STUDIES ON THE VIRULENCE 0F LEPTOSPI RA POMONA fr—vw—w 1w".— *‘3 4_. Then: for ”ac Degree of M. 5. MICHIGAN STATE UNIVERSETY Dietrich Charles Bauer 1957 rrw-r's: § 1‘. l l I V \. 4" STUDIES ON THE VIRULENCE or LEPTOSPIRg POKONA by DIETRICH CHARLES BAUER A THESIS Submitted to the College of Science and Arts hichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1957 AC'TOWLEDGMENTS I wish to express my appreciation to Dr. E. V. horse for his interest and guidance in this work. Thanks are due to Mrs. Athalie Lund- berg for her assistance. inwa— .i~—/— __~ I ’V'Her. ‘ —u.. i». 11‘ LINK”), O ed.“ :11. \- V52: '3 a, " u at,- . .1 L. D ‘a TABLE OF I. INTRODUCTION . . . . II. LITERATURE REVIEW . . III. MATERIALS AND METHODS IV. RESULTS . . . . . . V. DISCUSSION AND CONCLUSI VI. SUMMARY . . . . . . TABLES . . . . . . . . FIGURES . . . . . . . . REFEEIEI-CES o c o o o o o 0 NS CONTENTS firefli- HP AL. LIST OF TABLES TA BLE k’ AGE. 1. Titration of strain Wickard in hamsters, media passage no. 1 O O O O O O O O O O O 0 O 0 1+8 DJ . Titration of strain Wickard in hamsters, media passage no. u . . . . . . . . . . . . . . R9 3. Titration of strain Richard in hamsters media 9 Q Passaé;e IlCo \J o o o o o o 0 o o o o o o 0 50 ii. Titration of strain Richard in hamsters, media Eassdg€ DO. 10 o o o o o o o o o o o o o O 51 .5. Titration of strain Nicnard in hamste s, media paSSIALIE, :20. 12 o o o o o o o o o o o o o 0 S1. '5. Titration of strai Hicktru in hamsters, neuia fiassage no. 1f. 0 o o o o o o o o o o o o o 52 '7. Titration of strain Wiekard in hamsters, media passaé:e HO. 20 o o o o o o o o o o 0 o o 0 S3 53. Titration of strain Wickard in hamsters, media passage no. 2h . . . . . . . . . . . . . . S3 9. Titration of strain Wickard in guinea pigs, media passages l, u, 8 . . . . . . . . . . . . . . 5h If). Titration of strain Ohio in hamsters, meiia .passage no. 1 . . . . . . . . . . . . . . 5; ll. Titration of strain Ohio in hamsters, media passe-CDC Tit}. I“! o o o o o o o o o o o o o o 55 12. Titration of strain Ohio in hamsters, media passage no. 7 . . . . . . . . . . . . . . 56 13. Titration of strain Ohio in hamsters, media 35355858110. 11 o o o o o o o o o o o o o o 56 1h. Titration of strain Ohio in juinea size media . L J Passaéges 14. 51151 11 o o o o o o o o o o o o o 57 15. LDSO and ID 0 values for titrations of Wickard - and 01-11 strains 0 . . O O O . . O . . C 0 SB to. \ )‘k .. .‘t. LIST OF TABLES TA BLE r AGE 1. Titration of strain Wickard in hamsters, media passage no. 1 . . . . . . . . . . . . . . hB DJ . Titration of strain Wickard in hamsters, media passage 110. 1+ 0 o o o o o o o o o o o o o 4.9 3. Titration of strain Rickard in hamsters, media Q passage TIC. \J o o o o o o o o o o o o o 0 SO 2;. Titration of strain Wiciard in hamsters, media {LE-1850.56 1730. lo 0 o o o o o o o o o o o o o S]. S. Titration of strain Nicnard in hamsters, media paSSaL,£ ITO. 12 o o o o o o o o o o o o o o S]. 5. Titration of strain fiickzrd in hamsters, neaia passage no. .3 o o o o o o o o o o o o o 0 S2 7. TTitration of strain Wickard in hamsters, media passage no. 20 . . . . . . . . . . . . . . 53 Q . . . . .. we 'Titration of strain Wickard in hamsters, meCia passage n0. Pk o o o o o o o o o o o o o 0 S3 9- CFitration of strain Wickard in guinea pigs, media passakjes 1, 1+, 8 o o o o o o o o o o o 0 o 0 52+ 10- CPitration of strain Ohio in hamsters, media .passaae no. 1 o o o o o o o o o o o o o o 59 11- ‘Titration of strain Ohio in hamsters, media pE‘SSE-LDC 1’11“. I! o o o o o o o o o o o o o o 5: 12- Titration of strain Ohio in hamsters, media passage no. 7 . . . . . . . . . . . . . . 56 13' {Titration of strain Ohio in hamsters, media gvssage no. 11 . . . . . . . . . . . . . . 56 1 O O o O a U. TTitration of strain Ohio in guinea 9155, media passages h and ll . . . . . . . . . . . . . 57 IS. I¥DSO and ID 0 values for titrations of Wickard and Ohi strains . . . . . . . . . . . . . 55 Z1315. _: -1 I 9—40 'HI ..‘i i.» ..—E Y'” 1“ -_. LIST OF TAbLES - continued TABLE mar. 16. Tlfi effect Of the aLe of hamsters on susceptibility to L. scmona (uickard) . . . . . . . . . . . 59 17. Growth of E. pomona in vitro after 1, h and 8 media passages . . O C O C C O O O O O C O O O O C 60 18. In vivo adhesion of strain Nickard and er"thr00"tes 61 __ ____ a J 19. In vitro adhesion of leotosuiree and ervthroc‘tes __.. __..._._. L t V after incubation at 37 C for thirty minutes . . 62 20. 'The hemolysis of sheep ery hrocytes by various cultures of L. Eomona . . . . . . . . . . . 63 21. the hemolysis of erythrocytes from various animals by L. ggmona . . . . . . . . . . . . . . . . C\ \rl LIST OF FIGURLS PAdu 1. T%e effect of passage in nedia on the virulence of L. gomona for hamsters . . . . . . . . . . . 67 2. The effect of the size of the inoculum on the survival time of hamsters CD . . . . . . . . . . . 6 a r... at w P.» ru. 9P. .1. nay . .x .r F. '4. x ...v .CH 3 . .9.IU. s .Hv , . h pu. I. INTRODUCTION‘ In the study of infectious diseases, a Knowledge of tflie pathogenesis of the infection is of utmost importance. cupservations on the pathogenicity and virulence of the etio- loggical agent contribute to a betier understanding of patho- geriesis and the host-parasite relationship. There is a lack of agreement amend microbiologists on deffiinite meanings for the terms "pathogenicity" and "virulence". Frenauently the two terms are used as synonyms. This indicates the Jaecessity for prefacing a study of these properties by as- SigrLing the definitions to be used throughout this thesis. A usefflil distinction between the two terms has been made recently by Pfiiles (hO). He suggests that "virulence“ be used to refer to tune observed infective capacity of the pathoEen studied, wheri applied to the tissues of the host. The frequency with WhiCii virulent strains are found in a group of organiSms debervnines whether the term pathogenic is applied to that EPOUTD. "Pathogenicity" is therefore, no more than the dis- posifitixmlto virulence of a class of parasitic microbes. For a definition of "virulence", Dubos (21) has pre- sented the following: "Virulence is not a permanent intrinsic PPOIDExpty of a given species. It eXpPesseS Chly the ability or o o o o I 8-,given strain of the infective agent, in a certain growth & ‘. l \ U‘U‘L' VA ache-W tip). 566?. E '37 n .4 i A‘Nrfi LGI; Us phase, to produce a pathological state in a particular host, when introduced into that host under well defined conditions." The importance of Leptosflra gomona as a zoonotic agent has been well establisned (11;, 17). The epidemiological, epizootiological and clinical aspects of this leptospirosis have been described in detail, (66, 11,7, 52). However, informa- tion regardingthe microbic factors which determine the viru- lence of this serotype is limited. A lmowledée of these fac- tors would aid in a more complete understanding of the patho- genesis of leptospirosis. his study was undertaken to determine the virulence of two strains of Lepto_spira pomona for hamsters and guinea Pigs, and to ascertain the changes in virulence of the organisms by passage through artificial fluid media. During the course of these studies, attempts were made to demonstrate 1:2 m3 adhesion of leptospirae to erythrocytes. Observations on the relationship between the in y_i_t_r_~_9_ production of a hem01ysin and the virulence of Leptospira pornona were also made . II. LITERATURE REVIEW In 1936, Clayton, Derrick and Cilento (17) isolated a strain of leptospira from a farmer in Queensland, Australia which proved to differ serologically from known existing spmacies. This species was designated "Leptospira pomona" by Derrick (20) . Morphology Since morphological distinctions cannot be made between the ‘various Species of leptospirae (66), the description by Nognichi (h9) of Laptospira icterohaemorrhagiae is applicable F0 1;. pomona. He described the "spirochete" as being 7 to ho Micxnans in length with a diameter of about 0.25 microns. Spirnals occurred at approximately 0.5 micron intervals. The terntinal portions of the spirochete were described as pointed Witki one or both ends bent. Nobuchi observed that reproduction tool: place by transverse division. He could not recognize flaéyella, axial filaments, or membranes. The organism was re- 818tent to 10 percent saponin but dissolved readily in 10 percent bile salts. More recent investigations with the electron micrOSCOpe hav‘3 elucidated previously undetectable structures. Morton anti Jtnderson (hB) were unable to observe any internal structure in __I_:. icterohaemorrhagiae. However, Babudieri (2) concluded from his studies that leptOSpirae consist of a cylinder of homogeneous protoplasm, enveloped by a very thin anistic membrane, wrapped around a rigid central filament, the axistyle. In old cultures he found thin filaments, which he interpreted to be axistyles and residual masses which were freed by the destruction of the membrane and protoplasm. The axistyle was not affected by the papain, pepsin, or by freezing. Bessemans and coworkers (6) investibated the granules which are found in old cultures, or in cultures'subjected to temperature extremes, irrmune sera, or bile salts. By means or a pneumatic micromanipulator, single granules were isolated Which could give rise to normal leptospirae. The resistance Of these forms to noxious agents and heat was found to be no greater than that of leptospirae. Czekalowski (1?) considers these granular forms to be phases in the life cycle of lepto- SPiI‘ae. Other workers (2, 66) consider them to be merely de- generative products. Webster and Reynolds (69) were unable to find any dif- ference in the virulence for guinea pigs of long; and short forms of L. pomona. Cox and Larson (1U) have recently reported on the colonial morphology of leptospirae. They described two types of leptospiral colonies. The first type is smaller in diameter and more Opaque than the second which is larger in diameter and. translucent. Some strains were observed to produce colonies which developed irregular margins resembling the rough variant colonies of bacteria. Virulence of LeptOSpirae Many workers (66, 62, 23, 39, 15) have reported on the reduction in virulence of leptospirae due to cultivation in artificial media. Langworthy and Moore (39) transferred 3:. wrohaemorrnagiae at one to two week intervals in semi-solid medium. A perceptible decrease in virulence for guinea pigs was noted after two months, with a marked decrease in four to Six months. Chang (15) found that L. icterohaemorrhagiae lost its virulence for guinea pigs after three to four transfers in 8emi-solid and fluid media over a period of ISO to 200 days. F'aine (21;) reported a 50 per cent lethal dose of 10 leptospirae for a culture of L. icterohaemorrhagiae which had been trans- ferred 9 times in fluid media since its isolation from a guinea Pig. Fifty percent lethal dose values of L; x 105 cells for an 9 eighteenth culture passage and greater than 10 cells were ob- s61"ved for a strain which had been maintained 2 1/2 years in culture. £0. pomona was described by Webster and Reynolds (69) as losing its virulence for calves and guinea pigs after 3 to 1L mOnths in semi-solid medium. Alexander gt gl. (1) reported that a strain of Q. pomona lost its lethality for hamsters £1fter one year's passage in fluid medium. Rheinhard and Hadlow (52) found that L. p‘omona which had been maintained for three years in artificial medium, or which had undergone 100 serial passages in embryonating eggs was relatively avirulent for cattle. Several procedures have been proposed for the main- tenance of virulence of leptospirae g; m. Stavitsky (61) found the virulence of L. icterohaemorrhagiae to be maintained for 100 days by freezing liver blocks from infected guinea pigs at --20 C. Freezing of cultures and 10 percent liver suspensions were unsuccessful in preserving virulence. Lyo- Philiz‘ation of any of these materials did not maintain viru- lence. Chang (15) reported that the virulence of leptospirae COUld be maintained for 288 days when the organisms were grown in fresh liver enriched semi—solid medium. Bauer (2’4) observed that a culture of La. icterohaemorrhagiae was still virulent for Ellinea pigs after one year in semi-solid medium without transfer. An increase in virulence of leptospirae may be at: tained by passage through susceptible animals. In 1917 Stokes, Ryle, and Tytler (614.) reported that the virulence of 1"" aerohaemorrhagiae was increased by guinea pig passage. When guinea pigs were inoculated with blood from infected humans the average survival time of the animals was 10 days. After 11+ passages through guinea pigs the average survival time was LL . 33 days. Noguchi (50) found that one passage of Q. ictero- haemor-rhagiae through guinea pigs decreased survival time of the animals from 9-12 to 6-0 days. Hamdy and Ferguson reported (30) that the virulence of 32:. pomona for hamsters was increased by serial passage. After sixteen passages, death occurred four days after inocu- lation. At the twentieth passage death occurred on the third day. Van Thiel (66) reported that after eleven successive inoculations 01' _I:.. pomona into guinea pigs, death occurred in 2 out of 22 animals. Other workers have reported that attempts to increase Virulence by animal passage were unsuccessful. Bernkopf gt al. (5) passed a "bovine strain" of leptospirae through six pas- sages in calves with little virulence increase. Borg-Petersen (9) Could not detect an increase in the virulence of Lapto- éELIE 3212.2 when passed through guinea pigs and mice. Stavitsky (6U) examined extracts from cultures of 3' Qerohaemorrhagiae for the presence of substances associ- ated with virulence. He was unable to demonstrate hyaluronidase, fibI‘lr‘iolysin, leukocldin, or coagulase. However, Volland and Brede (67) allowed filtrates from cultures of various patho- genie species of leptospirae to act on the synovial fluid of an 0X. A decrease in the viscosity of this fluid, which is rich in hyaluronic acid, was noted. They interpreted their findings as indicating the presence of hyaluronidase in the culture filtrates. The presence of a leptoSpiral toxin had been suggested as early as 1917 when Inada (33) prOposed that toxins arise from the disintegration of the Spirochetes within the organs. Van Thiel (66) has described the clinical picture of lepto- spirosis as one of "general intoxication". Ferguson and co- workers (25) have proposed that the cause of fetal death and abortion in bovine leptospirosis is a hemolytic toxin. Stavitsky (60) has described the findings of Japanese workers, in which toxic substances for guinea pigs were found in cultures of leptospirae kept under anaerobic conditions at 37 C. for 2 to 3 days. However, Stavitsky (60) was unable to demonstrate this toxin. In 1956 Alexander SE 1;. (1) observed the presence of a hexnolysin in cultures of some species of pathogenic lepto- Spirae. The hemolysin was described as being soluble, non- dialy8able, thermolabile and oxygen-stable. No antigenicity Of the hemolysin could be demonstrated. Hemolytic activity was Observed with sheep, cow, and goat erythrocytes. This work was followed by the report of Russell (56) on the Presence of a hemolytic agent in cultures of L. pomona. Characteristics similar to those previously described (1) were attributed to this hemolysin. The hemolysin was most active for sheep erythrocytes. Human and rabbit erythrocytes were affected to some extent, while erythrocytes from guinea pigs showed little activity. Chang (15) has suggested that the virulence of lepto— spirae is closely associated with the ability to attack fresh liver substances. He demonstrated that avirulent strains, isolated from guinea pigs, and grown in fresh liver enriched 2—we- semi-solid medium regained some of their virulence for guinea a pigs. Stavitsky (62) has suggested that the surface potential of leptospirae may be associated with their virulence. How- ever, there is no agreement on the sign of the surface charge of these spirochetes. riligler and Aschncr (36) found from electrophoretic studies that L. icterohaemorrhagiae was posi- tively charged at the pH of blood. However, Brown and broom (11), in a study of the adhesion of colloids to leptospirae in the presence of immune serum, found leptospirae to be nega- tively charged. Stearns and RO€pA€ (63) have demonstrated that dissoci- ation forms of brucella have different electr0phoretic mobili- ties. The in vitro adhesion of leptospirae and other spiro- chetes to blood particulates and other bacteria has been ob— seI‘Ved. (ll, 10, 12, 38). This adhesion takes place in the presence of immune serum and complement (68). Lamanna (37) has I‘ecently reviewed serological adhesion and discussed its 01010gical signifi cance . 10 Studies of genetic variation of leptospirae and its relation to virulence are limited. Schlossberger (57) reported in 1950, the demonstration of induced mutation of E. ictero- haemorrhagiae. Using methods similar to those of Griffith (28) with pneumccocci, Schlossberger observed that when a heat- killed virulent strain and an avirulent strain of E. ictero- liaemorrhagiae where incubated together for 3 days and then inoculated into a guinea pig), that the animal died and the virulent strain was isolated. However, attempts by this worker to demonstrate this phenomenon using other combinations of viru- lent and avirulent strains were unsuccessful. Paine (21;) found that one passage of a strain of l_J. Eterohaemorrhagiae of reduced virulence through guinea pigs, Pastored the strain to its original virulence. Lie postulated that, passage of leptospirae through susceptible animals results in the selective multiplication of virulent mutants. Pathogenicity of g. pomona Naturally occurring 'nf‘ections produced by _I:. pomona have been reported in man (111,), cattle (3), swine (27, 8), horses (55), sheep (31, 5'8), and dogs (66). The pig appears to be the principal or usual host of L3 Komona (71). However, the clinical signs of the disease in. ~ . , . -r . swine are minimal, with the exception of abortion and death Of mewborn pigs (7). 11 The disease in cattle is characterized by abortions, decreased milk production, hemoglobinuria, anorexia and general depression (342, M3). ’ ’ L. pomona infections in sheep result in fever, inap- petance, and hemoglobinuria (L5, 55). Fever, temporary blindness, meningitis, and aching and swelling of the joints (66) are frequently reported for human g. pomona infections. Hoag 9.1.3. §_l_. (32) described experimental _I_i. pomona in- fections in 2-day old chicks. Clinical symptoms were lac-kings. Leptospiremia of about 9 days duration was observed. Antibooy PPOduction was weak in these animals. Gleiser and coworkers (26) reported that the inoculation 0f 17—day old chicken embryos with p. pomona produced death in 2'5 Clays after hatching. Ringen and Okazaki (53) reported that guinea pigs, ham—Steins, and white mice were readily susceptible; whereas young chicks were relatively resistant. The infection in hamsters is usually lethal (1); however, the virulence of L. W for guinea pigs is low (66, 70). 12 III. ICATIIRIALS AND METHODS The hifect of Radium rassage on the Virulence of g. pcmona ‘ (f' Two strains of Q. “omona were employed in this stuc L9. annona (strain Wickard) was isolated (h3) from the urine \ of 811 infected dairy cow and had been maintained in young guiruea pigs for three years. This strain produced a febrile "5 (D J) ~§“fl1se in guinea pigs. It was not lethal for hamsters. Sincxe its 150 ation, it had been used for experimental infec- ticnis; and was known to be pathogenic for cattle (hf), swine (d7) , sheep (hfi), goats (h7), and dOQS (16). ‘L. oomona (strain Ohio) was isolated from an infected 210G . b‘r L )‘r'. C r_ t t7,» ' ' 1t, )1‘71 "I n [A 1; .' W t (2t!- t: c. t J \IOI (stirs; 0‘- Ala “grlcu 'L-L u.‘ .LJAE. CI ...le L4 5L .Lon 0L mkxbstzer, Ohio. It had been maintained in hamsters and guinea 1 V1 13 and was lethal for hamsters. It had seen used to produce {w 6Xpet‘imental infections in sheep (hS), hogs (Q6), and QOQS (15). The culture medium used throughout this study was a mociihfied Chang's fluid medium (13) containing 0.01 per ocnt heiuciglobin (Difco) and 10 per cent sterile rabbit serum. E84313- screw cap culture tube contained approximately 10 ml of ”Edie. The leptospirae were transferred to fresh media when a ‘ r} I '\ o ‘ (llllture had reached a staéc Cl makimum growth. This deter- r‘ ii . -— o \ 0 ‘ 1"-’-"~:—L'tion was approx1mateo by Visual Observation of the turlaiétity of the culture and by periodic examinations OJ dariflfixeld microscopy. Eight to twelve dats were required for J madenLun growth to occur and approximately 5 x 108 cells per cc werwa cibserved. The inocula consisted of 3.1 to 0.3 ml of cul- turea. All cultures were incubated at 29 C.. Virulence determinations were made durin; the logarithmic growtfli phase of a culture. The concentration of leptosyiree in 5 'to 10 day old cultures was ascertained by darkfield micrcuscopy using & Petroff-Hauser sacterial countin“ b chamber. The (naunting grocedure consisted of placing a drop or culture on true ctuntin cell with a catillarx pipette and adjusting » - . U ‘Uus ccrver glass over the drOp. If the aneunt of fluid was insuihficient to cover the cell area or was in excess resultin 1” thEB sanele overflowing the cell, the cell was cleaned and a "s T f) O (J c. '3 o ‘3 0 Q ‘eated. When a satisfactory preparation had been (abtained the chamber was placed on the stage of the dark- fi? 1(' )4 Inicrescope and allows; to remain undisturbed for several TfiJfllte3s to minimize any movement of the fluid. At a H2OK Optiszl magnification, and darkfield illumination, the number of nytjlfl leptospirae in BC, 1/LOO square millimeter squares was d&3term1fled. The 80 squares represented a group of le Squar13s from each corner of the counting area and one group in ttus center. The number of leptospirae per square was cal- culained and multiplication of this figure by 2 x 107 have the nuaumer of organisms per cc. The entire counting procedure was re ,, _..._, _ petmted at least once more to assure accuracy. The difference 1’4 between two or more determinations gave an error of O to 5 percent of the averages. When the concentration of a sample was ‘too great for accurate counting, it was diluted so that from 2 to (.3 leptospirae were seen per square. No increase in accuracy was obtained by decreasing the motility of the organisms with formalin. Therefore, only viable, motile leptospirae were counted. The error of this method for deter- mining; bacterial concentrations is reported to be about 10 percent (70). When the number of leptospirae in the culture had been ascertained, ten fold serial dilutions were made. Dilution blanks containing; 9.0 ml of Chang's medium minus the rabbit serum and hemoglobin were used. In some studies dilution blanks-S containing 13.5 ml or 13.0 ml of diluent were employed. Five or seven Syrian hamsters were inoculated intra- Peritoneally with 1.0 cc of each dilution. In some titrations five guinea pigs were also inoculated intraperitoneally with 1-0 0c of each dilution. All hamsters and guinea pigs used were from animal colonies maintained in this laboratory and mom to be free of leptospiroses. Hamsters were from 3 to 5 weeks of age, while the guinea pigs were Li to 6 weeks old. Hamsters were observed daily for signs of illness or death. When a hamster became moribund, 0.2-0.5 cc of blood Was Obtained by cardiac puncture. The sample was placed in a drop of sterile heparin sodium solution and centrifuged for 5 minutes at 2000 rpm in a Servall Small Type A centrifuge. 15 A sample of the plasma was examined by darkfield microscopy for the presence of leptospirae. In some cases the number of leptospirae in the plasma was determined using the counting chamber. Twenty to thirty days following inoculation all sur- viving animals were sacrificed and a blood sample was obtained from each. A modified agglutination—lysis test (uh) was con- ducted for each serum sample usinb living E. 225223 antigen (strain Johnson). Any animal which died three or more days after inocula- tion, or any which survived and had a serum antibody titer of 10-2 or greater was considered to have been infected. The virulence of cells from each passage was exPresscd as that number of leptospirae which would kill 50 percent of the inoculated animals. This value will henceforth be referred to as the LDSO' The number of leptospirae infecting fifty per- cent of the inoculated animals will be termed the IDSO. LDSO and IDSO values were calculated by the method of deed and Muench (51, 5h). A. Virulence Determinations with Strain Wickard Virulence determinations with strain Wickard were con- ducted for medium passages l, h, 8, 10, 12, 16, 20, and 2h. The first passage was a 32-day old culture of blood from an eXperimentally infected sheep. The sheep had been inoculated With blood from a guinea pig in the leptospiremic state or the lo disease. Contaminants were not observed in this culture when inOCulations were made in Brain Heart Infusion Broth (Difco). The inoculum for the hamsters was centrifuged for 10 minutes at 2000 rpm in a Servall Small Type A centrifuge to remove gross particles prior to counting. Seven hamsters and five guinea pigs were used for each dilution. To determine if the cell free culture fluid was anti- genic and might interfere with the results, a portion of the culture was passed through a Seitz filter and then frozen and thawed twice in the refrigerator. LeptOSpirae were not visible after this treatment when viewed by darkfield microsc0py. Ten fold dilutions of the filtrate were made and hamsters inoculated as previously described. Five hamsters were inoculated with each dilution for passages h through 2h. Passages l, h, and 8 were also inoculated into each of five guinea pigs. Virulence determinations for passage 10 were made using both washed and unwashed leptospirae. The organisms were separated from the culture fluid by centri- fuging hO ml of culture for 35 minutes at 12,000 rpm in a Servall Superspeed Centrifuge, Type 58-1. The sedimented leptospirae were then washed three times in ho m1 of sterile Chang's buffer (pH 7.0-7.2). The buffer consisted of h.0 5 NaZHP0u.7H20, 0.8 g KH2POu’ and 8.0 3 NaCl in two liters of distilled water. The washed cells were resuspended in ho ml of buffer and a virulence determination made. The supernatant fluid from the first centrifugation was passed through a Seitz l7 -fi1ter and frozen and thawed twice in the refriberater. One cc of this preparation was injected intraperitoneally into each of three hamsters. Inoculation of the filtrate int: Chang's medium produced no growth of leptosoirae follow‘nw 30 days incubation at 29 C. Virulence determinations on media passages h and u were r repeated. The leptospirae were isolated f em another experi— nmnitally infected sheep. Virulenen Determinations with Strain Ohio I“. .. Hm Mn 4 ‘1‘ "r’ 1L8 flPFt hauls” yifbage e1 Strald QulO u&c a Q; “Ca old (mzlture from infected guinea pig blood. Virulence deter- ndfuations in hamsters were made on Guinfiza pigs were used for passagv C. CDhe Effect of Age of hamsters on Susceptibility t C _I:- . p magma Two titrations were made using hamsters of various ages,, The first study employed 7 week old and h week old hamst:ers. each age group was inoculated with 10 fold dilutions . C in the febrile stabe of leptOSpiros c. 0f tfllood from guinea pips blood has iseeond study employed 15 week and h week old hamsters. J «ll-u fTTWn .infected guinea pigs was again used for the inoculum. 9- Growth of Leptospirae in vitro after Passabe in media The number of leptospirae needed to initiate growth in CharLR;'s medium was determined for passages l, h and 8 of strain Nickard. Counts and serial dilutions were made as F‘I'GViO‘JSlY deseribed. The inocula consisted of 0.1 ml of fluid from each dilllttion blank and was transferred to each of five or seven tUb€3S of medium per dilution. The cultures were examined by fieluli'dln. slur. [1.4% . n f 17:» darkfield micrOSCOpy for the presence of leptospirae at 10, 20, and 30 days. The number of leptospirae needed to initiate Lrowtn in 50 percent of the inoculated tubes was then determined. Adhesion of Leptospirae to Erythrocytes A. la Yivg Adhesion The Jremise that lectosairae mi ht adhere to erythro- k L . C.) v ,ated using Juinea pigs V cytes during leptospiremia was investi, infected with the Wickard strain of g. pgmgna. Infected guinea pigs which had temperatures of 10h F to 106 F were bled aseptically. Heparin was used as an anti- coagulant. A portion of the blood was diluted ten fold, and each dilution inoculated into hamsters and Chang's medium as previously described. Five ml of the whole blood were centri- fuged for 15 minutes at 200» rpm in a Servall Small Type A centrifuge. The plasma was removed aseptically with a sterile capillary pipette. To it was added sterile Alsever's solution (59) to make a final volume of 5.0 ml. Ten fold dilutions of the plasma were made, and 5 hamsters and 5 tubes of Chang's medium inoculated with each dilution. The sedimented erythro- cytes were washed three times by resuSpending in 5.0 ml amounts of Alsever's solution and centrifugation for 15 minutes at 2000 rpm. After the final washing, the erythro- cytes were resuspended to 5.0 ml volume in Alsever's solution. Ten fold dilutions were inoculated both into hamsters and media. Twenty to 25 days after inoculation the hamsters were sacrificed and a blood sample obtained. An agglutination lysis test was made on the sera. Antibody titers of 10"2 were considered ixmticative of the presence of leptospirae in the inoculum. Culinires were examined after 10, 20 and 30 days of incubation. As a control procedure, 0.3 ml of a culture of virulent §.gxnnona (strain Wickard) was added to 3.0 ml of heparinized blocxi from a non-infected guinea pig. The same separation, waslring and dilution procedures were performed as described for 'the infected blood titration. Only Chang's medium was inOCiilated. B. Jhi vitro Adhesion 1. Two cc of blood were obtained by cardiac puncture froni a guinea pig having a Serum antibody titer of 10-5. blood was ssimilarly obtained from a non-infected guinea pig. One ml Of tliis was placed in heparin and centrifuged at 1000 rpm for Esrnidnutes. The plasma was removed from each sample with.a CEPilglary pipette. Guinea pig i.lasma, containing L. pomona antihnsdy, was added to one portion of the normal cavian erytkubocytes. r“he final volume equaled the original amount of blxzod or 1.0 ml. A similar amount of normal plasma was added. to a second portion of normal erythrocytes. Both erythlrocyte-plasma samples were incubated at 37 C. for M5 minutees. They were then centrifuged for 15 minutes at 1000 rpm arid the plasma removed. The erythrocytes were washed twice ‘with 2.0 ml amounts of Alsever's solution and resuspended to theeir original volume of 1.0 ml with fresh normal plasma. To eacflq sample was added 0.1 ml of a culture of g. pomona (strain Wickard). The culture contained approximately 1.6 x 108 organises per cc. The final concentration was therefore approximately 1.5 x 107 leptospirae per cc. The samples were incubated for 15 minutes at 37 C. Followinb in- cubation the samples were centrifuged at 1000 rpm for 15 minutes and the leptospirae in the plasma counted using) the Petroff- Hauser bacterial counting; chamber. The erythrocytes were di- luted approximately 1:10 and observed by darkfield microsCOpy for visible leptospirae-erythrocyte adhesion. 2. In a second £3. vitae study of adhesion, sheep erythrocytes and leptospiral antisera from sheep were used. The L. oomona antibody titer of the sermzi was 10's. The tech- nique was as follows: 0.1 r11 of culture of strain Wickard was added. to each of six tubes. The six tubes contained respec- tive 13;: 1) 0.9 ml of 0.85 percent saline, 2) 0.9 ml of waole blood, 3) 0.1 ml of erythrocytes in 0.53 ml of 0.35 percent saline, h) 0.1 m1 of erythrocytes in 0.3 1.11 of normal sheep sera which had been inactivated at a temperature of 55 C. for thirty minutes, 5) 0.1 m1 of erythrocytes in 0.1 ml of L. Pomona antiserum plus 0.7 ml of inactivated serum, and 6) 0.1 ml of g. pomona antiserum plus 0.? 911 of normal sheep sera. The tubes were incubateu at 37 C. for 30 minutes followed by centrifugation for 15 minutes at 1000 rpm. The number of leptospirae were determined for the supernatant fluid from each tube using; the 001111131119 chamber method. The erythrocytes 21 were diluted 1: 0 and viewed by darkfield microscow for le p tospirae-erythroc; te adhesion . 3. The adhesion of g. pomona (strain Wickard) to polystyrene latex spheres (PSL) was investigated. The spheres were obtained from the Dow Chemical Company, Midland, Michigan. The diameter of the (PSL) was 1.171 microns. A distilled water suspension containing 7.2 x 108 spheres per cc was used in this study. Chang's buffer, 0.1 ml, was placed in each of eleven tubes and 0.1 ml of strain Wickard culture was added to tubes 1, and 94-9. Tubes 1-9 contained 0.1 m1 of PSL suspenSion. TUbe 2 received 0.1 cc of normal sheep serum, while a similar amount of ovine L. pomona antiserum with an antibody titer of 10.5 was placed in tube 3. Tubes h-é contained ten fold dilutions of the normal serrm. Tubes 7-9 had similar dilutions 0f antiserum. Tube 10 contained 0.1 ml of culture plus 0.1 ml or antiserum. Tube 11 had 0.1 m1 of PSL and 0.1 ml of anti- serurn. The tubes were incubated for thirty minutes at 37 C. FOIlowing incubation 0.1 1:11 of PSL was added to tube 10 and _ 0.1 ml of culture to tube 11. A portion of each tube was then examined by darkfield micrOSCOpy for adhesion of leptospirae to the PSL, 22 In vitro Hemolysin Production in Jelation to Virulence Leptospirae were grown in the isotonic buffered medium described by Alexander §£_al. (l), augmented by the addition of 0.01 percent hemoglobin (Difco). .This medium was inoculated with 0.5 ml of material from cultures of both strains of Q. pomona which exhibited various degrees of viru- lence. The cultures were incubated at 29 C for 10 to 1h days. One to 3 days after growth had reached a maximum, the Irnaber of leptosoirae was determined using the bacterial cunt— 1ng chamber. The cultures were then placed in the refrigerator (h_C3.) until used. Normal ovine and bovine blood were collected by veni- Puncture, haparinized and preserved in Alsever's solution at I - . . ~ . 4 C- Hamster, rabbit and guinea pig blooo were obtained by cxnmiiac puncture and similarl, processed. ‘The erythrocytes of the \rarious swecies were prepared by wasning three times with isotconic buffered base (pd 7.h). The base consisted of th isotcbnic buffered medium minus rabbit serum and hemoglobin. The vyashed red blood cells were then resuspended in saificient isotCJnic base to make a 5 percent cell suspension by volume. A staihdard curve of percent hemolysis versus Optical density wastleatermined for the erythrocytes of each species. This was acconqolished bv adding 5.0 ml of distilled water to 5.0 n1 1) of the; above erythrocyte suspension. Centriiu¢ation of this nmterigal.at 2000 rpm did not ChJSC sedimentation of the red I blocud. cells. Therefore, 100 percent hemolysis of tie reu blCKDd cells had occurred. Appropriate amounts of henolysed fin . , V 0 fl 1' r . ' ease were mixed to give rad e: oi U erythrocytes and isotoni c frcnn. 10 to 100 percent hemolysis. The final volume in >ach dilnitxion was 1.0 ml. In order that sufficient amounts of sanuclxe were available for optical density determinations, 2.0 snl (bf‘ isotonic base were added to each dilution, to mane a totes]. volume of 3.? ml. Optical density determinations were thCll :uade in a Bausch and Lamb Spectronic 20 Colorimeter usind rmrtciieo.test tubes. Readings were made at a wave length of tdo ‘0 do 52C) urn. Percent hemol s was plotted versus optical density. Ten E. pgmgna cultures of varying degrees of virulence WeIWB 'tested for hemol5tic properties. The followinC is 6 list 0f"tlies cultures used, their origin, and the number of transfers irz Vuéciia since isolation from an animal prior to their beina te‘it‘tri for hemclytic preperties. 3.- Strain Wickard, from infected dairy cow, in medium m passage for three ye rs. 23. Strain Wickard, from infected guinea pig, third medium passage. L3.. Strain Wickard, from experimentally infected sheep, third medium passage. )Lo» Strain Uickard, from experimentally infected sheep, fourth medium passage. 53- Strain hickard, from experimentally infected sheep, ninth medium passaLe. 24 6). Strain Wickard, from experimentally infected sheep, tenth medium passage. 7’. Strain Wickard, from experimentally infected sheep, twenty-fifth medium passage. 0 c ‘ o 9 q ~. 0 o . 1 - o s . Strain Onio, from iniecteu guinea pig, fourtn neoium passage. ‘9. Strain Ohio, from infected guinea jig, sixth medium 1(fi O .1 0“" «w '.5 “a 1' a" r' '3 -t* v ‘ -‘ ‘ ° ”n “ ~ ' ‘° ..,.,,V a o otr'E. “1'1 .i.LO, rrCu- i;ll\.CLbu 01.11.1164; trig), blii’iitl‘. .ub‘Ulvlu Since cultures 1 and 2 had not been included in previous viruiZLence titrations 1.0 cc of each culture was inoculated intra— Eer113cwmmflJ§ into each of three hamsters to determine if these cu]. ture s were infective . 1 Two fold serial oilut’ons of the cultures were made in the i.sc&xmnc base. To 1.0 ml of each dilution, 1.0 ml of a S PCPCHSIIt suSpension of washed erythrocytes was added. As con- trOIJS 1.0 ml of the washed er3thrccytes were added to 1.0 ml Ofthte diluent and of the medium. The tests were incubated at 37 C. for four hours and then placed in the refrigerator (u C -) for twelve hours. Incubation has been reported to be necefssmary for complete hemolysis to occur (56). Following ilunlhmition, the tubes were gently aritated and centrifuged at 1LN3C>rmm1in an International Clinical T§pc Centrifuge model CL. One ml of the supernatant fluid was removed and placedi in.a colorimetric test tube. Two ml of isotonic base F.) VI was; sadded and the optical density determined. Optical densities melee: plotted versus dilution. From the standard curve 143 CDptLiczal density of a 50 percent hemolyzed erythrOCJte suspension Via}: cistermined. Using this value tee dilutior of culture nesceassery to groduce 50 percent hemolbsis of srytLroeytes was Elsczeastsined. This dilution was used to ex>ress the hemolfitic J pcrtenntialrflTzrculture. All ten cultures were tested usinb cots ~.-- 4. hauls L 5 ’3 erythrocytes. Two fold culture dilutions of 1:2 thrcn13h 1:102L were used for the tests em 10" n' shee' ervthro- q r I?) .2 o a cbrte 8, while culture dilutions of 1:2 throug? 1:123 were used Vflaerl lemster erythrocytes were exployed. Cow, rabbit and 5L11rmes.pig erbthrocytes were tested absinst cultures 1, 2, 7, arni '8. Dilutions from 1:2 through 1:16 were employed. IV . RESULTS The Effect of I’lediuga rassage on the Virulence of £1. gomona A. Virulence Determinations with Strain Wickard The results of virulence determinations using strain wj-C]ri€-tI‘d are presented in Tables 1-12. Figure 1 depicts the Changes in virulence which occurred followin C passage of the OPEBanisms in media. Unfortunately, titrations of the twelfth rzxeciia passage in hensters ard the repeated titration oi‘ the eiihtn media passes did not include a dilution of leptospirae DPOClucing, less than fifty percent infections. (Tables 5, and 3, respectively) For the calculation of LDSO and IDSO values 0f therse titrations it was essumed that the next highest ten f01d dilution of leptospirae would not have croducec any in- f(actions. Therefore, these values may be higher than the actual value. Examination of Tables 1-8 indicates that strain Wiel‘iard was not lethal for hamsters following one passage in "19618 . A lethal effect was observed in titrations of the fourth through twenty-fourth media passages. However, ham- StePS exposed to the first passage were infected as demonstrated by CleVéElobment of serum antibody. Therefore, the virulence of the f1I‘st passage was comparatively low. The relatively high Pt) degruee: of infectivity of this passafie is reflected by the IDSO valtxe <3f 3.8 organisms (Table 15). The virulence of the Nickard strain increased between the :fjxrst and fourth passages. A decrease in virulence was ohsxartred for the eighth passage followed by an increase in iiu1112nce through the twelfth nedinn transfer. The sixteenth, twerrtixeth and twenty~fourth passages indicated a s sad; reduc- tiori ijfi virulence (Figure 1). When the fourth and eighth Pasnsagges of strain Wickard, wnich had been isolated from another expeurignentally infected sheep (repeat titration), were titrated, arliner-ease in virulence occurred after the fourth passace. All hamsters and guinea pigs demonstrated antibody PPOCUJCltiOH when they were inoculated with a 10"2 dilution of twitze. frozen and thawed filtrate cf the first culture passaoc. Fiftfiy percent of the guinea pi;s and hamsters inoculated with a 1(3-i3 dilution of the filtrate experienced a serological PeSFNDzlse. Inoculation of higher dilutions did not produce am31-body in either species. The leptospirel protein of the 0611-13.33 culture may have resilted in antibody production f“? 'tlle eighth passaCe titration in guinea pigs. A comparison of the titrations of the sixteenth pas- 585639 in which a whole culture or washed cells were used as ino‘ruxlal'fable 6), indicates that the whole culture material posseSsed a lower LDgO value. Microscopic examination of the Le’r’t’aOSpirae after three vashin;s in buffer revealed that many of the organisms had become granular, and some cellular disiritxsbration had occurred. In the counting of these lepto- E$1P¥i69, attempts were is to include only those cr-=" .c A on.) C 3" w- whicll Jere intact and had retained their normal morphology and. nuotility. Hamsters inoculated with filtered, frozen and thavaecl superna e of the culture material did ot disiglab’ any :3 4|) obsearxzable signs of infection. Sera from these anima‘s did not contain demonstrable L. pornona antibo’y. Hamsters and guinea pigs were equally susceptible to 9&S88;9 inoculum, whereas hamsters were s1i;-w t1; of the first and fourth passabes. B. \Iirulence Determinations with Strain Ohio Chandes in virulence similar to tie se observed ior stxreijri Rickard were noted. Tables 13-17 represent tlie results Ci? satx*ain Ohio titrations. Figure 1 demonstrates the rapid dec1°e£ase in virulence from the first to the iourth The L11230 values of these two titrations were 1 and 15,039: PbSPE’cigively. The virulence of strain Ohio increased with the .Sezvento and eleventh passa‘Q as at v a rate similar to that .Of StHTEiin Wickard. A SO percent end point was not reached Witfl the f11"8t passage. The LDJC value was determir ed as previously 9 described. Hamsters and guinea pigs were esser .tiall, equally M4309 Fili.ble to iniection w th this strain. Figure 2 illustrates the relation311 I L 1 be tween the C¢~ ‘thr ino culum and the duration 0‘ sur'iVul of infected p;- hamsters. The plotting of these cats results in essentially a straight line. From the slope of this line t}1e generation time of L. oomone in vivo hay be calculated using the method of Youmans and Youmans (72). The generation time is equal to the log of 2 divided by the reciprocal oi the slope. Tiis method of oalculatinc generation times in vivo is based on the ass‘mption that then death occurs a constant.number of organ Mi .s is present. Studies with L. icterohaemorr agi ti U! s.) infection in guinea pi_ have indicated that this rela ion- ship is present (2h). In the present stu dub leptos ire 1 counts for plasma from meribond hamsters ranged from 3 x 10v 1" o . . 1 . . to 10 organisms per cc. Since the nunber oi leptospirae in (‘A the blood at death hrs been reported as being from SC to 70 percent of the total number of or(anisms in the body (25), tne range of organisms in the plasma probably represents a rela- tively constant leptospiral population in the hamster at the time of death. A comparison of the dosaLe-response curves of lepto- Spirae possessing comparatively high and low de rees of viru— lence, indicates that the lepes are relativelv equal but that death of an animal inoculated with organisms of lower virulence was delayed about 5C hours. The fact that the curve of the less virulent culture is not linear near the Lbso inoculum is 2 probably due to the interference of antibody. The calculated generation time of E. pomona in hamsters was found to be 7.5 hours The majority of hams ers died between A and 12 days d. following inoculation. Exemina ion of plasiria from hamsters revealed the presence 0.; (D F4 <4 ‘3 L 1 O which died within this perio of leptospirae. However, in a few cases hamsters died 12 to 18 days following inoculation. Leptospirae were not seen in the plasma of these animals, but antibody was present as de- termined by the agglutination lysis test. C. The Effect of Age on Susceptibility of hamsters to E. pomona Table 16 records the results of two determinations on the swsccptibility of G and 15 week old hamsters to g. pomona as compared with A week old animals. Eight week old hamsters were slightly more susceptible than the L week old animals. 3'3 dilution of guinea pig blood -3.u It was calculated that a 10- would infect 50 percent of those 3 weeks old, whereas a 10 dilution would produce the same percentage of infections in the A week old hamsters. Fifteen and L week old hamsters were equally susceptible. A dilution of guinea pig blood of lO-B'é was calculated to be the dilution which would produce 50 per- cent infection in each age group. D. Growth of Leptospirae in Vitro after Passage in Media Table 1? summarizes the results of this studg. The numser of organisms necessary to produce Lrowth in 50 percent ..’.“| '~ 0 a _ _ h F’ 0x the inoculated tubes is: 2.4 for tne first passabe, c0.) fOP the fourth passage, and 22.9 for the eighth passage. 31 Adhesion of Leptospirae and Erythrocytes .A. [in Vivo Adhesion The results of leptosyirae-erythrocyte adhesion Po stud es usinb strain Nickard are summarised in Table 16. Titration of the blood components in hamsters indicated that rae were in the 131 51a and washed H- 9-. equal numbers of leptoSp erythrocyte portions. When the titrztion was made in Chang's me ium, a slightly hiuher concentration of le tos irae was noted in the erythrocyte portion than in the plasma. The control study, employing normal guinea gi' blood to which ‘u p. the Wickard strain culture h" been added, indicated that the organisms were ental 1* dis tributed between the erythrocytes and {1‘18 Sirga 0 Microscopic examination of blood from sneep, éuinea piLS ‘ and hamsters ootained during the leptospiremic phase 01 ini‘eC- tion 04 id not reveal adhesion of leptospirae to erytiroc te s. Adhes ion oi leptospirae to ac*ronacytes was observed in 7 to 30 day old Chang's medium cultures of infected blood from various animals. Invest l“£ tion of adhesion by visual methods uresence of filamentous elegents I. is often complicated by the attached to, or projecting from, erythrocytes. The occurrence of these "pseudospirocnetes" and methods for distinguisnint them from true Spirochetes have been reported (29). \A) to B. '1g Vitro Adhesion 1. When the supernatart fluid was separated from the erythrocytes which had been incubated with normal plasma, it was found that it contained over 90 percent of the original number of leptOSpirae. Less than ten percent of the original number of organisms remained in the fluid removed from erythro- cytes incubated with plasma which contained é. pomona antibody. An examination of diluted preparations of sedimented erythro- (D cytes by darkfielo microscoyy, fail d to reveal leptospirae in either the preparation of res blood cells incubated with normal or with innune plasma.. 2. Table 23 summarises the results of the second . 'A‘ l‘ 1y . I'i.’ ‘fi.‘ A. trial to demonstrate in V1 r adhesion oi C) erythrocytes and leptospirae. Lo adhesion was demonstrated. If adhesion had taken place, tube 6 containing antibody and complement would have contained fewer leptospirae than tube 5 to which no con- plemeni was supplied. The fewer numbers of orbanisms in these tubes containinQ L. pomona antiserum was probably due to lysis Of the leptospirae by lytic antibody. Examination of resus- P6n81ons of the sedimented erytnroc; tee from each tube did not show erythrocyte leptosoirae adhesion. 3. Adhesion of leptospirae and polystyrene latex eneres was not demonstrated in any of the tests. The twenty—fifth media cases 6 of the Nickard strair cemon stra ted the greatest hencl,tic activity. The virulerce of this culture was relatively lot. fne fourth passaée of this strain produced agproximately one-third the hemol;sin of the twenty-fifth passage and yet was co ideraoly more virulent. Culture 2 (Table 20) wnicn was not lethal for hamsters, demonstrated more henolgtic activity than many of Ft], the more virulent (lethal) c:ltu_re:. inc culture of the wicharc Ho strain wh on had been in thi& for three ;cars groouced the 10 west ceicentration of hcnolysin. “h culture stinzlated antibody formation in hamsters. The tiire pzse awe of this strain which was isolated from a mi ep, also produced low hemolysin concentrations. (hi tires of tie relatively virulent V ninth and tenth pdSSQ;€S of the hiclard strain (repeat titra- tion) indicated only noderatc henolgtic ECtiVltJ. A definite relations hi) between virulence and hcmclgtic potency was also absent with strain Ohio. Cultures of the C1- h Dassa es of this strain or ibited approximately L s) iyth and eigh 0’! 501 larcent of the hemol;fl tic activity ca inn: virulent fourth gassage. A correlation between the henolytic botentiel and the age of the culture, number of orbar isms in the culture at the time of deactivation, or number of passa;es in media, was not evident. Table 20 suuiarizes the results of this study. ‘D 1 na cultures were hemo- ) OHIO _* Table 21 indicates that E. lytically active against cow, rabbit, and hamster erythro- cytes. Guinea pig erythrocytes were not susceptible to hemolysis. Cow and sheep erythrocytes had relatively eoual susceptibilities. Rabbit cells were slightly more sensitive than those from hamsters. Differences between the comparative hemolytic activity of the various cultures for the erythro- cytes cf the different animals and those of sheep were not ob- served. b) UL V. DISCUSSION AND CONCLUSIONS The Effect of Medium Passage on the Virulence of t. pomgga A and B. The rapid decrease in virulence observed for both L. pomona strains following cultivation in media is in agree- ment with previous reports (66, 71). However, the fact that increases in virulence may occur following several medium passages of the leptospirae has been less frequently reported. Van Riel has been cited (66, l) to have observed an increase in virulence of a strain of g. icterohaemorrhagiae for guinea pigs after 8 months cultivation in media. In the present study the increases occurred for the fourth and tenth passages of strain Wickard and for the seventh of the Ohio strain. The repeated titration using strain Wickard indicated that an in- crease in virulence had occurred for the eighth transfer. Alterations in virulence are usually eXplained as a result of changes in the number of virulent organisms in the inoculum rather than as an alteration in tne virulence of each organism (21, 2h). Continuous éuinea pig passage of strain Wickard may have lessened the original or inherent virulence for hamsters. The single passage of the organisms in sheep may have selected a few virulent leptospirae which when transferred 36 in media reproduced and were in sufficient numbers in the fourth passage to cause death of hamsters. The increases in virulence observed with both strains after h to 8 medium passages may be due to a mutation. The culture medium would seem to favor the proliferation of the virulent mutant over the less virulent organisms. The subse- quent decrease in virulence observed after 12 medium passages of strain Wickard indicates'either a loss of the ability of the mutant to outgrow the parent cells or that another muta- tion to a less virulent population had occurred. Hamsters which survived the inoculum usually failed to demonstrate serum antibodies. It is evident that the lethal leptospirae must have constituted the greatest percentage of individuals in these inocula. The only exception was observed for hamsters inoculated with the first medium passage. Regardless of the mechanism involved in the virulence changes of these two strains, the change does not seem to be complete and is reversible. Morse (hi) has observed that swine inoculated with the nonlethal strain Wickard shed leptospirae in their urine which occasionally killed hamsters. Hamdy and Ferguson re- ported an attenuation of a strain of L. pomona for cattle after several hamster passages (30). The fact that passage of strain Ohio in guinea pigs did not seem to effect its virulence for hamsters may be be- cause this strain was in continuous guinea pig passage for 37 about 8 months prior to the initiation of virulence determina- tions, as compared to about 3 years for strain Wickard. Therefore, the length of time that the strain is maintained in guinea pigs may determine whether alterations in_the viru- lence of the organism will take place. It is possible that strain Ohio and strain Wickward possess different virulence factors. Guinea pig passage may have an effect on just a cer- tain factor or factors. Recent observations have shown a_dif- ference in the in gigg hemolytic activity of the two strains for hamsters (65). This may indicate a dissimilarity in one or several factors associated with the pathogenic properties of these strains. When the LDSO values observed for washed and unwashed cells of the sixteenth passage were compared, it was difficult to assess the observed difference. This was due to the dis- ruption of the organisms during the washing procedure. Al- though abnormal cells were not included in the enumeration, it is very likely that organisms incapable of reproducing were included among those counted. Due to these factors it does not seem justifiable to attribute a greater virulence to the un- washed leptospirae. A soluble agent capable of prouucing ob- servable illness was not demonstrated in the cell free culture fluid of this passage. A straight line was obtained when the average survival time of infected hamsters was compared to the number of organ- isms inoculated (Figure 2). From the slope of the line the 38 in vivo generation time of both strains was calculated to be 7.5 hours. The generation time of L1. icterohemorrhagiae in guinea pigs has been reported as 8.3 hours (2‘4). The relation- ship would appear to be significant. The increase in the average survival time of hamsters inoculated with a less virulent culture is probably due to there being fewer virulent organisms in this inoculum. Since there seems to be a constant number of leptospirae in an animal at death, a greater period of time would be required for the fewer virulent organisms in this inoculum to multiply to the required population. The observation that some hamsters die 1-6 days after the leptospiremic phase of the disease and have demonstrable antibody in their sera is in accord with the suggestion by Paine (2h) that the lesions of leptospirosis may develop to such a degree that they cannot be repaired or compensation cannot occur. ' Hamsters appeared to be slightly more susceptible than guinea pigs to infection with leptospirae of the early medium passages of the Wickard strain. The findings are in contrast to the report of Ringen and Okazaki (53), who found the guinea pig to be the more susceptible laboratory animal. The disagreement may be due to the fact that these workers used a strain of Lg. pomona which had been cultivated in media for several years. Other factors may be involved’such as the strain of animals used or the strain of La. pomona employed. 39 C. The Effect of Age on the Susceptibility of Hamsters to _I:. pomona Only slight differences in susceptibility to L. pomona (strain Wickard) were observed for LL, 8, and 15 week old hamsters (Table 16!. When susceptibility is based on the period of survival of infected hamsters rather than the number of organisms needed to produce infection, 3-5 week old hamsters have been reported to be more susceptible than older animals (30). The age of hamsters which may be employed in the diagnosis of leptospirosis does not seem to be limited to weanlings if infection in these animals is determined by development of antibody. D. Growth of Leptospirae lg vitro after Passage in Media The results (Table 17) indicate that the number of leptospirae needed to initiate growth in Chang's medium in- creased as the organisms were transferred in media. These findings are not consistent with the theory that as bacteria are transferred in media they become adapted to the media by the selection of adapted variants. The results may be due to differences in the media used for each titration. The medium component that is least under experimental control is the rabbit serum. The increased number of organisms needed to promote growth after h and 8 medium passages may be associated with the fact that these 11,0 passages were lethal for hamsters in contrast to the first passage. Adhesion of Leptospirae and Erythrocytes A. In vivo Adhesion _I_a_. Qomona (strain Wickard) was equally distributed between the plasma and washed erythrocyte portions of in- fected guinea pig blood as determined by titrations in nam- sters. Titrations in media indicated a slightly higher con- centration of leptospirae in the erythrocyte portion. Equal concentrations of leptospirae were found in the erythrocyte and plasma portions of normal guinea pig blood to which the leptospirae had been added. Leptospirae were not observed adhering to erythrocytes during leptospiremia in guinea pigs, hamsters, and sheep. It was not possible, therefore, to unequivocally demon- strate that adhesion was commonplace during leptospiremia. The presence of leptospirae in the erythrocyte portion was probably due to some extent to the speed of centrifugation. It was felt that 2,000 rpm for 15 minutes was necessary to insure complete separation of the erythrocytes and plasma. Other factors may be involved such as an electrostatic attraction of leptospirae and erythrocytes or adherence of the organisms to leucxocytes and platelets present in the centrifugate. hl IL in vitro Adhesion l. The results of this experiment indicated a removal of over 90 percent of the leptOspirae by the antibody treated erythrocytes. However, leptospirae were not observed by darkfield microscopy when a systematic search was performed on diluted suspensions of the erythrocytes. Approximately 106 organisms per cc would have been present in the diluted sus- pensions, a number which is readily detectable microscopically. It would seem that a large number of leptospirae had been des- troyed. Lytic antibody present on the erythrocytes or incom- pletely removed by washing seems to be the likely cause of the destruction of the leptospirae. 2. The results of this experiment (Table 19) also seem to be influences by lysing of the leptospirae by antibody. Complement is necessary for $2 yitgg adhesion to take place (37) but does not seem to be required for the lytic action of leptospiral antibody (71). The approximately equal reduction in the number of organisms in all tubes of the series which contained antiserum.indicates that complement is not necessary for this reduction to take place. In all cases the reduction is probably due to lysing of the leptospirae. 3. Polystyrene latex spheres did not seem to be a suitable material for the adhesion of leptospirae. Lamanna (37) reported that adhesion of bacteria and organic colloids has been.observed. h2 in vitro Hemolysin Troducticn in Relation to Virulence The hemolytic potency of the cultures examined seemed to be independent of the virulence of the culture for hamsters. A culture of strain Wickard which was not lethal for hamsters produced a greater concentration of hemolysin than several other passages of the strain which were lethal. The culture which demonstrated the highest LDSO produced the greatest hemolysis. Determinations of LDSO were not made for each of the cultures examined. The virulence of these cultures was assumed to be represented by the curve shown in Figure l. Virulence changes may have occurred between the points on this graph. However, no more than 1 to 3 passages were made between viru- lence determinations and it does not seem likely that marked alterations occurred within these limits. An explanation for the differences in hemolytic ac- tivity of the various cultures could not be ascertained from the experimental results. However, the suggestion has been made that mutations of the leptospirae occurred during the course of the investigation. The changes in hemolytic potency may be the result of differences in the metabolic activity of the mutants and parent cells. The differences in susceptibility of erythrocytes from several animal species to the hemolytic factor of Q. pomona is in agreement with previous reports (56). ;g_vivo hemolytic #3 activity in hamsters did not appear to correlate with the in vitro hemolytic activity of the strain for hamster enthro- cytes. The previously mentioned hematological examinations (65) of strain Ohio infected hamsters gave no indication of a hemolytic type of infection. However, the Ohio strain pro- duced comparatively significant amounts of hemolysin igpyitgg. The results of the present study indicate that the virulence of g. pomona may increase when the organisms are transferred in Chang's medium. Future work concerning the effect of various media on virulence and comparative growth rates of virulent and avirulent strains in media are indicated. There is a possibility that an alteration in the virulence of Q. pomona may occur when the organisms are maintained in guinea pig passage. The inoculation of sheep with the nonlethal strain may favor the proliferation of a few lethal mutants. The ef— fect on virulence of successive passages of a given strain in a single host species may be responsible for alterations in infectivity and lethality fo the "passage species" and other species. This would be important in the epizootiology of leptospirosis. The recently reported medium for observing the colonial morphology of leptospirae (16) might facilitate the investigation of leptospiral variation. Unfortunately, repeated attempts in this laboratory to cultivate leptospirae, known to be virulent, on this medium were unsuccessful. It has been suggested that the hemolytic activity of the leptospirae is an attribute of a hemolytic toxin (1). The results of the present study did not unequivocally demon- strate a relationship between the hemolytic activity of Q. pomona and its virulence for hamsters. If the hemolytic ac- tivity is due to a toxin, it would seem that this is only one of several factors concerned in virulence. in order to assess the role of the hemolytic factor in the course of leptospirosis, the purification and characterization of the hemolysin will be required. Subsequent demonstration that the purified toxin (hemolysin) contributes pg; 33 to the course of the infection might then be afforded. MS VI. SUNhARY An investigation was made on the effect of medium passage on the virulence of two strains of g. pomona for hamsters and guinea pigs. Strain Wickard which originally produced only a mild infection in hamsters, was lethal for these animals after h passages in media. A decrease in viru- lence was observed in the early medium passages of both strains, followed by an increase in virulence after h to 8 transfers. After 12 passages, strain Wickard demonstrated a steady de- crease in virulence. Similar changes in infectivity for guinea pigs were noted. It was suggested that the changes in virulence may be the result of mutations of the leptospirae. Hamsters were slightly more susceptible to E. pomona than guinea pigs. Four, 7 and 15 week old hamsters were es- sentially equally susceptible to infection. The 22.1l12 adhesion of leptospirae and erythrocytes was not unequivocally demonstrated. in vitro studies did not reveal adhesion, although the presence of lytic antibody nay have influenced the results. The virulence of the various medium passages was not related to the ability to producL hemolysin.i§ vitro. The susceptibility of hamster erythrocytes to the hemolysin from 146 the various cultures did not seem to be associated with the ability of these organisms to produce a henolytic effect in hamsters. 1+7 TABLES Tables l-lh. *Average number of days after inoculation that animals survived. **Numerator = number of animals infected; Denominator = number of animals inoculated. 45.. C3 TABLE 1 TITRATION OF STRAIN WICKARD IN EAMSTEHS, MEDIA PASSAGE NO. 1 A ——_—-—--- “~——_ Number of Number of Number of Average Leptospirae Animals Deaths Survival per Inoculum Infected Time* 1.22 x 104 7/7** c - 1.22 x 103 7/7 0 - 1.22 x 102 7/7 0 - 1.22 x 101 6/7 0 . - 1.22 1/7 0 — M9 .. -- o o m\o m\o sea a om.m mo.a -- u- o o m\o m\o NOH a om.m flea a mo.fi m.oa -- e o m\m m\o moa x om.m mow a mo.H o.m m.mH m m m\m m\m sow a om.m non a mo.H e.ma e.HH m m t.m\m a*m\m moa w om.m sea a mo.H consensus consensus coapmapne coapmapae consensus soapswpaa soapmspae consensus pmodom chflmfiao pmmdom stflwaao pwmdmm HmsamHao peodom Hmsamfiao . ESHSQOGH m $35.9 9&9ch . a., a pappsm mwmnope to assess empowchsmwmmHe< “ma mmefiamopqmq J .02 WUdWma>adm owmao>< consensus ymofipsspne.. monumepaa eoapmspae chflwwao umodom chHmHao no Amafidz vopoomcH mamefic< no acnfisz scapegoae soapwapae pmodom chfimaao EdadoocH nod mdaHdmOpdoA ho 909552 w m mum49. .mdm «Ham; «mmmemd¢m 2H QMdMOHB KHdem m0 ZOHBH>a5w ommao>< no AmQEsz Ho hopsdz mo honedz 0a .02 flo¢mm105 3.8 " u " 1,509 1,509 I?“ " 8 " 235,885 235,885 ‘ " 10 " 2h,500 16,9h9 é " 12 " 709* 709* 1 " 16 " 968 968 L——3 " l6 (washed " 1,716 1,716 cells) n 20 " 7,760 7,760 " 2h " 271,200 271,200 " a (repeat) " 1,600 1,212 " 8 (repeat) " 1,202 261* " 1 Guinea pig -- 8.2 n u " -- 6,823 n 8 " -- 235,885 Ohio 1 Hamster 0.9* 0.9% " h " 15,051 15,051 " 7 " 8,607 8,607 " 11 " 29 29 " h Guinea pig -- 15,051 " ll " -- N7 *50 percent endpoint not reached. These values repre- sent the highest possible value which could be calculated from the data. 59 TABLE 16 THE EFFECT OF THE AGE OF HANSTERS ON SUSCEPTIBILITY T0 L. POMONA (WICKARD) Dilution Number of Number of of Infected Four Week Old Eight Week Old Guinea Pig Blood Hamsters Infected Hamsters Infected -1 - e:- c’ £1- 10 5/5 2/5 -2 10 5/5 5/5 -3 10 M5 5/5 -1 F, 10 L 0/> 2/5 10'5 0/5 0/5 Dilution Number of Number of of Infected Four Neck Old Fifteen Week 01d Guinea Pig Blood Hamsters Infected Hamsters Infected -2 :6 66 10 5/5 5/5 - 6H6 1O 3 3/3 5/5 4+ 10 1/5 1/5 16-5 o/s 0/5 -6 10 0/5 0/5 *Numerator = number of hamsters infected; Denominator 8 number of hamsters inoculated. aw Two hamsters escaped from the cage. Thus only 3 hamsters remained for infectivity determinations. ' —'—-—-—Q—-—-——v 4 1 , e . 5 i .7 r .I ..IN«A.1 i u. 61 H. u.l ‘ZIunathI .fi .6; .00pwa5oocH 0005p 90 900555 N 9000:5EOC0D mnpzo9m £003 00950 no 900E5C u 900090E5ze m\o 00.5 m\H 0.0 . 5\m 00H m\m . HOH a 00.5 m\m HOH x 0.0 5\5 HOH a mm.H m\m ‘ N0H x 00.5 m\m N0H a 0.0 5\5 N0H x mm.H m\m 00H m 00.5 m\m 00H x 0.0 5\5 00H x mm.H w +. n . x .. K . 6m\m 00H 0. 5 6L\m 00H 0 0 *5\5 00H mm H £03090 E5H5005H 900 593090 E5H5oosH 905 £03090 S5H5005H 90d wconnm 0005B 009Hamopdeq mstoCm 0035B 009Hnmopdoq wcHZonm 0095B 009Hdmoud0q 90 900552 90 902852 _ 90 900853 9o 900852 no 900352 90 900852 m 0memmwm : awammwm H 0M0mmmm '1‘ 00040000 «Home 0 50¢ a .H 00504 omeHa mm «20200 .H no 053000 5H mqm Mm mMHMoommBMmm Amman ho mHmMHozmm mme ON mqmdB .0ocHenopo0 007-- .0 09dem 8099 0090H0009pKM# m 0M0mmmm 00008 90 wonasw SO99 OHSO CwmhmeH HmnH O O O O mm mm m0 O CO 00 OO OOHNO.0 oo\0OHx0.H 0000 OH 0 0w00000 0900E 0muH O O O O HH Hm m0 H9 m0 OOH 0OHKO.H 00\ OHK0.H 0000 OH 0H0 mocHsm 5009 0 0H0O :Hmnpmo : 0m00000 0900§ “muH O O O O OH O0 m9 O0 OO OO :Ome.H oo\0OHxO.O 0900 OH 0H0 mocHsm £009 oHnO chnpmm mflmhaoa0m £90090» 0m ‘2... do 0mN ._ ... ... 0.2; «0... an... 0:. 0; .02. 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J , 4 iouqut-O.Ao.ot a... ..o. .o‘OAQOo. nacbl 55.90 T1§1II1086. f v§vool ¢.ov o... ..o. u... .‘ou v1.- Obo.w..rb «“1t ‘oo. a 401. I... i..- .... .o.. ...o L '0' 01910. coo. ....vo vvvnnAu!.o|;w‘:o1|19.o-vfi .0.. no! ‘00.} 9.0.4-... odov t... :q p 4 a 4 1‘50. ...o v... ...o+.... on L J 1:. .9..YL¢.‘ am.&A+ 7.... ...o 14'"... ...c .... otoi‘...u o... ....A...., 70.4 o a A ¢ 9! 10.0. - 0A 44 0., o . p u u . . . u . v10; v61. .1 1-10 u ‘31. o . ¢ n o . 4 ~ . o c . H001 OOoQAOOAC . o. ..oovl¢1uk’ toIO11 omnivAY9quc 00.. V..t v 10. 11. REFERENCES Alexander, A. D., Smith, 0. H., Hiatt, C. W. and Gleiser, A. C. Presence of hemolysir in cultures of pathogenic leptospires. Proc. Soc. Haber. Biol. and 163.,‘2_ (1956): 205-211 0 Babudieri, B. The morphology of the genus Leptospira as shown by the electron microscope. J. Hyg., Q1 (19h9): 390-392 0 Baker, J. A. and Little, R. B. Leptospirosis in cattle. J. Exp. Med., g2 (19u8): 295-303. Bauer, J. 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