ABSTRACT PATHOLOGY OF EXPERIMENTAL LEPTOSPIRA POMONA INFECTION IN HAMSTERS by Mohamed-Tewfik Fawi Abdu A series of experiments using 91 hamsters was conducted to study experimental Leptospira pomona (strain Ohio) infection. In addition to gross and microscopic pathological studies; serological, bacteriological, hematological and clinical observations were made. Marked central nervous system disturbances were noticed as early as day 6 (post-inoculation) in hamsters which received the high dose (approximately 2, 500 organisms), and on day 8 in animals inoc- ulated with the low dose (approximately 100 organisms). The first microscopic renal lesions were observed in the renal cortices of hamsters inoculated with the high dose by day 2. These lesions consisted of albuminous degeneration, vacuolization and some necrosis as evidenced by pyknosis of the epithelial cells in the proxi- mal and distal convoluted tubules with a few intertubular and peri- corpuscular lymphocytic foci. By day 6, in addition to these lesions, hyaline casts were seen in the lumens of the renal tubules. There was also partial disappearance of tubular cells with areas of inter- tubular hemorrhage. The liver had areas of albuminous degeneration and hepatic cell individualization. Mohamed-Tewfik Fawi Abdu Demonstrable microscopic renal lesions were present by day 6 in animals inoculated with the low dose. These lesions consisted of a few intertubular and pericorpuscular foci of lymphocytic infiltration. By day 7, in addition to the lymphocytic foci, the renal tubular epithe- lium had marked albuminous degeneration with vacuolization, and some of the Bowman's capsules appeared to be slightly thickened. There were also numerous hyaline casts in the lumens of the tubules with a few areas of perivascular lymphocytic infiltration. The liver had areas of hepatic cell individualization with perivascular lympho- cytic infiltration in the areas of the portal triads. By day 9, early proliferation of fibroblasts and leukocytic infiltration were present in the portal triad regions. Gross renal lesions consisting of greyish-white foci approxi- mately 0. 5 mm. in diameter on the cortical surface were observed in hamsters inoculated with the high dose starting on day 5. 'Similar lesions were seen in hamsters inoculated with the low dose starting on day 7. No significant microscopic lesions were seen in the brains of infected hamsters, even in those showing marked central nervous system disturbances. The concentration of hemoglobin and the packed-cell volume decreased to levels below normal on day 7 in hamsters inoculated with the high dose. On post-inoculation day 8, the hemoglobin concentration Mohamed-Tewfik Fawi Abdu and packed-cell volume values reached a level considerably higher than normal which was indicative of hemoconcentration. Only terminal hemoconcentration was noticed on days 8 and 9 in animals which re- ceived the low dose. Blood nonprotein-nitrogen determinations showed that there was a progressive rise starting on day 7. Terminal values of 424 mg. /100 ml. or approximately ten times the normal levels of 40 mg. /100 ml. were recorded. The dramatic rise in blood nonprotein nitrogen was believed due to acute renal failure. Central nervous system dis- turbances were postulated to be a result of the severe uremia. All blood cultures from infected animals were positive including cultures made from infected animals showing significant antibody titers. The first detectable antibody production was on day 5 in the case of hamsters inoculated with the high dose, and on day 8 in hamsters inoculated with the low dose. Leptospira pomona was found to be present in the kidneys of hamsters inoculated with the high dose starting on day 4, and in the brain starting on day 6. In the case of hamsters inoculated with the low dose 1'1: pomona was present in the kidney tissue starting on day 6 and in the brain starting on day 8. PATHOLOGY OF EXPERIMENTAL LEPTOSPIRA POMONA INFECTION IN HAMSTERS By MOHAMED-TEWFIK FAWI ABDU A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department of Veterinary Pathology 1962 Dedicated to My Father and Mother. iii AC KNOW LEDCEMENTS The author wishes to express grateful acknowledgements to the following: To Dr. C. C. Morrill for his guidance, patience and under- standing; To Dr. S. D. Sleight who kindly contributed his time, sug- gestions and supervision; To Dr. R. F. Langham for his guidance, assistance, and advice; To Drs. W. E. Field and D. A. Schmidt for their technical assistance and suggestions; To The Ministry of Animal Resources, Republic of the Sudan, and United States Operations Mission to Sudan for their encourage- ment and financial assistance; To Mrs. A. Lundberg, Mrs. N. Anderson, Mrs. D. Palombo, Mrs. J. Bradley, and Mrs. S. Hansen for their technical assistance. TAB LE OF CONTENTS INTRODUCTION . .................................... LITERATURE REVIEW ................ . .............. MATERIALS AND METHODS EXPERIMENTAL RESULTS Clinical Hematological Serological Bacteriological Pathological DISCUSSION ...... . ...... . SUMMARY AND CONCLUSIONS . . . . . . . . . ..... TABLES 1 THROUGH 4 . . . . FIGURES 1 THROUGH 17 . . . REFERENCES ........... . iv 14 14 14 15 16 16 19 24 28 32 49 Table 4a. 4b. LIST OF TAB LES Summary of average hematological values ............ Differential leukocyte count average values ........... Antibody titers for I; pomona in sera of infected hamsters ....................................... Summary of guinea pig inoculations with homog- enized tissues from hamsters inoculated with the high dose .................................... Summary of guinea pig inoculations with homog- enized tissues from hamsters inoculated with the low dose .................................... 3O 31 32 vi LIST OF FIGURES Figure Page 1. Kidney at day 2. A. Pyknotic nuclei in tubular cells. B. Lymphocytes in the intertubular and pericorpus- cular areas. C. Vacuolization of tubular cells. x720 . . 32 2. Kidney at day 6 demonstrating hyaline casts in lumen of tubules x250 ..................................... 33 3. Kidney at day 6. A. Vacuolization of tubular cells. B. Hyaline casts in lumen of tubules. x720. ........... 34 4. Kidney at day 6. A. Hyaline casts in lumen of tubules. B. Pyknotic nuclei in tubular cells. C. Vacuolization of tubular cells. D. Partial disappearance of tubular epithelium. E. Intertubular hemorrhage. x7 20 . . . ..... 35 5. Kidney at day 6. A. Lymphocytes in intertubular area. B. Pyknotic nuclei in tubular cells. x7 20. . . . . . . . . . . . . . 36 6. Liver at day 6 demonstrating hepatic cell individualiza- tion (high dose). x720 . . . . . . . . . ........ . ..... . ....... 37 7. Kidney at day 6 demonstrating lymphocytes in pericor- pusculararea. x720. ......... 38 8. Kidney at day 7 demonstrating numerous hyaline casts in lumen of tubules. x250 ....... . ...... . . . . ......... 39 9. Kidney at day 7 showing marked vacuolization of tubular cells. x720 ............... ...... 40 10. Kidney at day 7. A. Vacuolization of tubular cells. B. Slight thickening of Bowman's capsule. x720. . . . . . . . 41 ll. Kidney at day 7 demonstrating perivascular lymphocytic infiltration. x720 ................ . ...... . . . . . ...... 42 12. Liver at day 7 demonstrating hepatic cell individualiza- tion(1ow dose). x720 43 Figure l3. 14. 15. 16. 1?. Liver at day 7 demonstrating perivascular lymphocytic infiltration in the area of portal triads. x7 20 . . . . . . . . Liver at day 9. A. Hepatic cell individualization. B. Proliferation of connective tissue in areas of portal triads. x250 . .. ...................... . ...... Liver at day 9 demonstrating lymphocytic infiltration in area of portal triad. x7 20 ........... . . . . . ...... Liver at day 8 demonstrating leptospirae. x 1,150 . . . Kidney at day 9 demonstrating leptospirae. x 1,150 . . . vii Page 44 45 46 47 48 INTRODUCTION Leptospirosis, caused by Leptospira pomona, is a disease of considerable economic and public health importance (25, 44). This disease in domestic animals is important not only because of the effects on the animals (30, 32), but also because infected hosts may serve as reservoirs of infection for man (6). The possibility of using the Syrian hamster as the animal of choice for isolation of leptospirae has been investigated by several workers (24, 33, 34). Trier (42) observed that central nervous system disturbances were produced in hamsters after inoculation with 1;.pomona (strain Ohio). The purpose of this experiment was to study the pathological manifestations of L. pomona (strain Ohio) in hamsters with particu- lar reference to central nervous system disturbances. LITERATURE REVIEW Leptospira pomona was first isolated from a dairy farmer in Queensland, Australia, by Clayton 9.13.1; , in 1936 (12). Natural infections produced by _I:__ pomona have been reported in man (8), cattle (1), swine (5, 17), horses (36), sheep (20, 37) and dogs (45). Serological surveys have shown that _I__:_ pomona infections are distributed in all states of the United States (5, 6, 9, 18, 30, 35, 37). It has been estimated that the loss from leptospirosis in farm animals is greater than 100 million dollars per year (44). The great- est share of this loss is due to bovine leptospirosis. Although mor- tality may not be high, the financial losses may be considerable due to abortions, loss of milk yield in dairy cattle and weight loss in beef cattle (27). Within the last 10 years there has been a remarkable change in opinion regarding the importance of leptospirosis in human and animal health. It is now considered a major public health problem. Many wild and domestic animals living in close association with man are capable of transmitting the infection either by direct or indirect contact (25). Swine are apparently the main natural host of _I:_ pomona (46). The majority of cases in swine are subclinical, with the exception of abortion and death of newborn pigs (7). Experimentally, it has been shown that swine can transmit _I._J._ pomona infection to other pigs, cattle, sheep and goats (ll, 31, 32). Leptospira pomona infection in cattle is characterized by abor- tions, decreased milk yield, anorexia, general depression and hemo- globinuria (30). A naturally occurring case of bovine leptospiral meningitis with clinical symptoms was described by Hoag and Bell (21). The disease in sheep results in inappetence, fever, hemoglobin- uria and abortion (31, 37). Langham St El: (23), in their study of experimental ovine lepto- spirosis, found that the principal macroscopic changes in _L. pomona infected lambs were greyish-white circumscribed foci and streaks in the renal cortex. Some of these lesions extended through the cortex into the medulla. Microscopically the lesions consisted of focal infiltrations of lymphocytes, some plasma cells and macrophages. These lesions were usually peritubular, perivascular, or pericor- puscular. The central nervous system of some of these lambs showed one or more of the following: scattered areas of increased oligoden- droglial and microglial cells in the cerebrum, meningitis with lymph- ocytic infiltration, and perivascular hemorrhages. Morse_e_3_t_a_l. (29), in their study of experimental caprine lepto- spirosis, found that the gross pathological alterations in goats were confined to the kidneys as greyish-white foci extending throughout the depth of the cortex. Microscopically these foci consisted of accumu- lations of lymphocytes, plasma cells and a few macrophages. Some goats in the experiment showed microscopic brain lesions. The cere— bellum, cerebrum and medulla showed perivascular cuffing with lymph- ocytes, plasma cells, macrophages and edema. In some brain sections there was an increase of oligodendroglia, while others showed lesions in the medulla oblongata with numerous gitter cells. Sleight _e_ta_l_. (40), in their study of the early pathogenesis of L. pomona infection in young swine, observed macroscopic lesions in the kidneys similar to those previously described. Microscopically, the majority of these lesions were rather discrete, consisting mainly of infiltrations of small numbers of lymphocytes between tubules. Other lesions included hyaline casts, hydropic degeneration and pyknosis of tubular cells and an increase in collagenous fibers. Some animals showed brain lesions which were located principally in the cerebrum and consisted of meningoencephalitis characterized by perivascular lymphocytic infiltrations. Sleight and Lundberg (41) isolated I:_ pomona from brain tissues of experimentally infected swine as long as 18 days after inoculation. They found that leptospirae were present in the kidneys from post- inoculation day 4 until the experiment was terminated at day 45. Morton (33) in 1942 was the first one to report on the suscepti- bility of Syrian hamsters to leptospirosis. He showed that subcutan- eous injections of L_ icterohaemorrhagiae into 3'to 5-week old hamsters resulted in death of the animals 5 to 8 days after inoculation. Subcutaneous inoculation of _I_4. canicola into 3-to 5—week old hamsters failed to bring death, but cultures made from heart blood were posi- tive for leptospirae up to 96 hours after inoculation. Randall and Cooper (34) in 1944 reported that the Syrian hamster was the animal of choice for the isolation of leptospirae. Both 1_J. canicola and I: icterohaemorrhagiae killed hamsters in 5 to 10 days. Larson (24) in his investigations of experimental leptospirosis in hamsters, found that intraperitoneal inoculation of a 10% emulsion of liver and kidney tissue from a guinea pig infected with _I__,, canicola produced death of the animals within a week after inoculation. At necropsy, hemorrhages were found in the lungs. Examination of the peritoneal fluid under dark-field illumination showed many active leptospirae. Inoculation of a 10% emulsion of liver and kidney tissue from a mouse injected with 1.; icterohaemorrhagiae into hamsters caused death of the animals 5 to 8 days after inoculation. At necropsy, leptospirae were detected in the tissues by dark-field illumination. The tissues were icteric and hemorrhages were present in the abdominal muscles, lungs and kidneys. Uhlenhuth (43) infected Syrian hamsters with aspirated periton— eal fluid of a guinea pig inoculated with .12 canicola. The hamsters became sick and died in 7 to 12 days. Icterus occurred shortly before death. At necropsy, hemorrhages in the lungs, kidneys and liver were observed. Doljansky (14) reported that Syrian hamsters did not show any clinical signs nor lesions when inoculated with certain strains of I: grippotyphosa. However, a serological examination showed a rise in agglutinin titer and the hamsters were found immune to infection against pathogenic strains of_L_. grippotyphosa. Hamdy and Ferguson (19) studied the effect of serial passages on the virulence of I: pomona (Hardacre strain) for hamsters. The animals recovered from early passages but their kidneys were grossly involved. As passages progressed, animals died within 4 days after inoculation. At necropsy there were hemorrhages in the lungs, and the liver and the kidneys were congested. Microscopically, pulmonary hemorrhages and infiltration with lymphocytes and monocytes occurred, accompanied by broken alveolar walls. There was necrosis of hepa- tic cells. The kidneys showed hemorrhages in the tubules with some hyalinization of the glomeruli. In the chronic phase of the disease only the kidneys were involved. There was an increase of interstitial con- nective tissue and degeneration of the epithelium of the tubules. Some glomeruli were shrunken while others were edematous. Bauer (2) reported that hamsters were susceptible to infection with k pomona (strain Ohio). He found that there was an inverse relationship between the size of the inoculum and the duration of survival of infected hamsters. He also observed that 4-, 8- and lS-week-old hamsters were nearly equally susceptible to I: pomona. Bauer (3) later observed hematological changes when hamsters were inoculated intraperitoneally with 1". pomona (Strain L W). The blood changes occurred 72 hours after inoculation. There was an increase in the urea values and in the bilirubin levels, while a decrease in hemoglobin and hematocrit readings was observed. Dif- ferential leukocyte counts indicated a relative neutrophilia and a cor- responding relative lyrnphocytopenia. Fizette (15) studied the relationship between the leptospirae and the animal hoSt using golden hamsters infected with a highly virulent strain of 1". pomona. Blood taken from the diseased ham- sters in the terminal stages of the infection showed a slight increase in blood clotting time. The administration of a wide range of dosages of different forms of vitamin K failed to bring about the decrease of hemorrhages in the infected animals. According to Trier (42), central nervous disturbances were produced in hamsters as early as the fifth day after inoculation with L. pomona (strain Ohio). The head was held back, the legs stiffened in extensor rigidity and paralysis occurred. In some cases the animals trembled violently. There was considerable hematological change. Minimal levels of 9. 3 gm. /100 ml. of hemoglobin and 4, 910, 000 erythrocytes /cu. mm. were observed. A leukocytosis occurred but the author was of the opinion that this might have been the result of hemocentration. I: pomona (Ohio strain) was shown to be lethal by day 7 after inoculation. Burk (10) reported that strains of leptospirae highly virulent for hamsters gave rise to a fatal acute leptospirosis. Strains of moderate virulence produced a chronic leptospirosis with kidney localization and positive serological tests after 21 days. Lewis and Gray (26) established an acute fatal leptospirosis in gerbils when 1000 or more virulent I: pomona organisms were given. Animals inoculated with less than 1000 organisms did not develop a lethal infection but a chronic infection was established. MATERIALS AND METHODS The strain of 1:. pomona used in these experiments was first isolated from porcine urine in Ohio (39). Lately it has been main- tained in continual guinea pig passage to assure retention of its virulence. In the first experiment 20 hamsters 4 to 6 weeks old were used. Sixteen animals were each inoculated intraperitoneally with an inoc- ulum which consisted of 0. 5 ml. of a 1:1 saline dilution of L. pomona infected guinea pig blood collected at the peak of the febrile response. Due to the shortage of guinea pigs, titration to determine the number of organisms present per ml. of guinea pig infected blood at the height of pyrexia was not done. However, previous studies in this laboratory have shown that approximately 104 organisms per m1. are present in the infected blood of guinea pigs at the climax of pyrexial response (1050 to 1060 F.) (42). Therefore, it is calculated that each hamster received approximately 2, 500 organisms. These ham- sters are designated from here on as high—dose animals. The remaining 4 hamsters were used as controls and inoculated with 0. 5 ml. each of a 1:1 saline dilution of normal guinea pig blood. Each animal was put, in an individual cage, with controls being separated from the infected ones. 10 The killing date for each animal was predetermined by random number assignment, 3 infected hamsters and one control being killed on alternate days starting on day 2. (Here and hereafter, the day referred to is the post-inoculation day.) Some animals having become moribund, it was necessary to kill them before their assigned day. By day 8 the remaining infected animals died. In the second experiment, 24 hamsters 4 to 6 weeks old were used. Nineteen of these animals were inoculated and the remaining 5 served as controls. The inoculum was similar to that used in the first experiment and these animals are included in the high-dose group. Isolation procedures were as previously described. Three infected animals and one control were killed on alternate days starting with day 3. Animals were killed before their assigned day as soon as they were observed to be moribund. By day 8 the remaining infected animals had been killed. In the third experiment, 18 hamsters 4 to 6 weeks old were inoculated intraperitoneally with 1:. pomona (Ohio strain). The inoculum consisted of 1 ml. of a 1:100 saline dilution of infected cavian blood for each hamster. By calculation, each hamster thus received approxi- mately 100 organisms. These hamsters are designated from here on as the low-dose animals. Four hamsters, serving as controls in this experiment, were each inoculated intraperitoneally with 1 ml. of 11 sterile saline solution. Isolation procedures were as previously described. The sacrificing date for each hamster in this group was predetermined by random number assignment; three infected animals and one control were to be killed on days 9, 12, 15, 18, 24 and 30. Animals were killed before their assigned day as soon as they were recognized as moribund. The first of the infected hamsters died on day 8. By day 9, the remaining infected animals died or were killed. In the fourth experiment, 25 hamsters 4 to 6 weeks old were used. Twenty of these animals were artificially infected and the remaining five were used as controls. The inoculum was similar to that used in the third experiment and these animals are included in the low-dose group. Isolation procedures were as previously described. The killing date for each hamster in this group was predetermined. Starting on day 3, 2 infected animals and 1 control were killed daily, except that no control was killed on days 5 or 7. Moribund animals were killed before their assigned day as soon as they were observed to be in that state. The animals in the four experiments were anesthetized with ether and bled aseptically by cardiac puncture using heparinized syringes and needles. A few drops of the collected blood were used in inoculation of Stuart's medium (Difco) enriched with 10% rabbit serum, using 2 . . . 0 tubes of medium for each animal. The cultures were incubated at 30 C. 12 for a period varying up to 4 weeks and examined for the presence of leptospirae employing the dark-field microscope. The remaining blood was used for serological and hematological studies. After bleeding, the animals were sacrificed by administering an overdose of ether and were necropsied. Serological examinations were conducted by the modified micro- scopic agglutination-lysis test (16, 29), using 1:. pomona (strain Johnson) live antigen. Hematological studies were done on hamsters of the last three experiments to establish hemoglobin values, packed cell volumes, and differential leukocyte counts (4, 13). In addition, blood nonprotein nitrogen (4, 13) and total leukocyte counts were run on some hamsters of the third experiment and all hamsters of the fourth experiment. When volumes of individual blood samples were not sufficient for determining n onprotein nitrogen, pooled blood samples were used. Tissues were saved from the kidneys, liver, spleen, lung, skeletal muscle, brain and spinal cord of each animal. These tissues were placed in one or more of the following fixatives: Zenkers fluid, 10% neutral formalin solution and Carnoy's fluid. The sections were stained by one or more of the following staining procedures: hematoxylin and eosin for general characteristics, oil red O for fat, Best's carmine stain for glycogen, Warthin-Starry method 13 for spirochetes and cresyl echt violet for cellular details of brain substance (28). Samples of kidney and brain tissue were saved for guinea pig inoculation from every group of animals killed, including the con- trols. The tissues were homogenized in 0. 85% sterile sodium chloride solution to give approximately a 10% tissue suspension. One guinea pig was inoculated intraperitoneally with 1 ml. kidney suspen- sion, and another was injected intraperitoneally with 1 ml. brain suspension from each group killed, including the controls. Blood from these guinea pigs was obtained for serological study 14 days after inoculation. The inocula were considered to contain leptospirae if the sera of the inoculated guinea pigs contained antibodies for 1:. pomona at a dilution of 102 or higher (29, 39). 14 EXPERIMENTAL RESULTS Clinical The only significant clinical symptoms observed were the marked central nervous system disturbances. These were noticed as early as the 6th day in hamsters which were inoculated with the high dose and on the 8th day in animals inoculated with the low dose. The animals showed hyperexcitability with dorsal head flexion, lordosis and tone- clonic spasms. Hematological Hematological data obtained during the course of the experiments are summarized in Table 1. In animals inoculated with the high dose, the hemoglobin concentrations and packed-cell volumes were normal up to day 6. On day 7 the hemoglobin and packed-cell volume values were markedly lowered, but on day 8 the hemoglobin concentrations and packed-cell volumes reached levels of 18. 1 gms. /100 ml. and 49. 8 per cent respectively. Hamsters with hemoconcentration were moribund and were showing the central nervous system disturbances listed above. In hamsters which were inoculated with the low dose, the hemo- globin concentrations, packed-cell volumes and total leukocyte counts were found to benormal up to the 7th day. On the 8th and 9th days 15 the values of the above-mentioned three constituents rose. These hamsters were also moribund and showed the central nervous system disturbances. Blood nonprotein nitrogen determinations were done on blood samples obtained from animals inoculated with the low dose. The values of blood nonprotein nitrogen remained for the most part with- in the normal range (40 mg. /100 ml.) up to the 6th day. On day 7 the blood nonprotein nitrogen rose to a value of 124 mg. /100 ml. and reached a maximum of 424 mg. /100 ml. by day 9. Results of differential leukocyte counts are given in Table 2. The high-dose animals had a relative neutrophilia and lymphocytopenia starting on day 7. The low-dose group showed a relative and absolute neutrophilia and lymphocytopenia starting on day 6. Serological Table 3 summarizes the agglutination-lysis test results both in the control and infected hamsters. The first detectable antibody pro- duction was on day 5 in the case of hamsters inoculated with the high dose and on day 8 in hamsters inoculated with the low dose. The maxi- mum agglutination-lysis reactions were present in the 1:100 serum dilution on day 8 in the animals inoculated with the high dose. l6 Bacteriological Leptospirae were demonstrated in tissues of the infected ham- sters by inoculation of guinea pigs with homogenized hamster tissues (Table 4). These findings indicated that _I:‘_ pomona was present in the kidneys of the hamsters inoculated with the high dose from day 4 until the animals died or were killed, and in the brain starting on day 6 and thereafter until the animals died or were sacrificed. In the case of hamsters inoculated with the low dose, the organisms were present in kidney tissue starting on day 6 and thereafter until the animals expired, and in the brain from day 8 until the animals died or were killed. All blood cultures from infected animals were positive includ- ing cultures made from infected animals showing significant antibody titers. Pathological A. Gross A few greyish-white foci mainly located in the renal cortices were observed in hamsters inoculated with the high dose starting on day 5. These lesions were approximately 0. 5 mm. in diameter. In hamsters inoculated with the low dose, a few scattered greyish-white foci principally in the renal cortices were first seen in hamsters sacrificed on day 7. 17 There were no other gross lesions observed which could be attributed to L. pomona infection. No significant gross lesions were observed in the control animals. B. Microscopic The first microscopic renal lesions were observed in the cor- tices of hamsters inoculated with the high dose killed on day 2. These lesions consisted of albuminous degeneration, vacuolization and some necrosis as evidenced by pyknosis of the epithelial cells in the proxi- mal and distal convoluted tubules with a few foci of lymphocytes in the intertubular and pericorpuscular areas (Figure 1). On day 6, in addi- tion to the lesions described above, hyaline casts were in the lumen of the renal tubules. There was also partial disappearance of tubular cells with areas of intertubular hemorrhage (Figures 2, 3, 4 and 5). The liver had areas of albuminous degeneration and hepatic cell individualization (Figure 6). In hamsters inoculated with the low dose, the first microscopic lesions were present in the renal cortices of animals killed on day 6. The lesions consisted of a few foci of intertubular and pericorpuscular lymphocytic infiltration (Figure 7). On day 7, in addition to the lymphocytic foci, the renal tubular epithelium had marked albuminous degeneration with vacuolization. Some of the Bowman's capsules appeared to be slightly thickened. There were also numerous hyaline 18 casts in the lumens of the tubules with a few areas of perivascular lymphocytic infiltration (Figures 8, 9, 10 and 11). The liver had areas of albuminous degeneration, and hepatic cell individualization with perivascular lymphocytic infiltration in the areas of the portal triads (Figures 12 and 13). By day 9, early proliferation of fibro- blasts and lymphocytic infiltration were in the portal triad regions (Figures 14 and 15). No significant microscopic lesions were seen in the brains of the infected animals, neither in sections stained with hematoxylin and eosin nor in the sections stained with cresyl echt violet. No significant microscopic lesions were seen in the control animals. Leptospirae were found in the Warthin-Starry stained sections of the kidney and liver in hamsters inoculated with the low dose and killed on days 8 and 9 (Figures 16 and 17 respectively). No lepto- spirae were demonstrated in the remainder of the stained tissues. 19 DISCUSSION The hamsters used in these experiments were found to be sus- ceptible to infection with _L_ pomona (strain Ohio). This strain has previously been observed to be lethal for hamsters by several work- ers (2, 3, 42). In addition Trier (42) noted that severe central nervous disturbances were produced by this strain in hamsters but no attempt was made to study the pathological changes. As previously stated, it was estimated that approximately 2, 500 leptospirae were present in the inoculum given to the hamsters re- ceiving the high dose and 100 organisms were in the inoculum given to those hamsters which received the low dose. It has been calculated that the I. D. 50 for hamsters using I: pomona (strain Wickard) is 3. 5 organisms (3). Bauer (2) reported that there was an inverse relationship be- tween the size of the inoculum and the duration of survival of infected hamsters. In the present experiment, hamsters inoculated with the high dose were moribund and died earlier than those inoculated with the low dose. The shorter course of infection was evident not only in survival time but also in the earlier appearance of gross and microscopic lesions, in earlier positive blood cultures and in earlier detectable antibodies. The strain of L_ pomona used in these experiments did not induce a chronic infection in any of the experimental hamsters. By day 9 the 20 hamsters which received approximately 100 organisms died or were moribund. It would have been desirable to induce a chronic infection in order to possibly demonstrate brain lesions, and chronic kidney and liver lesions. Lewis and Grey (26) were able to induce a chronic infection in gerbils by giving fewer than 1, 000 organisms. Gerbils receiving larger numbers of leptospirae died during the acute phase of the disease. By receiving smaller number of organisms the animals evidently were able to produce sufficient antibody before the leptospirae had reached a concentration which would have been lethal. The organ- isms present in the renal tubules survived and produced a Chronic infection. This diversity in results between hamsters and gerbils may be due to the difference in host susceptibility and/or variations in the virulence of the infecting organisms. On day 7, the concentration of hemoglobin and packed-cell volume decreased to levels below normal in hamsters inoculated with the high dose. This suggests that some hemolysis took place around the seventh day after infection. This reaction was probably caused by the release of leptospiral hemolysin in the blood. This observation has been reported by other investigators (2, 42). Starting on day 8, increases in hemoglobin concentration and packed-cell volume were noticed. This was considered an indication of hemoconcentration which may have been a result of dehydration due to the effects of the disease. 21 The relative and absolute neutrophilia and lymphocytopenia which were observed starting day 7 (high dose) and day 6 (low dose) have been observed by Bauer (3). The blood cultures were all positive through all days of the experiments which indicated that the agent was still present in the blood at the time of death or sacrifice of the animals. Although antibody production was detected by the agglutination-lysis test as early as the 5th day in animals receiving the high dose, and starting on day 8 in animals inoculated with the low dose, antibody production was evidently not in sufficient concentration to enable the hamsters to overcome the overwhelming leptospiremia. Although renal lesions were seen as early as day 2 in hamsters inoculated with the high dose, no leptospirae could be demonstrated by guinea pig inoculation from these hamsters. The inability to demonstrate organisms by guinea-pig inoculation may be due to the possibility that these lesions are a sequel to the leptospiremia. The failure to isolate leptospirae from renal tissue may also be due to the possibility that an infective dose of the organisms was not present in the inoculum. No gross or microscopic lesions were seen in lungs of infected animals in the experiments. This finding contradicts the observation by some workers (19, 24, 43) who found gross and/or microscopic lesions in lungs of infected hamsters. 22 The greyish-white foci in the kidneys which were seen as early as day 5 (high dose) and starting on day 7 (low dose) have been described in other species (11, 23, 38). The microscopic appearance of the kidney sections demonstrated extensive degenerative changes; some necrosis, many hyaline casts and lymphocytic infiltration. However, the lymphocytic infiltration was not as extensive as has been reported in other species (23, 31). The renal changes were the most logical explanation for the dramatic rise in blood nonprotein nitrogen. Therefore the term uremia to characterize the terminal phase of the syndrome could be justified. The lesions in the livers of the infected animals consisted of albuminous degeneration with hepatic cell individualization in animals infected with both dosage levels. However, proliferation of fibroblasts with lymphocytic infiltration were found in hamsters infected with the low dose and which were killed on day 9. This may be an indication of the passing of the lesion into the subacute stage. Brain lesions in leptospirosis have been described in man (22) and in domestic animals (31). These lesions have been found to occur in subacute or chronic infections with _I_.l_ pomona. The nervous symptoms accompanying L pomona infection in humans and other domesticated animals are due to encephalomeningitis characterized by perivascular lymphocytic infiltrations. The absence of brain or 23 spinal cord lesions in hamsters may be due to the overwhelming leptospiremia which terminated in death before giving the animals a chance to go into the subacute or the chronic stage of infection. The nervous symptoms in infected hamsters with I: pomona were likely due to uremia resulting from acute renal failure. When these experiments were started it was hoped that some relationship could be established between the nervous signs and histopathologic changes shown by hamsters with leptospirosis and those shown in humans and other domestic animals suffering from this disease. The nervous symptoms in infected hamsters with I; pomona were thought to be due to uremia resulting from acute renal failure and animals died before lesions of meningoencephalitis could be seen. In man and other domestic animals the nervous symptoms are associated with inflammatory changes in the brain and meninges. 24 SUMMARY AND CONC LU SIONS A series of experiments using 91 hamsters was conducted to study experimental Leptospira pomona (strain Ohio) infection. In addi- tion to gross and microscopic pathological studies; serological, bac- teriological, hematological and clinical observations were made. Marked central nervous system disturbances were noticed as early as day 6 in hamsters which received the high dose (approximately 2, 500 organisms) and on day 8 in animals inoculated with the low dose (approximately 100 organisms). The first microscopic renal lesions were observed in the renal cortices of hamsters inoculated with the high dose by day 2. These lesions consisted of albuminous degeneration, vacuolization and some necrosis as evidenced by pyknosis of the epithelial cells in the proxi- mal and distal convoluted tubules with a few intertubular and pericor- puscular lymphocytic foci. By day 6, in addition to these lesions, hyaline casts were seen in the lumens of the renal tubules. There was also partial disappearance of tubular cells with areas of intertubular hemorrhage. The liver had areas of albuminous degeneration and hepatic cell individualization. Demonstrable microscopic renal lesions were present by day 6 in animals inoculated with the low dose. These lesions consisted of a few intertubular and pericorpuscular foci of lymphocytic infiltration. 25 By day 7, in addition to the lymphocytic foci, the renal tubular epi- thelium had marked albuminous degeneration with vacuolization. Some of the Bowman's capsules appeared to be slightly thickened. There were also numerous hyaline casts in the lumens of the tubules with a few areas of perivascular lymphocytic infiltration. The liver had areas of albuminous degeneration and hepatic cell individualization with perivascular lymphocytic infiltration in the areas of the portal triads. By day 9, early proliferation of fibroblasts and leukocytic infiltration were present in the portal triad regions. Gross renal lesions consisting of grayish-white foci approxi- mately 0. 5 mm. in diameter on the cortical surface were observed in hamsters inoculated with the high dose starting on day 5. Similar lesions were seen in hamsters inoculated with the low dose starting on day 7. No significant microscopic lesions were seen in the brains of infected hamsters, even in those showing marked central nervous system disturbances. The concentration of hemoglobin and the packed-cell volume decreased to levels below normal on day 7 in hamsters inoculated with the high dose. On post-inoculation day 8, the hemoglobin concentration and packed-cell volume values reached a level considerably higher than normal, indicating hemoconcentration. Only terminal hemocon- centration was noticed on days 8 and 9 in animals receiving the low dose. 26 Blood nonprotein-nitrogen determinations showed that there was a progressive rise starting on day 7. Terminal values of 424 mg. /100 m1. , approximately ten times the normal levels of 40 mg. / 100 m1. , were recorded. The dramatic rise in blood nOnprotein nitrogen was believed to be due to acute renal failure. Central nervous system disturbances were postulated to be a result of the severe uremia. All blood cultures from infected animals were positive, includ- ing cultures made from infected animals showing significant antibody titers. The first detectable antibody production was on day 5 in hamsters inoculated with the high dose, and on day 8 in hamsters inoc- ulated with the low dose. Leptospira pomona was found to be present in the kidneys of hamsters inoculated with the high dose starting on day 4, and in the brain starting on day 6. In the case of hamsters inoculated with the low dose, 1; pomona was present in the kidney tissue starting on day 6 and in the brain starting on day 8. From this investigation it was concluded that: (1) acute renal failure appeared to be the primary cause of death; (2) insufficient antibody production to overcome leptospiremia was postulated to be a contributory factor in the death of hamsters; (3) there were no detectable brain lesions which could be correlated with the clinical nervous system disturbances; (4) the central nervous system disturbances were postulated to be a result of uremia; (5) no chronic infection could be induced with I: pomona (strain Ohio) due to the lethal effects of this strain. 27 TABLE 1 SUMMARY OF AVERAGE HEMATOLOGICAL VALUES 28 J J Num be r of Hgb Pcv Wbc an animals included Controls 14. 5 46. 1 7, 210 43 7 High Dose PI day 3 14. 1 45. 3 * * 2 PI day 5 l3. 6 43. 8 * ’1‘ 3 PI day 7 9. 5 36. 3 ’1‘ * 3 PI day 8 18. l 49. 8 ’1‘ * 2 Low Dose PI day 3 14.0 45.4 6,810 52 2 PI day 4 l3. 4 43. 4 7, 650 80 3 PI day 5 13. 2 43. 5 7, 000 50 3 PI day 6 13. 5 43. 7 7,150 42 3 PI day 7 l3. 7 43. 9 6, 000 124 3 PI day 8 19.5 51.6 13,210 410 3 PI day9 18.6 50.9 11,910 424 3 * Not attempted Hgb = hemoglobin in grams per 100 m1. of blood Pcv = packed-cell volume expressed as per cent Wbc = leukocytes per cmm. an =non protein nitrogen in mg. per 100 ml. of blood P I post-inoculation High Dose = 0. 5 ml. of a 1:1 dilution of infected guinea pig blood 1 m1. of a 1:100 dilution of infected guinea pig blood Low Dose 29 econ boa 0.33 mucdoo ou>oox52 130R. v“. N NeNN 2 NHNN i e o o N .38 E N eemN NN NNNN Ne N o o N .38 E E 82 NN oweN Ne a o o s .33 E z NNNN NN Name we N a o e Nee E e oflN Ne eeNN Na N o o N .38 E N SN 3 NEVN Ne - N o e N .33 E N EeN a... NNFN 2. H o o N Nee E Ammop 30$ when—mam: pouoomcH N ON Ne N o o N New E N ON 1. 3 N o o N .38 E N om Ne N o o N .38 E a No 1. N N o o N .38 E 2.5% :35 whoumEms @3035 N AcNNe oe NNNN N a o o 20.380 3.3 8.30694 o>fimaom oudHOmNL/w winged 10.83: :33 SEE ou>ooc02 poucoa nocmmom uOmmm ouewoonmecnd HEmonouon poucoEwom uwmmcoz mHDJ<> MO§H>< l .HZDOO HHWOOMDMA AdflfiZMMMhhHQ N Him m<.H. 30 TABLE 3 ANTIBODY TITERS FOR 11:. POMONA IN SERA OF INFECTED HAMSTERS PI Daya Dose used (Agglutinin titerb 2 High dose - 3 H - 4 " - 5 " lo1+ 6 " lo2+ ,7 H 102++ 8 H 102 +++ 3 Low dose - 4 " _ 5 " - 6 " .. 7 " - 8 H 101+ 9 " lo2+ a PI = post-inoculation b The titer is expressed as the logarithm of the reciprocal of the highest dilution of serum in which leptospirae were agglutinated or lysed. + = 25 per cent agglutination or lysis ++ = 50 per cent agglutination or lysis +++ = 75 per cent agglutination or lysis TAB LE 4a SUMMARY OF GUINEA PIG INOCULATIONS WITH HOMOGENIZED TISSUES FROM HAMSTERS INOCULATED WITH THE HIGH DOSE 31 Day after infection Brain Kidney 2 — - 3 - _ 4 - + 5 - + 6 + + 7 + + 8 + + TABLE 4b SUMMARY OF GUINEA PIG INOCULATIONS WITH HOMOGENIZED TISSUES FROM HAMSTERS INOCULATED WITH THE LOW DOSE Day after infection Brain Kidney 3 - - 4 - - 5 - - 6 - + 7 - + 8 + + 9 + + + indicates that the serum of the guinea pigs contained antibodies for .1: pomona at a dilution of 10'2 or higher two to three weeks after inoculation with the respective tissue suspensions of the infected hamsters. - indicates that the serum of guinea pigs was negative. p . 6 Fig. 1. --Kidney at day 2. A. Pyknotic nuclei in tubular cells. B. Lymphocytes in the intertubular and pericorpuscular areas. C. Vacuolization of tubular cells. x720. 32 33 ‘su. *fi \ J ' c r 5 I“ #, x . , ~96 ’ “fir ‘ i, . .a,. , ‘ ' -fl . .. ~.. / &. . 461/4 ,. KW/ '\ f ‘ Fig. 2. --Kidney at day 6 demonstrating hyaline casts in lumen of tubules. x250. 34 A. Vacuolization of tubular cells. Fig. 3. --Kidney at day 6. B. Hyaline casts in lumen of tubules. x720. 35 - Fig. 4. —-Kidney at day 6. A. Hyaline casts in lumen of tubules. B. Pyknotic nuclei in tubular cells. C. Vacuolization of tubular cells. D. Partial disappearance of tubular epithelium. E. Intertubular hemorrhage. x720. area. Fig. 5. --Kidney at day 6. B. Pyknotic nuclei in tubular cells. x7 20. A. Lymphocytes in intertubular 36 38 Fig. 7. --Kidney at day 6 demonstrating lymphocytes in pericorpuscular area. x720. 39 Fig. 8. --Kidney at day 7 demonstrating numerous hyaline casts in lumen of tubules. x250 . 40 Fig. 9. -—Kidney at day 7 showing marked vacuolization x7 20. of tubular cells. 41 Fig. 10. --Kidney at day 7. A. Vacuolization of tubular cells. B. Slight thickening of Bowman's capsule. x720. 42 Fig. 11. --Kidney at day 7 demonstrating perivascular lympho- cytic infiltration. x720. Fi 1.g 12. nivd iud aizl atio n(lowdos e). x.720 -ivL aaeontdy7dm nsraineaicttghpt ell 43 44 Fig. 13. --Liver at day 7 demonstrating perivascular lympho- cytic infiltration in the area of portal triads. x720. 45 w \ u. I. a _ K a \ 1 A. Hepatic cell individualization. l4. --Liver at day 9. Fig. x250. V. ‘ u. .. I . \ O ./ .h'” a L . . . rdwkul 1.11”,” V. 4. , .. .. l - c i . \‘_ .... v 9 JV B. 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