyzed in the cold against PBS until no sulfate ion could be detected in the dialysate when tested with a 10 per cent barium chloride solution. Merthiolate (Lilly) was added until a final concentration of 1:10,000 was reached and the solution cflfcrude hemolytic extract was stored at 4 C until further use. Alternative method g§_hemolysin preparation An alternative method of hemolysin preparation was also employed in these studies. The technics used were identical to those above except the crude hemolytic extract was dialyzed 2 hours against distilled water and 72 hours against frequent changes of 0.005 M phosphate buffer, pH 8.1, ionic strength 0.005, in order to precipitate the euglobulin portion of the hemolytic extract. Following this precipitation the con- tents of the dialysis tubing were centrifuged at 10,000 rpm for 15 minutes at 4 C, again in the 858 angle head. The re- sulting euglobulin precipitate and the supernatant fluid were each tested for hemolytic activity. 15 HemoLytic activity assay method The method of Bauer and Morse (1958) was employed for the assay of hemolytic activity and was as follows: A series of two-fold hemolysin dilutions was made in PBS to a total volume of 1 ml per tube. Dilutions ranged from 1:2 to 1:512. To each tube 1 m1 of a l per cent suspension of washed sheep erythrocytes was added. The tubes were incu- bated for 30 minutes in a 37 C water bath followed by 60 minutes at 4 C. The tubes were then centrifuged in an I.E.C. Model CL centrifuge with a 215 head for 5 minutes at 1,500 rpm to sediment the erythrocytes. The Optical density of the supernatant fluid was determined in a Bausch and Lomb Spectronic 20 colorimeter at 540 mu. In each instance a set of controls was employed. The positive hemolysis control consisted of 1 ml of a saponin solution plus 1 m1 of the erythrocyte suspension. The erythrocyte control consisted of 1 ml of PBS and 1 ml of the erythrocyte suspension. The optical density of the hemolysis control was assigned a value of 100 per cent hemolysis. The optical densities of the hemolysin dilutions were converted to per cent hemolysis. The reciprocal of the dilution producing 50 per cent hemo- lysis was designated as the number of hemolytic units (HU) per m1 of hemolysin. 16 Electrophoresis For the electrophoretic studies a Shandon Universal Electrophoresis Apparatus and VoKam Constant Voltage/ Con- stant Current D. C. Power Supply were used. Cellulose acetate strips (Colab), 2.5 x 12 cm, were used as the sup- porting medium. The principal electrophoretic buffer em- ployed was Oxoid barbitone acetate buffer (Colab) and was prepared by dissolving 8.8 grams of buffer crystals in one liter of distilled water. The pH of this buffer was 8.6 with an ionic strength of 0.07. Tris-E.D.T.A.-Boric Acid buffer (Aaronson and Gronwal, 1957) with a pH of 8.6 and an ionic strength of 0.05 was also used. An electrophoretic run consisted of applying 0.005 ml of the sample of crude hemolytic extract to a cellulose acetate strip impregnated thoroughly with buffer solution and fastened securely in position on the strip supports of the electrophorator. The sample was applied directly above the negative electrode. All electrophoretic analyses were carried out at constant amperage at a current level of one milliampere (1 mA) per strip. The runs were performed at 4 C to minimize heating effects. The duration of each run was 2 hours with the barbitone acetate buffer and 45 min— utes with the Tris-E.D.T.A.—Boric Acid buffer. At the end 17 of the designated times the current was switched off and the strips were removed with forceps and placed immediately in a 0.2 per cent Ponceau 8 (National Aniline) in 3 per cent trichloracetic acid solution and stained for approximately 10 minutes (Colab, 1960). The strips were thoroughly rinsed in a 2 per cent acetic acid solution to remove any unad- sorbed dye and dried between sheets of paper toweling. In cases where staining was insufficient with Ponceau S, a 0.001-.002 per cent Nigrosin stain was used (Smith, 1960; Colab, 1960). In an effort to determine the effects of electrophoresis on the hemolytic activity of the crude hemolytic extract a number of strips were removed from the electrophorator, sec- tioned into three equal pieces, and transferred directly to blood agar plates, consisting of blood agar base and de— fibrinated bovine blood. These plates were incubated at 37 C and observed a total of 48 hours for hemolysis. As a hemolysis control 0.005 ml of the crude hemolytic extract was introduced directly onto the surface of a con- trol blood agar plate and incubated under the same conditions. Gel filtration The gel filtration column was prepared by introducing approximately 30 grams of dry Sephadex G—200 into a 18 sufficiently large volume of PBS and allowing it to swell and settle. The excess buffer was decanted to remove the "fines" and the mixture was then introduced gradually into a 4 x 60 cm chromatographic column and allowed to remain undisturbed until the gel completely settled. A disc of filter paper was canfikflly laid in place on top of the gel to help prevent agitation of the column contents. A three liter reservoir containing PBS was placed above the column and connected to it by rubber tubing fitted with a clamp which served to regulate the flow of buffer into the system. Buffer was introduced carefully onto the top of the gel to a depth of approximately three inches. The flow rate of the column was adjusted so that the amount of buffer entering the top of the column was equal to the amount of fluid leav- ing the bottom of the column. The apparatus was allowed to undergo final settling by permitting the buffer to flow through the system overnight at 4 C. Before any analytical work could be performed the column required standardization to determine when molecules of a known size would leave the system. Normal chicken serum was chosen as the standardizing agent. The flow of buffer from the reservoir was stopped and the column was allowed to drain until the level of the l9 buffer was even with the filter paper disc covering the gel. Ten ml of chicken serum were carefully introduced onto the filter paper pad with a Pasteur pipette and allowed to absorb into the top of the column. When the level of sample in- troduced equaled the height of the paper disc the supply of buffer from the reservoir was carefully reintroduced into the system, again to a depth of about three inches. The flow rate was readjusted and the sample passed through the gel into collection tubes. All fractionation procedures, including chicken serum and hemolysin preparations, were carried out at 4 C. The various fractions resulting from the column treat- ment were collected in 18 x 150 mm test tubes contained in an R.S.Co Model 1205A fraction collector. These fractions were analyzed in a Bausch and Lomb Spectronic 20 colori- meter at 750 mu for their protein content as determined by the Lowry modification of the Folin technic (Lowry, t E13: 1951). Electrophoretic analysis of the normal chicken serum fractions Determination of the electrophoretic mobility of the resulting fractions of normal chicken serum following 20 treatment with gel filtration was also undertaken. Random samples of 0.005 ml each were taken from the collection tubes and subjected to cellulose acetate electrophoresis, using barbitone acetate buffer. At the end of the 2 hour electrophoretic run the strips were removed and stained for 12 hours in a 0.001-0.002 per cent Nigrosin stain. An identical amount of normal chicken serum was frac- tionated electrophoretically in the same manner and stained 10 minutes with Ponceau S. The strip of normal chicken serum and strips from the randomly selected fractions were then compared. Based on known molecular weights of various serum com- ponents (Putnam, 1960), as well as the electrophoretic mo- bility of the various fractions as compared to whole normal chicken serum, it was easily determined when different mo- lecular weight entities made their exit from the column. The methods used in standardizing the column were re- employed in the analyses of the crude hemolytic extract. In this case 10 ml of sample were introduced onto the column and fractions were collected and analyzed for pro- tein content. Those collection tubes having a high protein content were pooled and concentrated by pervaporation at 21 4 C. These preparations were then analyzed for possible hemolytic activity. Effect 9f_p§_gp_the stability 9§_hemolysin The stability of the crude hemolytic extract at various pH levels was also determined. Routine hemolytic assays were performed as before, except the pH of the buffer diluent and erythrocyte suspension was changed in each assay. pH values ranging from 5 to 9 were employed. Con- trol systems were subjected to the same pH variation. Effect 9f_storage 9p_the stabilipy.g§_hemolysin The stability of the hemolytic activity of the crude hemolytic extract was also examined. Samples were stored at 4 C and -20 C and examined for hemolytic activity at 7, l4 and 60 day intervals. Lipase determinations The protein fractions resulting from gel filtration of the crude hemolytic extract were analyzed for lipase activity. The test was performed in the following manner: (Sigma, 1961). l. A substrate consisting of 2.5 ml water; 3.0 ml Sigma Lipase Substrate, Stock No. 800-1; and 1.0 ml Tris LW , 22 Buffer, Stock No. 800-2 was prepared for each sample to be analyzed. 1.0 ml of sample was added to each substrate tube. Tubes were shaken vigorously for 5 seconds and in— cubated for 6 hours in a 37 C water bath. At the end of the incubation period the contents of each tube were poured into clean 50 ml Erlen- meyer flasks. 3.0 ml of 95 per cent ethyl alcohol were added to each flask, followed by the addition of 4 drops of thymolphthalein indicator. The contents of each test flask, plus a blank con- trol flask, were titrated against 0.05 N NaOH until a faint blue color appeared. The number of ml necessary to titrate the blank was subtracted from the amount necessary to titrate the sample. The difference between these two numbers represented the values of lipase present. The 0-40 per cent ammonium sulfate fractions from strain strain J and from uninoculated Stuart's medium were also analyzed for lipase activity in the same manner. 23 B. Studies 9p_0ther Toxic Factors Organisms Again, two strains of Leptospira pomona were chosen as test organisms. Strain LW and strain J were grown as before. Following maximum culture turbidity each culture was filtered through a Seitz-type filter to remove the organisms. Fractionation procedures Ammonium sulfate was added to each culture filtrate un- til a 40 per cent saturation level was reached. The solu- tions were then allowed to stand 12 hours at 4 C. The resulting precipitates were, as before, centrifuged at 10,000 rpm for 30 minutes at 4 C and resuspended to 5 per cent of the original volume in PBS. To the remaining fil- trates ammonium sulfate was added until a 60 per cent saturation level was achieved. Centrifugation and re- suspension of this precipitate were carried out in an iden- tical manner. Ammonium sulfate was again added to the remaining filtrates until an 80 per cent saturation level was reached, centrifugation and resuspension repeated, and, finally, ammonium sulfate was added to the remaining fil- trates until 100 per cent saturation was attained. These 24 procedures resulted in obtaining four fairly concentrated protein fractions from each culture. Uninoculated Stuart's medium containing 10 per cent inactivated rabbit serum underwent identical fractionation and served as a medium.control, supposedly void of any toxic factors. Each of the fractions was dialyzed against PBS until no trace of sulfate ion was detected in the dialysate. Each of the twelve fractions--four from the culture filtrate of strain LW, four from the filtrate of strain J and four from the uninoculated Stuart's medium--was assayed for hemolytic activity. Merthiolate was added and the solutions were stored at 4 C until further use. Tissue culture experimentation Bovine fetuses were obtained through commercial meat packing sources. The kidneys were removed aseptically and placed in Hank's balanced salt solution (BSS) (Cunningham, 1960) containing penicillin and streptomycin in suitable concentration. All solutions employed in tissue culture experimentation contained these two antibiotics as an added safeguard against possible bacterial contamination. 25 Cortical tissue was removed aseptically and placed in sterile petri dishes containing BSS. The tissues were then minced with scalpels, resulting in a finely diced tissue suspension. After repeated rinsings in BSS the suspension was placed in BSS containing 0.25 per cent trypsin 1:250 (Difco) and trypsinized 1 hour at room temperature. The cells were allowed to settle in the flask and the trypsin solution was carefully decanted. Fresh trypsin solution was added and the mixture was again trypsinized an addi- tional 30 minutes at 37 C. Following trypsinization the cells were washed in BSS and centrifuged at 1,500 rpm for 5 minutes. This washing and centrifugation procedure was repeated twice and the resulting mixture was filtered through sterile gauze. The filtrate was again centrifuged at 1,500 rpm for 5 minutes to sediment the cells. Approx- imately 0.4 m1 of the packed cells were added to 200 ml of growth medium, which consisted of BSS solution containing 0.5 per cent lactalbumin hydrolysate plus 10 per cent ster- ile bovine serum. Five m1 aliquots of this mixture were added to sterile 2—ounce prescription bottles, resulting in a total of 40 separate cultures. These were incubated at 37 C and observed routinely for monolayer formation. 26 Following monolayer formation the growth medium was re- moved and the cells were maintained by the addition of a maintenance medium consisting of BSS solution containing 0.5 per cent lactalbumin hydrolysate plus 2 per cent sterile bovine serum. The monolayers were then subjected to the following in- oculation procedures: Varying amounts, ranging from 0.1 ml to 0.5 ml, of the leptospiral culture filtrate fractions and the uninoculated Stuart's medium fractions were intro- duced onto the monolayers. A total of 36 monolayer cultures were inoculated. A set of controls was also employed in these experiments and consisted of introducing sterile Stuart's medium containing 10 per cent inactivated rabbit serum nto two monolayer cultures. The two remaining cul- tures were uninoculated. The cultures were then incubated further at 37 C and observed for cytopathic effects. Chicken embryo experimentation Embryonating chicken eggs were obtained through a com- mercial poultry raising concern. The two routes of inocu- lation chosen were yolk sac and intravenous. For yolk sac studies inoculations of 0.5 ml into 7-day—old embryos were em— ployed° Intravenous studies, utilizing 0.05 ml inoculums, were performed on 15-day-old embryos (Cunningham, 1960). In each trial a minimum of 10 embryos was inoculated with each of the 12 protein fractions prepared. This resulted in a total of 120 embryos for each trial. The eggs were incubated an additional 6 days in the case of the yolk sac inoculation route, and 5 days for the intravenous route in a Jamesway Model 258 incubator at 38 C and observed daily. Heat lability studies All fractions toxic to tissue cultures and/or embryonat- ing eggs were suspended in a 56 C water bath for 5, 10, 15, 20 or 30 minutes. At the end of the designated times an aliquot of each fraction was removed from the bath, cooled, and injected into embryos via the yolk sac route. The em— bryos were incubated and observed as before. Electrophoresis Fractions of the culture filtrates, other than the crude hemolytic extract, which were lethal to the tissue culture monolayers and/or the embryonating chicken eggs were ana- lyzed electrophoretically on cellulose acetate strips. The entire electrophoretic procedure employed in these partic- ular experiments was identical in all respects to that used in analysis of the crude hemolytic extract, including amount 28 of sample analyzed, time of electrophoretic run, applied field strength, buffers and staining technics. Sterility controls In all embryonic work a series of sterility controls were utilized. These consisted of inoculating an amount of sample, equal the amount injected into the embryonating chicken eggs, into thioglycollate broth (Difco) tubes. These tubes were incubated at 37 C and observed for 72 hours for evidence of bacterial growth. Treatment 9L_the 60-80 per cent fractions with Sephadex G—50 A 2.5 x 40 cm chromatographic column of Sephadex G-50 was prepared in a manner identical to that of the G-200 column used in the investigation of the hemolysin. Frac- tions of culture filtrates, other than the crude hemolytic extract, found to be lethal to embryos, were passed through this column at 4 C. Protein analyses of the resulting fractions were performed as before. The collection tubes under each protein peak were pooled and pervaporated at 4 C. The resulting concentrates were stored at 4 C until further use. 29 Lipase determinations The fractions resulting from the gel filtration of the 60-80 per cent culture filtrate fractions were analyzed for lipase activity. The same procedure used in the ana- lysis of the hemolysin fractions was re-employed in these particular studies. RESULTS Treatment of the various culture extracts and uninocu- lated Stuart's medium with ammonium sulfate gave rise to the following fractions: Strain LW_ 0-40 per cent ammonium sulfate fraction 40-60 per cent " " " 60-80 per cent " " " 80-100 per cent " " " Strain J_ 0-40 per cent " " " 40-60 per cent " " " 60-80 per cent " " " 80—100 per cent " " " Uninoculated 0-40 per cent " Stuart's medium 40-60 per cent " " " 60—80 per cent " " " 80-100 per cent " " " For purposes of clarification the results will be di- vided into two parts: First, the studies with crude hemo- lytic extract (strain LW 0-40 per cent fraction); and second, studies on the other toxic factors. Hemolytic activity pf the euglobulin precipitate 9f the 0-40 per cent ammonium sulfate fraction 9: strain LW_ Routine hemolytic assays demonstrated that almost all of the hemolytic activity associated with the crude hemolytic 3O 31 extract from strain LW was present in the euglobulin pre- cipitate. Hemolytic titers of this precipitate gave values of 128 HU/ml, while those of the supernatant fluid were only 2 HU/ml. Results are listed in Table 1. Table 1 Comparison of hemolytic activity of the euglobulin precipitate and supernatant fluid from the 0-40 per cent ammonium sulfate fraction of strain LW culture filtrate 1 Sample Hemolysin dilution in PBS (pH 7.2) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 Euglob. ppt 0 Super- natant + Greater than 50 per cent hemolysis - : Less than 50 per cent hemolysis Hemolysin studies Lg_tissue culture Bovine embryo kidney monolayers were completely destroyed within 24 hours following introduction of the crude hemolytic extract. The cells had been completely disrupted from the wall of the culture flask and were floating within the cul- ture medium. The phenol red indicator had not changed 32 appreciably from its original red color, thus indicating that very little, if any, cellular respiration had occurred. Control cultures in these experiments responded normally and continued to maintain themselves. Cells from these cultures appeared normal when stained with Giemsa stain, while those subjected to the hemolysin preparation pos- seSsed deeply stained pyknotic nuclei and shrunken cytoplasm. Hemolysin studies with embryonating chicken eggs Introduction of the hemolytic extract from the culture filtrate of strain LW, either by yolk sac or by intravenous injection, killed all embryos approximately 96 hours post- inoculation. Criteria for death were the apparent curling and dwarfing of the embryo, as well as lack of any visible motion. Death was verified in each case by gross examina- tion of the exposed embryo. Results are listed in Tables 4, 5, and 6. Those embryos dead within 24 hours postinoculation were considered traumatized. Controls employed in these experiments were identical to those used in the tissue culture studies. All control embryos were unaffected. 33 Standardization gf_the Sephadex G—200 column Analysis of protein fractions obtained when 10 ml of normal chicken serum was eluted from the column gave rise to three distinct protein peaks. The first peak included molecules having molecular weight of 200,000 or greater, while the second and third peaks contained molecules of approximately 200,000 to 160,000 molecular weight and molecules of less than 160,000 molecular weight, respectively. These findings were verified by electrophoresis of random samples chosen from collection tubes as compared to elec— trophoresis of normal chicken serum. It was ascertained from these studies that the first peak contained primarily alpha alpha and beta globulins, while the second peak 1' 2 contained a single beta globulin and gamma globulins. The third peak was composed primarily of serum albumin. The results are represented graphically in Figure 1. Gel filtration Q§_the crude hemolytic extract As seen in Figure 2, elution of the crude hemolytic extract from the column again led to three distinct protein peaks, as determined by the Lowry modification of the Folin technic. Analysis showed that hemolytic activity was pre- sent only in the fraction under the first peak. This 34 indicated the hemolytic activity was associated with a molecule, or molecular unit, of molecular weight 200,000 or greater. No other eluted fraction exhibited any hemolytic activity. Heat lability 9f_the hemolysin Experiments determining the heat lability of the crude hemolysin extract indicated that the hemolytic activity was destroyed by heating for 5 minutes at 56 C. Experi- ments with embryonating chicken eggs and routine hemolytic assay technics gave identical results in this respect in that no embryos were killed with heat-treated hemolysin, and no hemolytic activity was observed i§_vitro. Results are listed in Table 3. Storage stability Qf_the hemolysin §L_4_§_ Beginning with an original hemolytic titer of 256 HU/ml the titer dropped to 128 HU/ml at the end of 7 days and finally reached a low point of 16 HU/ml after 60 days. Re- sults are shown in Figure 3. Storage stability 9£_the hemolysin §L_-2 .g The original titer of 256 HU/ml dropped to 128 HU/ml after 7 days. At the end of 60 days the titer still 35 remained at 128 HU/ml. Results are show in Figure 3. pH_stability 9f_the hemolysin The pH range of hemolysin stability is between pH 7.0 and 8.0 with an apparent optimum value of pH 7.2. A sharp drop in hemolytic activfly occurred on either side of this value, as seen in Figure 4. Electrophoretic analysis 9f_the hemolysin Results in these experiments were inconclusive, in that no differences in the electrophoretic mobility of the 0-40 per cent ammonium sulfate fractions of strain LW, strain J or uninoculated Stuart's medium were apparent. Also, no hemolysis occurred when the electrophoretic strips containing the crude hemolytic extract were incu- bated on the blood agar plates. Hemolysin not subjected to electrophoresis, however, did produce a detectible hemolysis within 24 hours after its application to the control blood agar plate. Lipase activity gf_hemolysin Crude hemolytic extract from strain LW contained 7.1 Sigma-Tietz lipase units per ml. Of the three protein fractions resulting from the gel filtration of this crude 36 hemolytic extract significant lipase activity was demonstrated in the first of these three peaks. This indicates that the lipase has a molecular weight of 200,000 or greater. This first peak contained 7.2 Sigma-Tietz lipase units per ml, as opposed to 4.8 and 4.4 for peaks two and three, respectively. The control blank contained 4.3 Sigma-Tietz lipase units per ml. Values greater than 1.5 units over the value of the blank are considered significant. Lipase activity p§_the 0-40 per cent ammonium sulfate fractions 9f_strain g_and uninoculated Stuart's medium The 0—40 per cent fraction of strain J contained 4.9 Sigma- Tietz lipase units per ml. The identical fraction of uninocu- lated Stuart's medium contained 4.5 lipase units per ml. Results of the above data are presented in Table 7. Studies 9p_ammonium sulfate culture fractions other than the 0-40 per cent fraction from strain LW (crude hemolytic extract) Hemolytic activity 9£_the various fractions No hemolytic activity was observed in any fraction when investigated by routine hemolytic assay technics, as seen in Table 2. 37 Table 2 Hemolytic activity of ammonium sulfate fractions from strain LW, strain J and uninoculated Stuart's medium Culture , Ammonium sulfate concentration filtrate 0-40% 40-60% 60-80% 80-100% LW + — — — J _ - _ _ Uninoc. Stuart's - — - - medium Tissue culture studies A common toxic factor for bovine embryo kidney monolayer tissue cultures was found in the 60-80 per cent ammonium sulfate fractions of the culture fluids of both strain LW and strain J, but was not found in the 60—80 per cent frac- tion of uninoculated Stuart's medium. This toxic factor acted within 48 hours postinoculation. Criteria for cyto- pathic effect were identical to those employed in the studies of the effect of hemolysin on tissue cultures. 38 Embryonating egg studies The same toxic manifestation which appeared in the tis— sue culture studies was also observed in embryonating chicken eggs. The criterion here, as in the studies with hemolysin, was death of the embryo. Death resulted in an average of 7 out of a total of 10 untraumatized embryos with the 60-80 per cent ammonium sulfate fraction from strain LW, and in 5 out of 9 untraumatized embryos with the identical fraction of strain J. Death occurred approximately 96 hours post- inoculation. No such toxic effect was noted when the 60- 80 per cent ammonium sulfate fraction from uninoculated Stuart's medium was employed. A11 embryo controls and sterility controls remained normal. Heat lability studies Heating for 10 minutes at 56 C negated the toxic effect of all fractions. Controls consisted of embryos inoculated with unheated fractions. Sterility controls were also em- ployed. All were normal. The results are shown in Table 3. 39 Table 3 Embryo lethality following yolk sac inoculation of 56 C treated toxic ammonium sulfate fractions onium Length of heat treatment (minutes) Unheated sulfate t 1 fraction 5 10 15 2o 30 C0“ r0 3 LW 0-40%* 10/10 10/10 10/10 10/10 10/10 1/10 LW 60-80% 7/10 9/9 9/9 9/10 10/10 5/10 J 60-80% 7/10 10/10 10/10 8/9 9/9 4/10 *crude hemolytic extract Key: Numerator: Number of embryos surviving. Denominator: Total number of embryos inoculated (10) less those traumatized. Electrophoretic analysis In the electrophoretic analysis of the 60—80 per cent fractions of strain LW, strain J and uninoculated Stuart's medium there was no significant difference in their respective migration patterns when barbitone acetate buffer, ionic strength 0.07 was used. However, when the Tris-E.D.T.A.- Boric Acid buffer system, ionic strength 0.05, was used a line common to the 60-80 per cent fractions of both strain LW and strain J was observed. This line was not found in the 40 identical fraction of uninoculated Stuart's medium. Results of these electrophoretic examinations are shown in Figures 5 and 6. Lipase activity 9f_the 60-80 per cent ammonium sulfate fractions 9f_strain LW, strain J_and uninoculated Stuart's medium No significant lipase activity could be demonstrated in any of the protein peaks resulting from gel filtration with Sephadex G-50. Results are shown in Table 7. 41 Table 4 Embryo lethality following yolk sac inoculation of culture filtrate ammonium sulfate fractions (Trial #1) Culture Ammonium sulfate concentration filtrate 0-40% 40-60% 60-80% 80-100% LW 0/10 8/9 3/10 10/10 J 8/9 9/9 4/9 10/10 Uninoc. Stuart's 9/9 9/9 9/9 6/6 medium Key: NUmerator: Number of embryos surviVing. Denominator: Total number of embryos inoculated (10) less those traumatized. 42 Table 5 Embryo lethality following yolk sac inoculation of culture filtrate ammonium sulfate fractions (Trial #2) Culture Ammonium sulfate concentration filtrate 0-40% 40-60% 60-80% 80-100% LW 0/10 9/10 4/10 10/10 J 10/10 10/10 6/10 10/10 Uninoc. Stuart's 10/10 9/10 10/10 10/10 medium Key: Numerator: Number of embryos surviving. Dengminator: Total number of embryos inoculated (10) less those traumatized. 43 Table 6 Embryo lethality following intravenous inoculation of culture filtrate ammonium sulfate fractions Ammonium sulfate concentration Culture filtrate 0-40% 40-60% 60-80% 80—100% LW 2/5 5/5 3/5 5/5 J 4/5 5/5 3/8 4/5 Uninoc. Stuart's 5/5 4/5 5/5 5/5 medium Key: Numerator: Number of embryos surviving. Denominator: Total number of embryos inoculated less those traumatized. 44 Table 7 Lipase activity of various ammonium sulfate fractions Lipase activity ractlon (slgma-Tietz units/ml) Control blank 4.3 Gel filtrated hemolysin Protein peak #1 7.2 II II #2 4 . 8 II in $3 4. 4 0-40 per cent fractions Strain LW 7.1 Strain J 4.9 Uninoculated Stuart's medium 4.5 Gel filtrated 60—80 per cent fractions Strain LW Protein peak #1 4.3 II II #2 4. l H II #3 4. 3 Strain J Protein peak #1 4.3 II II #2 4. 4 " ” #3 4.1 Uninoculated Stuart's medium Protein peak #1 4.0 " " #2 4.3 II II #3 4 . 3 45 OHH OOH om H0985: was» cofluomum om on 00 om ov om oomlw xmpmnmwm QmDOHSu wmmmmmm Hmumm Esumm cwxoflso HmEHoc mo msofluomum mnu mo mflmhamcm samuoum H wnsmfim J 0.0 wuflmcmp Hmufludo m.o N.H OHH HOQESQ wasu sofluomum OOH m om On 00 om 0v om 4 A a d - a oomlw xmpmnmmm Amsounu mmmwmmm Hmumm Acofluumnm mummasm EDHQOEEM Howie 3Q Camuumv cflmhaoamfi OUDHU mo mcofluumuw on» m0 mammamsm camuoum m musmflm 0.0 muflmcwp amoeumo m.o 47 Figure 3 Storage stability of hemolysin at 4 C and at -20 C 2.4 — ‘ Log HU/m1* —-—- --—: Storage at 4 C : Storage at -20 C 1 l l 0.4 0.8 1.2 Log time (days) *HU/ml: hemolytic units/milliliter A 48 Figure 4 Measurement of hemolytic activity Lg_vitro with varying pH 2.0 F Log HU/ml* 0.4 ’ pH *HU/ml: hemolytic units/milliliter 49 Figure 5 Electrophoretic migration patterns of the 60-80 per cent ammonium sulfate fractions in barbitone acetate buffer. A = strain LW; B = strain J; C a uninoculated Stuart's medium 50 Figure 6 Electrophoretic migration patterns of the 60-80 per cent ammonium sulfate fractions in Tris-E.D.T.A.-Boric Acid buffer A = strain LW; B = strain J; C = uninoculated Stuart's medium DISCUSSION Studies 9Q the crude hemolytic extract Based on the analysis of crude hemolytic extract from the culture fluid of strain LW by Sephadex G-200 gel fil- tration a molecular weight of 200,000 or greater has been assigned to the hemolysin molecule, or molecular unit. From evidence presented here one cannot definitely state whether the hemolysin is a single molecule or a biological dimer or polymer. There may be a single, large molecule to which is attached a smaller "schlepper" unit, the two of which work in combination to produce the hemolytic activity demonstrated. It is not beyond reason to assume that the hemolytic activity may be manifest in an aggregation of several smaller biologi— cal monomers, the total molecular weight of which is greater than 200,000. Ultracentrifugation studies may be of benefit in settling this dispute in that a single, large sedimenta- tion peak followed by one or more trailing peaks would indicate the presence of a relatively large molecule with several small molecules attached. Certain studies have led to the conclusion that the hemolysin may be a molecular unit, rather than a single molecule. Sleight and Dardas (1960, unpublished data) 51 52 subjected the crude hemolytic extract to DEAE cellulose chromatography. Two of the resulting fractions, one fairly large and the other a smaller trailing fraction, both pos- sessed hemolytic activity when analyzed by the standard hemolytic assay method. This apparently indicates that a fairly readily adsorbed molecule, as well as a less readily adsorbed molecule, are active in the production of hemolytic activity. These results, however, were not repeated with any degree of significance. Detection of hemolysin using cellulose acetate electro- phoresis rendered no concrete findings. No hemolytic activity was found in any of the resulting fractions. Here it seems possible that, if a molecular unit existed, the presence of an applied electric field induced the separation of the molecular species, resulting in the inactivation of the hemolysin. The possibility also exists that the hemolysin may have migrated completely off the electrophoretic strip during the course of the run. That the hemolytic unit is of large molecular dimensions is shown both in the studies with Sephadex G-200 and by the presence of hemolytic activity in the euglobulin precipitate of the 0—40 per cent ammonium sulfate fraction of strain LW, as opposed to the pseudoglobulin supernatant. Euglobulins 53 have molecular weights of upwards of 1,000,000 (Fruton and Simmonds, 1958). The fact that a greater percentage of hemo- lytic activity is found within this euglobulin precipitate is in disagreement with Bauer (1958), but its demonstration in these current studies is of a definite correlative nature. In previous unpublished studies (Belding and Lacey, 1962) the antigenicity of the hemolytic unit was tested in rabbits with rather unencouraging results. Two out of five rabbits inoculated with the hemolytic extract produced only a slightly detectible amount of antibody to the hemolysin as measured by the Ouchterlony technic (Ouchterlony, 1948). The possibility of performing L-plate studies as further correlative evidence of the molecular size of the hemolysin was not considered feasible due to the lack of knowledge regarding relative concentrations of the antigen (hemolysin preparation) and antibody (rabbit anti—hemolysin antiserum). Lillevik (1962, personal communication) suggested that the hemolysin may be a lysolecithin. Studies to determine the' validity of this assumption were not performed, but it is known that lysolecithins are powerful hemolytic agents which rapidly lyse erythrocytes, and are considered responsible for the harmful physiological effects of certain snake venoms (west and Todd, 1962). 54 The basic structure of a lysolecithin, however, does not endorse a chemical relationship between a lysolecithin and the hemolytic phenomenon associated with certain leptospiral serotypes. Sephadex gel filtration has indicated that the hemolysin unit has a molecular weight of 200,000 or greater. The basic structure of a lysolecithin is given below in Figure 7. Figure 7 Schematic representation of the structure of a lysolecithin cHZOH R —-cooca o l + CHz-O-P-O-(CH2)2-N(CH3)3 OH Exclusive of the R—group, the molecular weight of the above compound is 285. If hemolysin is a lysolecithin, and pose sesses a molecular weight of 200,000 or greater, then the dimensions of the R—group must be astronomical. Logic erases the possibility of an R—group of such proportions. Also, lysolecithins are not precipitated by ordinary protein precipitation technics. 55 The possibility exists that the hemolysin is a muco- protein but this seems unlikely in that mucoproteins, too, are not easily precipitated by ordinary protein fractionat- ing methods, and are quite heat stable (White eL_aL,, 1959). The demonstration of leptospiral lipase activity by Berték and Kemenes (1960) has been confirmed. This ac— tivity was noted only in the culture filtrate from strain LW, a hemolysin producer, and not in the culture filtrate from strain J, a non—hemolysin producer. The above results would tend to implicate the lipase directly with the hemo- lysin suggesting, perhaps, that the hemolysin and the lipase are the same chemical entity. Certain lipases, specifically phospholipase A, are known to be powerful hemolytic agents (Fruton and Simmonds, 1958). However, Bertok and Kemenes (1960) noted that the lipase had properties which differed markedly from the hemolysin, e.g., the greater heat stability of the lipase, and its earlier appearance and longer per- sistence in culture filtrates. Also, lipase activity has been demonstrated in both pathogenic and non-pathogenic strains. The possibility exists that the hemolytic activity may be due to the mutual effect of a lipase and some other loose- ly bound chemical component. In non—hemolytic and in non- pathogenic strains only lipase activity is present. Lack 56 of hemolytic activity may be due to the failure of these organisms to produce this additional chemical component. In the gel filtration of the crude hemolytic extract both lipase activity and hemolytic activity occurred in one protein fraction alone. Since Sephadex avoids the use of ion—exchange principles and involves only a filtration proc- ess, the intact "lipase-component” unit produced both lipase activity and hemolytic activity. When subjecting this "lipase-component" unit to ion exchange resins or to elec- trophoresis the ”lipase—component” unit may be disrupted, leaving only lipase activity present. This is a tempting speculation and warrants further biochemical investigation. In previous experiments, as well as in the present work, the degree of purity of the hemolysin was questionable. For example, when beginning with one liter of culture filtrate containing the hemolysin, and subjecting it to the fraction- ation, chromatographic and concentration methods employed in these experiments, a final volume of two ml of "concentrated hemolysin" resulted, based on Lg_vitro hemolytic assay methods. The exact chemical nature of the hemolysin has not been undertaken in these studies, primarily due to the insuf- ficient quantities of purified hemolysin available. Once obtained in a relatively pure form the basic chemical 57 properties of the hemolysin should be studied. Amino acid analyses and ultracentrifugation studies would be adequate starting points in this respect. Studies 2p other toxic factors The original impetus for this work came from the ex- periments of Lundberg (1962, unpublished data). After extraction of the hemolysin from the culture filtrate, ali— quots of approximately 10 ml of the remaining filtrate were injected intraperitoneally into young lambs. These lambs were observed daily for any possible toxic effects of the culture filtrate. Only slight temperature elevations were observed during the testing period. These were attributed to proteins in the rabbit serum medium supplement. It seemed feasible, however, that other toxic factors could be present and that effects were being masked due to dilution in such a large volume. It was decided to further fractionate this remaining filtrate and concentrate the resulting fractions in a manner similar to the methods used in the preparation of hemolysin. The presence of a toxic factor in the 60-80 per cent ammonium sulfate fraction of both strain LW and strain J has been demonstrated. This factor was lethal to tissue 58 cultures and embryonating chicken eggs. This fact that this toxic factor was lethal to these Egg assay systems, however, does not mean that this same toxic effect would be operative in another system; namely, an animal infected with either strain LW or strain J of Leptospira pomona. Under the con- ditions of experimentation used in this study, however, the experimental results with the embryonating eggs were re- peated with nearly identical findings, thus indicating some degree of significance. The electrophoretic analyses using cellulose acetate as the supporting medium gave mixed results. Use of the barbi- tone acetate buffer system produced electrophoretic strips with nearly identical migrational patterns, within experi- mental limits. However, when a buffer system of lower ionic strength was employed a new protein band was noted which was common to both strain LW and strain J. This fraction mi- grated slightly ahead of the albumin fraction and was not present when the same fraction of uninoculated Stuart's medium was tested. This new band may be a serum pre-albumin although, if this were the case, it should also appear in the uninoculated medium. On the basis of these findings it is possible that this new band could be the toxic factor pres- ent in these culture filtrate fractions. 59 One might question whether the death of the tissue cultures and the embryos could be due to incomplete dialysis of the culture filtrate fractions. This is unlikely, how- ever, since dialysis was carried out identically for each of the culture filtrate fractions, as well as for fractions of the uninoculated Stuart's medium. If dialysis was not complete, and the death of the tissue cultures and embryos was due to the presence of ammonium sulfate, the uninocu— lated Stuart's medium should also have been lethal to the assay systems. This, however, was not the case. Again, the exact nature of this toxic factor may well be determined once a fairly pure sample is obtained. In addition to biochemical analyses, a thorough study on its gross pathological and hematological effects should be under- taken. At present, work is in progress attempting to fractionate this toxic factor further through use of gel filtration technics. Also, in addition to the further studies on the exotoxic factors of the leptospires an investigation into the realm of possible endotoxic factors should be performed. SUMMARY Further characterization of the hemolysin from strain LW of Leptospira pomona has been carried out, primarily through gel filtration and cellulose acetate electro- phoresis. On the basis of the gel filtration experi- ments a molecular weight of 200,000 or greater has been assigned to the hemolysin. This hemolysin has also been shown to be a euglobulin which is stable at or near pH 7. The hemolysin is destroyed by heating for 5 minutes at 56 C, and also by electrophoresis. An alternative method of hemolysin preparation, in- volving euglobulin precipitation technics, has been presented. This method of preparation resulted in obtaining a more concentrated hemolysin. A lipase with a molecular weight of 200,000 or greater has been observed in the 0-40 per cent ammonium sulfate fraction of strain LW. No lipase activity was noted in analogous fractions of strain J or uninoculated Stuart's medium. 60 61 Studies on other possible leptospiral exotoxic factors have been undertaken through electrophoresis and also by tissue culture and embryonating egg technics. The presence of a toxic factor, other than hemolysin, present in the 60-80 per cent ammonium sulfate fractions of culture filtrates of both hemolytic and non-hemolytic strains of L, pomona has been demonstrated. The assay media for this study were bovine embryo kidney mono- layers and embryonating chicken eggs. This toxic effect was not noted in the identical ammonium sulfate fraction of uninoculated Stuart's medium. Further biochemical investigation into these toxic factors is suggested. BIBLIOGRAPHY Aaronson, T. and A. Gr6nwal. 1957. Improved separation of serum proteins in paper electrophoresis. Scand. J. Clin. Lab. Invest., 25 338. Alexander, A. A., O. H. Smith, C. W. Hiatt and C. A. Gleiser. 1956. Presence of hemolysin in cultures of pathogenic leptospires. Proc. Soc. Exptl. Biol. Med., 2L: 205-211. Alston, J. M. and J. C. Broom. 1958. "Leptospirosis in Man and Animals," lst ed., E. and S. Livingstone Ltd., London. Baker, L. E. 1929. The chemical nature of the substances required for cell multiplication. II. The action of glutathione, hemoglobin, and ash of liver on the growth of fibroblasts. J. Exptl. Med., 42; 163. ----- , and A. Carrel. 1925. Lipoids as the growth inhib- iting factor in serum. J. Exptl. Med., fig; 143. Bauer, D. C. and E. V. Morse. 1958. Variation and hemo- lysin production in relation to virulence of Leptospira pomona. Proc. Soc. Exptl. Biol. Med., 28; 505-508. ----- , L. N. Eames, S. D. Sleight and L. C. Ferguson. 1961. The significance of leptospiral hemolysin in the patho- genesis of Leptospira pomona infections. J. Infect. Dis., 108: 229-236. Belding, R. C., and R. B. Lacey. 1962. Unpublished data. Michigan State University, East Lansing, Michigan. Bertok, L. and F. Kemenes. 1960. Studies in the lipase- system of leptospirae. I. Tributyrinase activity. Acta. Microb. Acad. Sci. Hung., 1: 251-259. Bjork, W. and J. Porath. 1959. Fractionation of snake venom by the gel filtration method. J. Acta.Chem. Scand., L3: 1256—1259. 62 63 Byrne, R. J., W. Jahnes and C. A. Gleiser. 1955. Studies on experimental avian leptospirosis. Cornell Vet., 45; 290-296. ----- , 1955. Avian leptospirosis: Studies on chick embryo culture. Cornell Vet., 45: 297-304. Carter, H. E. 1957. "Experimental Biochemistry," lst ed. Stipes Publishing Company, Champaign, Ill. \ Consolidated Laboratories, Inc. (Colab). 1960. "Electro— phoresis with Oxoid Cellulose Acetate Strips." Con- solidated Laboratories, Chicago Heights. Cunningham, C. H. 1960. "A Laboratory Guide in Virology," 4th ed., Burgess Publishing Company, Minneapolis. Dosser, Y. M. 1961. Preparation of monolayer cell cultures of tissues of various animals. Problems of Virology, 6: 361-368. Dozsa, I., F. Kemenes and T. Szent-Ivanyi. 1960. Suscep- tibility of the red blood cells of ruminants with a four—chambered stomach (Pecora) to the haemotoxin of pathogenic leptospires. Acta.Vet. Acad. Sci. Hung., L9; 35-44. Dubos, R. J., Edit. 1958. "Bacterial and Mycotic Infec- tions of Man," 3rd ed., 820 pp. J. B. Lippencott Co., Philadelphia. Ebeling, A. H. 1921. Fibrin and serum as a culture medium. J. Exptl. Med., 33: 641. Epstein, W. V. and M. Tan. 1961. Chromatographic study of human serum by gel filtration. J3 Chromatog. 65 258-263. Fischer, A., T. Astrup, G. Ehrensvard and V. Oehlenschlager. 1948. Growth of animal tissues in artificial media. Proc. Soc. Exptl. Biol. Med., 61; 40. Flodin, P., B. Gelotte and J. Porath. 1960. A method for concentrating solutes of high molecular weight. Nature, 188: 493—494. 64 Flodin, P., B. Gelotte and J. Porath. 1960. A method for concentrating solutes of high molecular weight. Nature, 188: 493-494. Flynn, F. V. and P. DeMayo. 1951. Microelectrophoresis of serum proteins. Lancet, LL: 235. Fruton, J. S. and S. Simmonds. 1958. "General Biochemistry," 2nd ed., 1077 pp. J. Wiley & Sons, Inc., New York. Fukushima, B. and S. Hosoya. (In Van Theil, P. H.) 1948. "The Leptospiroses." Universitaire Pers Leiden, Leiden. Gay, F. R. and R. Thompson. 1929. Attempts to cultivate vaccinia virus in growing chick embryos. Proc. Soc. Exptl. Biol. Med., 26: 556-559. Gelotte, B. and A. B. Krantz. 1959. Purification of pep- sin by gel filtration. Acta. Chem. Scand., $1?) 2127. Goldberg, H. S. 1959. "Antibiotics: Their Chemistry and an-medical Uses," lst ed., 608 pp. D. Van Nbstrand Company, Inc., New York. Goodpasture, E. W. and G. J. Buddingh. 1933. Human immunization with a dermal vaccine cultivated on the membranes of chick embryos. Science, 18; 484-485. Hoag, W. G., W. S. Gochenour, and R. H. Yager. 1953. Use of baby chicks for isolation of leptospires. Proc. Soc. Exptl. Biol. Med., 8;; 712-713. Imamura, S., K. Kuribayashi and M. Kameta. 1957. Studies on toxins of pathogenic leptospira. Jap. J. Microb., 1: 43-47. Kemenes, F. 1958. Die durch pathogene Leptospiren verursachte hamolyse und ihre pathologische bedeutung. Acta. Vet. Acad. Sci. Hung., 8: 143-150. Killander, J., J. Ponten and L. Roden. 1961. Rapid pre- paration of fluorescent antibodies using gel filtration. Nature, 192: 182-183. 65 Kohn, J. 1957. A cellulose acetate supporting medium for zone electrophoresis. Clin. Chim. Acta., 2(4): 297. Lillevik, H. A. 1962. Personal communication. Michigan State University, East Lansing, Michigan. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Lundberg, A. L. 1962. Unpublished data. Michigan State University, East Lansing, Michigan. Matheka, H. D. and G. Wittmann. 1961. Desalting virus suspensions by gel filtration. Zbl. Bakt., 182: 169- 178. (English translation.) Ouchterlony, O. 1948. Lg_vitro method for testing the toxin producing capability of diphtheria bacteria. Acta. Path. et Microb. Scandinav., 25; 186-191. Overbreek, J. Th. G. and J. Lijklema. 1959. Electric potentials in colloidal systems, in Electrophoresis. Ed. by M. Bier. Academic Press, New York. Owen, J. A. 1956. Determination of serum protein fractions by zone electrophoresis. Analyst, 8;; 26. Pharmacia Fine Chemicals. 1959. "Sephadex in Gel Filtra- tion." Uppsala, Sweden. Porath, J. and P. Flodin. 1959. Gel filtration: a method for desalting and group separation. Nature, 183: 1657- 1659. V Putnam, F. W., Edit. 1960. "The Plasma Proteins, Vol. 1," lst ed., 420 pp. Academic Press, New York. Raffel, S. 1961. "Immunity," 2nd ed., 646 pp. Appleton— Century-Crofts, Inc., New York. Ringen, L. M. and W. Okazaki. 1956. The susceptibility of several different laboratory animals to infection with Leptospira pomona. J. Infect. Dis., 22: 60-62. 66 Rivers, T. M. and F. L. Horsfall, Edit. 1959. "Viral and Rickettsial Infections of Man," 3rd ed., 967 pp. J. B. Lippencott Co., Philadelphia. Rogols, S., W. B. Fizette and E. H. Bohl. 1959. Studies on the relationship of a phospholipase to the hemolysin of Leptospira pomona. Am. J. Vet. Res., g9; 592-596. Rous, P. and F. S. Jones. 1916. A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J. Exptl. Med., 2;; 549-555. ----- and J. B. Murphy. 1911. Tumor implantation in the developing embryo. Experiments with a sarcoma of the fowl. J. A. M. A., 56; 741—742. Russell, C. M. 1956. A "hemolysin" associated with lepto- spirae. J. Immunol., 11; 405-409. Sigma Chemical Company. 1961. The titrimetric determina- tion of serum lipase. Sigma Techn. Bull. No. 800. Sigma Chemical Company, St. Louis, Missouri. Sleight, S. D. and R. B. Dardas. 1960. Unpublished data. Michigan State University, East Lansing, Michigan. ----- ,and R. F. Langham. 1962. The effects of Leptospira pomona hemolysin on pregnant ewes, cows, and sows. J. Infect. Dis., 111: 63-77. Smith, 1., Edit. 1960. "Chromatographic and Electro- phoretic Techniques. Vol. II. Zone Electrophoresis," lst ed., 215 pp. Interscience Publishers, Inc., New York. Stavitsky, A. B. 1948. Characteristics of pathogenic spirochetes and spirochetosis with special reference to the mechanisms of host resistance. Bact. Rev., L;: 203-255. Stevenson, W. D. H. and M. D. E. Butler. 1933. Dermal strains of vaccinia virus grown on the chorio—allantoic membrane of chick embryos. Lancet, 255: 228-230. 67 Swim, H. E. and R. F. Parker. 1955. Preservation of cell . cultures at 4 C. Proc. Soc. Exptl. Biol. Med., 82; 549. West, E. S., and W. R. Todd. 1962. "Textbook of Biochem- istry," 3rd ed., 1423 pp. The Macmillan Company, New York. White, A., P. Handler, E. L. Smith and D. Stetten. 1959. "Principles of Biochemistry," 2nd ed., 1149 pp. McGraw—Hill Book Co., New York. Wolff, J. W. 1954. "The Laboratory Diagnosis of Lepto— spirosis," lst ed., Charles C. Thomas, Springfield. Woodruff, A. M. and E. W. Goodpasture. 1931. The suscep- tibility of the chorio—allantoic membrane of chicken embryos to infection with the fowl pox virus. Am. J. Path., 2: 209-222. Youngner, J. S. 1954. Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. Proc. Soc. Exptl. Biol. Med., 85; 202. IGAN STATE UNIVERSITY LIBRARIES 03015 51 ”'IIIIIIImu 1293 IU